Assessment Report - Europadissemination.echa.europa.eu/Biocides/ActiveSubstances/... ·...

Regulation (EU) No 528/2012 concerning the making available on the market and

use of biocidal products

Evaluation of active substances

Assessment Report

Margosa extract, cold-pressed oil of azadirachta indica seeds without shells extracted with super-critical carbon dioxide

Product-type 19 (Repellents and attractants)

January 2017

eCA: Germany

Margosa Extract, cold-pressed oil of Azadirachta indica seeds without shells extracted with super-critical carbon dioxide

Product-type 19 April 2017

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CONTENTS

1. STATEMENT OF SUBJECT MATTER AND PURPOSE ............................................... 4

1.1. Procedure followed ................................................................................................................................ 4

1.2. Purpose of the assessment report .................................................................................................. 4

2. OVERALL SUMMARY AND CONCLUSIONS ................................................................... 5

2.1. Presentation of the Active Substance ........................................................................................... 5 2.1.1. Identity, Physico-Chemical Properties & Methods of Analysis ................................................... 5 2.1.2. Intended Uses and Efficacy ..................................................................................................................... 6 2.1.3. Classification and Labelling ..................................................................................................................... 6

2.2. Summary of the Risk Assessment ................................................................................................... 8 2.2.1. Human Health Risk Assessment............................................................................................................ 8

2.2.1.1. Hazard identification and effects assessment .......................................................................... 8 2.2.1.2. Exposure assessment and risk characterisation ................................................................... 12 2.2.1.3. Risk Characterisation ....................................................................................................................... 12

2.2.2. Environmental Risk Assessment ......................................................................................................... 14 2.2.2.1. Fate and distribution in the environment ................................................................................ 14 2.2.2.2. Hazard identification and effects assessment ........................................................................ 16 2.2.2.3. PBT Assessment and Exclusion Criteria ................................................................................... 17 2.2.2.4. Exposure assessment and risk characterisation ................................................................... 20

2.3. Overall conclusions ...............................................................................................................................24

2.4. Requirement for further information related to the reference biocidal product ....24

2.5. List of endpoints .....................................................................................................................................24

APPENDIX I: LIST OF ENDPOINTS ..................................................................................... 25

Chapter 1: Identity, Physical and Chemical Properties, Classification and Labelling ....25

Chapter 2: Methods of Analysis .................................................................................................................30

Chapter 3: Impact on Human Health ......................................................................................................31

Chapter 4: Fate and Behaviour in the Environment .......................................................................33

Chapter 5: Effects on Non-target Species ...........................................................................................36

Chapter 6: Other End Points ......................................................................................................................37

APPENDIX II: LIST OF INTENDED USES ........................................................................ 38

APPENDIX III: LIST OF STUDIES ........................................................................................ 39



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1. STATEMENT OF SUBJECT MATTER AND PURPOSE

1.1. Procedure followed

This assessment report has been established as a result of the evaluation of the active substance margosa extract, cold-pressed oil of azadirachta indica seeds without shells extracted with super-critical carbon dioxide as product-type 19 (Repellents and attractants), carried out in the context of the work programme for the review of existing active substances provided for in Article 89 of Regulation (EU) No 528/2012, with a view to the possible approval of this substance.

Margosa extract, cold-pressed oil of azadirachta indica seeds without shells extracted with super-critical carbon dioxide (CAS no. 84696-25-3) was notified as an existing active substance, by Terra Nostra GmbH, hereafter referred to as the applicant, in product-type 19.

Commission Regulation (EC) No 1062/2014 of 4 August 20141 lays down the detailed rules for the evaluation of dossiers and for the decision-making process.

On 26th of April 2006, the German Competent Authority received a dossier from Terra Nostra GmbH. The evaluating Competent Authority (eCA) refused the dossier as incomplete for the purpose of the evaluation on 31st of October 2006.

After that Terra Nostra Registration Service Srl, hereafter referred to as applicant, informed the Commission of their intention to take over the role of participant as regards the active substance/product type combination.

On 31st of May 2010, the German Competent Authority received a dossier from the applicant. The eCA accepted the dossier as complete for the purpose of the evaluation on 31st of August 2010.

On 3rd December 2015, the eCA submitted to the Agency (ECHA) and the applicant a copy of the evaluation report, hereafter referred to as the competent authority report.

In order to review the competent authority report and the comments received on it, consultations of technical experts from all Member States (peer review) were organised by the the "Agency” (ECHA ). Revisions agreed upon were presented at the Biocidal Products Committee and its Working Groups meetings and the competent authority report was amended accordingly.

1.2. Purpose of the assessment report

The aim of the assessment report is to support the opinion of the Biocidal Products Committee and a decision on the approval of margosa extract, cold-pressed oil of azadirachta indica seeds without shells extracted with super-critical carbon dioxide for product-type19, and, should it be approved, to facilitate the authorisation of individual biocidal products. In the evaluation of applications for product-authorisation, the provisions of Regulation (EU) No 528/2012 shall be applied, in particular the provisions of Chapter IV, as well as the common principles laid down in Annex VI.

For the implementation of the common principles of Annex VI, the content and conclusions of this assessment report, which is available from the Agency web-site shall be taken into account. 1 COMMISSION DELEGATED REGULATION (EU) No 1062/2014 of 4 August 2014 on the work programme for the systematic examination of all existing active substances contained in biocidal products referred to in Regulation (EU) No 528/2012 of the European Parliament and of the Council. OJ L 294, 10.10.2014, p. 1


Product-type 19 March 2017

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However, where conclusions of this assessment report are based on data protected under the provisions of Regulation (EU) No 528/2012, such conclusions may not be used to the benefit of another applicant, unless access to these data for that purpose has been granted to that applicant.

2. OVERALL SUMMARY AND CONCLUSIONS

2.1. Presentation of the Active Substance

2.1.1. Identity, Physico-Chemical Properties & Methods of Analysis

The active substance margosa extract is a CO2-extract derived from cold-pressed Neem Seed Oil without shells (Azadirachta indica) using the manufacturing method developed by the applicant. It was clarified that the extraction including shells influences significantly the composition of the final extract. Furthermore, the shell is the nutrient medium for Aspergillus, which produced aflatoxins. Therefore, the extraction without shell is regarded an important criteria of the manufacture and it included in the description name of the substance.

Margosa extract is a plant extract and consists mainly of the limonoids azadirachtin A, azadirachtin B, nimbin, salannin, together with co-extracted fatty acids (bound in glycerides) and a small amount of water. Margosa extract may contain traces of aflatoxins. Based on the rules set out for the identification and naming of substances the name of UVCB substances should include the manufacturing process and the species from which it is extracted. Therefore, the extract is named margosa extract, cold-pressed oil of azadirachta indica seeds without shells extracted with super-critical carbon dioxide. However, within this report the short form margosa extract will be used for convenience.

Since margosa extract (UVCB) as such is regarded as the active substance, the physico-chemical parameters are in general determined on the whole extract and not on the individual constituents. Margosa extract is thermally stable up to 340°C. The vapour pressure should be 3.8 x 10-7 hPa at 20°C. Furthermore, calculated values of the vapour pressure for limonoids and azadirachtins are given in the LoEP: The water solubility was calculated for some significant constituents. The partition coefficient was determined for some significant constituents. Sufficiently validated analytical methods for the determination of azadirachtin A, the other limonoids and other constituents in the technical material are available. The content of aflatoxins is determined An acceptable residue analytical method is available for detecting the analytical lead compounds nimbin and salannin in air. Sufficiently validated primary and confirmatory methods for detecting residues of the analytical lead compounds nimbin and salannin in soil, drinking water and surface water are missing and should be provided. Other analytical methods are not required, because no relevant residues are expected in food and feeding stuffs. Because the active substance is not classified as toxic or very toxic, residue analytical methods in body fluids and tissues are also not required. Identity, Physico-chemical Properties and Method of Analysis of Margosa extract (REPRESENTATIVE PRODUCT)

Since the representative product is equal to the active substance, the information on the active substance applies.

Missing information regarding:

▫ Acidity/alkalinity and if necessary pH value (1% in water)

▫ Storage stability — stability and shelf-life. Effects of light, temperature and humidity on technical characteristics of the biocidal product; reactivity towards container material



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(the possible increase of the aflatoxins content during storage should be addressed in this context)

▫ Technical characteristics of the biocidal product, e.g. wettability, persistent foaming, flowability, pourability and dustability

will have to be submitted within the process of biocidal product authorization (see section 2.4).

2.1.2. Intended Uses and Efficacy

The assessment of the biocidal activity of the active substance demonstrates that it has a sufficient level of efficacy against the target organism(s) and the evaluation of the summary data provided in support of the efficacy of the accompanying product, establishes that the product may be expected to be efficacious.

Margosa extract is intended to be used as a repellent to deter ants (e.g. Lasius niger) from entering buildings. It is aimed at non-professional users to be used in private houses. The intrinsic repellent efficacy of the active substance was proven in a simulated use trial conducted with the black garden ant (L. niger). Margosa extract was applied in a 2cm wide line, and the ants were deterred from crossing that line, even though the only food and water sources were behind the treated area. The intended use is the application of a biocidal product containing margosa extract on doorsteps and windowsills in a 2cm wide line, serving as a barrier for ants trying to enter the house from the outside. The application rate was 4 g margosa extract per linear meter on open-pored beech plywood, and 1.6 g margosa extract per linear meter on acacia terrace board. More specific testing should be assessed during product authorization, as the substance used in the efficacy trials was 100% margosa extract.

The mode of action responsible for the repellent activity of margosa extract is not known. As the extract contains a mixture of limonoids, it is probably a combined effect of these that leads to a repellent effect. Since margosa extract is a non-lethal repellent, it does not exert a selective pressure for developing resistance. Ants that are repelled from entering a premise are free to feed in different, unprotected areas. Development of resistance is not expected.

In addition, in order to facilitate the work of Member States in granting or reviewing authorisations, the intended uses of the substance, as identified during the evaluation process, are listed in Appendix II.

2.1.3. Classification and Labelling

Currently, a harmonised classification according to Regulation (EC) No 1272/2008 (CLP Regulation) is not available for margosa extact.

The toxicological properties of margosa extract, cold-pressed oil of azadirachta indica seeds without shells extracted with super-critical carbon dioxide as evidenced in the submitted toxicity studies do not require classification and labelling. The applicant has proposed considering the safety waring “Keep out of reach of children”. In the light of the reported intoxications of children with neem oil, the RMS supports the use of this precautionary statement.



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Table 2-1 Proposed classification of active substance based on Regulation (EC) No

1272/2008

Hazard classes, hazard categories, hazard statements

Wording

Classification - -

Remark:

None proposed

Table 2-2 Proposed labelling of active substance based on Regulation (EC) No

1272/2008

Labelling Wording Hazard pictograms, signal words, hazard statements precautionary statements

P102 Keep out of reach of children

Classification and Labelling of the biocidal product Margosa Extract

Table 2-3 Proposed classification of the biocidal product based on Regulation (EC)

No 1272/2008

Hazard classes, hazard categories, hazard statements

Wording

Classification None None

Remark:

Classification of the biocidal product is not required.

Table 2-4 Proposed labelling of the biocidal product based on Regulation (EC) No

1272/2008

Labelling Wording Hazard pictograms,

signal words,

hazard statements

precautionary statements

None None



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Remark:

Classification and labelling of the biocidal product is not required

2.2. Summary of the Risk Assessment

2.2.1. Human Health Risk Assessment

2.2.1.1. Hazard identification and effects assessment

Margosa extract is a CO2-extract derived from cold-pressed neem seed oil without shells (Azadirachta indica) using the manufacturing method developed by the applicant and acts as a repellent against worker ants. As a botanical extract it belongs to the group of substances with unknown or variable composition, complex reaction products or biological material (UVCB) with unspecified molecular and structural formula. The total content of limonoids was determined to be 2.7 ± 0.4 % including azadirachtin A. While in other margosa extracts azadirachtin A is regarded as lead compound, margosa extract in this CAR is considered different in composition and properties from other margosa extracts (e.g. NeemAzal, Fortune Aza, ATI-720 = NPI 720). This also applies to the content of aflatoxins which is much lower in this margosa extract (aflatoxin B1: < 2 µg/kg (0.0000002% w/w; sum of aflatoxins B1, B2, G1, G2: < 4 µg/kg (0.0000004% w/w). is, therefore, considered another substance. Consequently, studies performed with one of the above-mentioned extracts are not considered in this CAR and read across to those extracts is not applicable. Likewise, toxicity studies with neem products found in the open literature were considered not relevant for margosa extract due to different starting material or extraction procedures.

The specification of margosa extract is covered by the batches of margosa extract as tested in the toxicity studies. Waiving Concept

Chronic and carcinogenicity studies with margosa extract have not been performed. The waiving of such studies is deemed acceptable in view of the lack of pertinent findings in genotoxicity tests and repeat dose studies.

According to “Guidance on information requirements” – Guidance on regulation (EU) No 528/2012… “The long-term toxicity study (≥ 12 months) does not need to be conducted if:

• Long-term exposure can be excluded and no effects have been seen at the limit dose in the 90-day study, or

• A combined long-term repeated dose/carcinogenicity study (8.11.1) is undertaken”

As only mild adverse effects (absolute and relative liver weight increases) were observed in the 90-day study in rats up to approx. 1000 mg/kg bw/day and long-term exposure is not expected according to the use scenarios submitted by the applicant, omission of carcinogenicity study is justified. Therefore, a carcinogenicity study is deemed not necessary because the overall conclusion from the genotoxicity tests is that margosa extract is not genotoxic. Furthermore, the subacute and subchronic feeding studies did not reveal any finding that could be correlated with a pre-neoplastic event or lead to the expectation of different or more severe effects after chronic exposure. The effects in both studies were limited to liver changes. The subchronic



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NOAEL/LOAEL for liver weight increase (450/960 mg/kg/day) is almost 500/1000 times higher than the highest predicted human exposure. Tumour promotion as result of adaptive liver enlargement can therefore be ruled out at doses that humans are likely to be exposed to as a result of use as repellent containing Margosa extract.

Studies on the effects of Margosa extract on fertility and postnatal development of offspring have not been conducted. A two-generation reproduction study is not deemed necessary, since human exposure is low when compared to the subchronic oral NOAEL of 450 mg/kg bw/day. Reproductive organ weights were not altered in this study and histopathological investigation did not reveal any abnormalities of these or any other tissues that might affect reproduction. This suggests that Margosa extract has no potential for reproductive toxicity at doses approaching the limit dose of 1000 mg/kg bw/day. Considering that exposure during or after use of Margosa extract is only 1.28/1.19 mg/kg bw/day in adults and 1.37 mg/kg bw/day in toddlers, there is no concern for reproductive or developmental effects associated with the use of Margosa extract as repellent against worker ants.

In addition, a prenatal toxicity study in a second species (rodent) is not considered necessary, based on the results of a dermal study in rabbits where no embryo- or foetotoxicity was revealed at any dose, even in the presence of local maternal toxicity.

In general, the limitations of the submitted data package were considered by applying an additional assessment factor for uncertainties in the derivation of reference values for margosa extract.

Absorption, Distribution, Excretion, and Metabolism

No studies on absorption, distribution, metabolism and excretion were submitted. Such ADME studies are performed with radioactively labelled compounds.

Margosa extract contains only small amounts of limonoids. As the active substance is a complex mixture of various compounds, margosa extract is regarded as active substance in accordance with the “Guidance Document on Botanical Active Substances Used in Plant Protection Products“ (SANCO/11470/2012- rev.8, 20 March 2014). Hence, results from ADME studies with labelled azadirachtin A are not meaningful for margosa extract.

For risk assessment purposes, absorption of the active substance margosa extract from oral and inhalation exposure is assumed to be 100 % since actual data are not available. For dermal absorption default values according to EFSA guidance (EFSA 2012) are applied.

Acute Toxicity

Margosa extract is not acutely toxic when administered orally, dermally or by inhalation. It is not irritating to the skin or eyes of rabbits following single application and did not sensitise the skin of guinea pigs. Since irritation of the skin was not observed in a primary dermal irritation study, the conditions for classifying the active substance as irritating to the skin are not met.

Short-term Toxicity

Slight, reversible increases in liver weight were the only finding in 28-day and 90-day feeding studies with margosa extract in rats. This effect is not considered in the 90-d study as liver weight increases were up to 18 % compared to controls. The NOAEL was 990 mg/kg bw/d in the 28-day study and 450 mg/kg bw/d in the 90-day study. In a 28-day dermal study with margosa extract in rats, no systemic effects were observed. Slight erythema with or without desquamation was noted in all males and females receiving



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500 mg/kg/day towards the end of the first week of dosing. In the dose groups receiving 1000 mg/kg/day incidence and time of appearance were similar and the grading ranged from slight to severe. The skin irritation disappeared in both groups during the second week of dosing and no further changes became apparent after that timepoint. Thus, classification for skin irritation following repeated exposure is not considered required.

Genotoxicity

Margosa extract did not show a mutagenic potential in bacteria and in Chinese hamster lung fibroblasts (V79 cells). Slightly positive effects for clastogenicity were observed with multiple damaged metaphases at the highest tested concentration (5000 µg/mL). This effect was observed in the presence of metabolic activation in the in vitro chromosomal aberration test in V79 cells. However, margosa extract was not genotoxic in the in vivo micronucleus test in mice exposed at dose levels up to and including 2000 mg/kg bw. In conclusion, based on the results of in vitro and in vivo genotoxicity tests, including adequate positive and negative controls, margosa extract is unlikely to pose a genotoxic risk to humans.

Chronic Toxicity/ Carcinogenicity

No chronic or carcinogenicity study has been submitted for margosa extract. The waiving of such a study is deemed acceptable in view of the lack of pertinent findings in genotoxicity tests and repeat dose studies (see also waiving concept).

Reproduction Toxicity

After dermal application of margosa extract to pregnant rabbits, local skin irritation occurred in all treated groups. Severity of the observed erythema and oedema was high in the highest dose group. It was considered that classification for skin irritation following repeated exposure of margosa extract was clearly demonstrated but severity in the relevant doses was considered not sufficient for classification according to CLP. Consequently, STOT-RE for skin irritation was not proposed. A slight, dose-related tendency towards reduction of maternal body weight gain was observed. Net body weight loss was observed in mid and high dose females but did not attain statistical significance. Overall, reductions in body weight gain in the mid and high dose groups were not accompanied by any sign of toxicity and the differences in terminal body weights were only marginal. This effect is, therefore, considered not to be biologically relevant. No embryo- or foetotoxicity was apparent. Small foetuses in all groups, including the control, were found mostly in litters of larger size and it appears that the higher proportion of such litters, rather than the treatment, contributed to the slightly increased number of small foetuses in the high dose group. Thus the maternal and the developmental NOAEL is 800 mg/kg bw/d, corresponding to the highest dose tested. Neither a prenatal toxicity study in a rodent species nor a two-generation study have been conducted for margosa extract (see also waiving concept).

Neurotoxicity

No specific studies for neurotoxic properties have been submitted. Available repeat dose studies in rats did not show any neurotoxic effects.

Further Studies

None



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Medical Data

No reports on adverse effects of margosa extract are available. Medical surveillance of an unknown number of plant workers occupationally handling with margosa extract did not reveal any obvious toxicity in humans. Reviews of the open literature on various neem products with unknown composition demonstrates evidence of poisoning incidents and side-effects in the use of neem products but are considered not applicable to margosa extract, "Margosa Oil" or "Neem Oil" is used as a traditional medicine in Asia and Africa. In case reports severe intoxications are described in children predominantly following oral administration of “Margosa Oil” as a home remedy for the treatment of various diseases (e.g. common cold, deworming). Margosa extract exerts no acute toxicity up to the limit dose of 2000 mg/kg bw in rats. In addition, no signs of toxicity were observed in repeated dose studies in rats (up to 90 days) and rabbits (treatment day 6-28) following oral (rats) and dermal (rats and rabbits) exposure. Hence, poisoning from margosa extract up to the limit dose of 2000 mg/kg bw is not to be expected.

Summary & Conclusion

Margosa extract is of low toxicity after acute and short-term/subchronic exposure and requires no classification/labelling according to Regulation (EC) No 1272/2008. Dermal irritation was noted after repeated application of the extract to the skin in two species (rats, rabbits), but not in the rabbit following single application. Thus, the conditions for classifying the active substance as irritating to the skin are not met In addition, the pregnant rabbits exhibited a reduction in body weight gain that may be attributed to maternal local irritation as the conceptuses remained unaffected.

The following systemic Acceptable Exposure Levels (AELs) are proposed:

Acute exposure: No endpoints relevant for an AELacute are seen in the single-dose toxicity studies. Due to the lack of acute toxicity, the AELacute was derived from the 28-d study in rats following oral exposure as a conservative approach. The NOAEL of 990 mg Margosa extract/kg bw/d from the 28-day oral rat study is considered appropriate to derive an AELacute. Slight increases of liver weights were observed which were not considered adverse. Applying an assessment factor of 200 and assuming 100 % oral absorption, a systemic acute Acceptable Exposure Level (AELacute) of 5 mg/kg bw/d is proposed for short-term exposure to margosa extract. Medium-term exposure: Repeated-dose studies up to 90-d and prenatal toxicity studies are taken into consideration to derive the AELmedium-term. The NOAEL of 450 mg Margosa extract/kg bw/d from the subchronic (90 day) oral rat study is considered appropriate to derive an AELmedium-term. Applying an assessment factor of 200 and assuming 100 % oral absorption, a systemic medium-term Acceptable Exposure Level (AELmedium-term) of 2.30 mg/kg bw/d is proposed for medium-term exposure to margosa extract. Long-term exposure: Chronic toxicity studies were waived based on the absence of any treatment-related adverse effects in repeat dose studies in rats and the absence of genotoxic effects mutagenicity assays. The most relevant NOAEL was observed in the subchronic (90 day) oral toxicity study in rats and it appears reasonable to use this value for the derivation of the AELlong-term. Applying a combined assessment factor of 400 (2 x 2 x 100) and assuming 100 % oral absorption, a systemic long-term Acceptable Exposure Level (AELlong-term) of 1.1 mg/kg bw/d is proposed for long-term exposure towards margosa extract.



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ADI/ARfD:

If margosa extract is applied in accordance with the intended use no residues of margosa extract are to be expected in food of plant or animal origin. Therefore, no ADI and ARfD are derived.

2.2.1.2. Exposure assessment and risk characterisation

Exposure of Professionals

The biocidal product is a consumer product. It is not expected that professionals with a profession that does not involve biocides (e.g. housekeeper) use the product in their work environment. In that case, the rapporteur assesses that the pattern of use is similar to the pattern of consumers.

Therefore, exposure and risk of professionals are estimated to be in the same range as of non-professionals (see chapter: “Exposure of Non-professionals” and “Risk Assessment for Non-Professionals”).

Exposure of Non-Professionals and the General Public

The biocidal product margosa extract (100% active substance) is applied on doorsteps and windowsills as a barrier against ants and brushing with an application rate from 20 mg/cm2 in materials like open-pores beech plywood to 53 mg/cm2 in smoother materials like pre-treated terrace acacia board. Exposure may occur directly during application (including brush cleaning) or indirectly by contact with treated surfaces. The most relevant secondary exposure scenarios are exposure of adults by contact with bare feet and exposure of toddlers by touching treated surfaces with hands and subsequent oral ingestion. Primary exposure

Application by brushing (inc. brush cleaning) Systemic inhalation exposure: 5.31 x 10-5 mg/kg bw/d

Systemic dermal exposure: 1.28 mg/kg bw/d

Total systemic exposure: 1.28 mg/kg bw/d

Secondary exposure

Exposure of adults by contact with bare feet Systemic dermal exposure: 1.19 mg/kg bw/d Total systemic exposure: 1.19 mg/kg bw/d Exposure of toddlers by dermal and oral contact Systemic dermal exposure: 0.95 mg/kg bw/d Systemic oral exposure: 0.42 mg/kg bw/d Total systemic exposure: 1.37 mg/kg bw/d

2.2.1.3. Risk Characterisation

Risk Assessment for Professionals

The biocidal product is a consumer product. It is not excluded that professionals with a profession that does not involve biocides (e.g. housekeeper) use the product in their work environment. In that case, the rapporteur assesses that the pattern of use is similar to the



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pattern of use of consumers.

Therefore, exposure and risk of professionals are estimated to be in the same range as of non-professionals (see chapters: ‚Exposure of Non-professionals‘ and ‚Risk Assessment for Non-Professionals‘).

Safety Measures for Professionals

See Chapter: Safety Measures for Non-Professionals

Risk Assessment for Non-Professionals and the General Public

Summary risk assessment for non-professional primary exposure to margosa extact

Exposure scenario Exposure adults

(mg/kg bw/d)

AEL/NOAEL (mg/kg bw/d)

Exposure (% of AEL)

MoE

Medium-term exposure - application of MARGOSA EXTRACT by brushing (incl. brush cleaning)

Inhalation 5.31 x 10-5 2.3/450 0.002 8474576

Dermal 1.28 2.3/450 56 352

Total 1.28 2.3/450 56 352 Summary risk assessment for secondary exposure of the general public to margosa extact

Exposure scenario Exposure (mg/kg bw/d)

AEL/NOAEL (mg/kg bw/d)

Exposure (% of AEL)

MoE

Adults Acute exposure - contact to treated surfaces with bare feet

Dermal 1.19 5.0/990 24 832

Total 1.19 5.0/990 24 832

Toddlers Acute exposure - contact to treated surfaces with hands when crawling

Dermal 0.95 5.0/990 19 1042

Oral 0.42 5.0/990 8 2357

Total 1,37 5.0/990 27 723



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Safety Measures for Non-Professionals

Primary and secondary exposure of non-professional users and the general public (including pets and domestic animals) to margosa extract after regular treatment with the biocidal product containing 100% of the active substance is acceptable with respect to human health. Specific safety measures are not required.

Based on information provided by the applicant the biocidal product dried on rough surfaces like plywood within 24 h due to absorption into the wood. However, on acacia wood it remains liquid. Consequently, labelling of the biocidal product with “Keep children away from treated surfaces until dried” is proposed.

Residues in food are not expected from the intended use. To ensure that residues in food do not occur, the following labelling is required: “Avoid direct contact or contamination of food and feedstuff.”

2.2.2. Environmental Risk Assessment

The technical a.s. margosa extract is gained by CO2-extraction from cold pressed oil from neem tree (Azadirachta indica A. Juss.) seed without shells. With 97% in total, the main components of margosa extract in Pt19 are fats and fatty acids, existent mainly as trigycerides of oleic acid, stearic acid, linoleic acid and palmitic acid. Limonoids (belonging to the chemical group of triterpenoids) are representing the second group of relevant constituents of margosa extract, with less than 2% in total. The representative product assessed within this report is identical to the a.s., therefore only the a.s. had to be evaluated within the environmental effects assessment and not a formulated b.p.

While it is known which components mostly contribute to the intended efficacy in PT19, it can also be deduced that mainly the limonoids should be regarded as relevant for (potential) adverse effects on non-target organisms in the environment. The limonoids from Neem tree are known to act as antifeedant and growth disruptor towards insects. Therefore the accompanying chemical analysis of the effect studies is based on salannin as the limonoid with the highest proportion in margosa extract. This also applies to recalculations to mean measured concentrations, if required. In addition, the effect assessment was supported by the physico-chemical properties of salannin for applying the equilibrium partitioning method.

The use of a non-labelled test substance and the non-feasibility of synthesising reference compounds result in an unavoidable limitation of findings concerning the fate and behaviour of margosa extract i.e., the impossibility to determine the pathway of degradation of the test substance and the quality and quantity of its degradation products in a number of environmental fate studies (e.g. soil and water/sediment degradation, hydrolysis, aqueous photolysis).

2.2.2.1. Fate and distribution in the environment

Biodegradation

Based on the results of a closed bottle test (OECD 301D) using margosa extract (identical with the product) the active substance was proved to be readily biodegradable fulfilling the 10-day window criterion (>60% degradation within 10 days). However, the study does not allow to draw conclusions if and to which extent the relevant compounds degrade. Based on the result of QSAR analysis none of the components azadirachtin, nimbin, salannin and stigmasterol can be considered as readily biodegradable. Considering the results of QSAR analysis and according to a decision of BPC WG ENV (III-2016), a degradation rate of k = 0 h-1 for biodegradation in sewage treatment plants is applied in the environmental exposure assessment of the active substance.



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Regarding the freshwater compartment, the rate constant for biodegradation in surface water (kbiowater) is set to zero as well due to the lack of information on the biodegradation half-lives of the relevant components. A study about the degradation behaviour of azadirachtin A and B in an outdoor mesocosm experiment, provided by the applicant, was considered only as supportive information.

Both isomers showed only low tendency to partition into bottom sediments accompanied by the dissipation of azadirachtin A and B from the water phase (DT50 of 25 and 45 days, respectively).

Also for the soil compartment, only supportive information is available from a published study, in which the degradation of azadirachtin A and B was investigated in a sandy loam at temperatures of 25°C and 15°C using a light regime for each environmental chamber of 16 h light and 8 h dark. Converting the DT50 values gained from this study according to an average EU outdoor temperature of 12°C results in a maximum DT50 of azadirachtin A of 56 days and for azadirachtin B for 75.3 days. Based on this, it can be assumed that azadirachtin A and B – if potentially entering the soil – would dissipate/be degradable. Since the tests were conducted with non-labelled material, neither mineralisation nor the formations of bound residues were quantifiable. Again, in the environmental exposure assessment of the active substance for the soil compartment no degradation was assumed due to the lack of knowledge about specific degradation rates of the relevant components.

Abiotic Degradation All three constituent limonoids of margosa extract are susceptible to hydrolysis, though the determined half-lives vary greatly. The hydrolysis rate was highest for azadirachtin (DT50 = 363.9 h at standard conditions of pH 7 and 12°C), whereas the mean hydrolysis half-life of nimbin was 1783.2 h (pH 7 and 12°C). The lowest hydrolysis rate was determined for salannin, corresponding to a DT50 of 22063.1 h (pH 7 and 12°C). Hydrolysis of the three limonoids is pH-dependent and in most cases an alkaline pH increases the hydrolysis rate, even though some results deviate from this observation. In Summary, hydrolysis might contribute to the degradation of azadirachtin and nimbin under environmental conditions, whereas it is negligible for salannin dissipation.

Photolysis in water is irrelevant for the dissipation of the three limonoids, as they do not exhibit significant absorption in the visual light spectrum. However, the limonoids are susceptible to indirect photolysis in the atmosphere with calculated half-lives of 1.3 h up to 1.7 h. But as the volatilisation of the three limonoids is negligible due to the very low vapour pressure and Henry’s Law coefficient, these processes are of minor importance for the environmental fate.

Distribution and Mobility

The adsorption behaviour to soil or sewage sludge of the constituent limonoids in Margosa extract was investigated using the HPLC method procedure according to the OECD Guidline 121. The determined arithmetic mean values for the Koc are 144 L/kg, 809 L/kg and 1766 L/kg for azadirachtin, nimbin and salannin, respectively.

Bioaccumation

A bioconcentration factor for the aquatic compartment was estimated on basis of salannin, which provides the highest log Kow of 3.5 out of the limonoids. Based on this, BCF for fish were estimated and below 100 L/kg wet weight for the limonoids (salannin: BCF = 95 L/kg ww). The calculated BCF value, BCFfish = 95 L/kg wet weight, indicated that limonoids have a low potential to bioaccumulate in aquatic organisms and would pose no unacceptable risk of biomagnification in the food chain.



16

2.2.2.2. Hazard identification and effects assessment

Aquatic compartment

A base data set of effect studies for the aquatic compartment is available for the environmental effect assessment. All tests were conducted with the pure a.s. Based on the complex composition and low solubility in water of margosa extract, the definition of a ‘difficult substance’ in accordance to OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures was met and due to the low solubility of the test substance, acetone has been used as a solvent. The stability of the test substance within the tests was monitored by analysing the concentration of salannin and, if required, recalculating from nominal to mean measured concentration based on the concentration of salannin.

One acute study with fish was provided for the test substance margosa extract. The study is considered as key study for fish. After 96 h and based on mean measured concentrations, a LC50 of 11.2 mg/L has been calculated (95 % c.i.: 9.7 – 12.8 mg/L).

One acute study with Daphnia magna was performed with the test substance margosa extract. The study is considered as key study for invertebrates. For 48 h, an EC50 > 128 mg/L has been calculated based on mean measured concentrations.

One 72 h growth study with the green algae Desmodesmus subspicatus was performed with the test substance margosa extract. The study covers both acute and long-term endpoints and is considered as key study for algae. After 72 h, a NOEC of 1.05 mg/L and an EC50 > 237 mg/L has been calculated based on growth rate and mean measured concentrations.

Fish are representing the most sensitive species in the aquatic compartment for margosa extract. Since short-term data from each of the three trophic levels are available, an assessment factor (AF) of 1000 should be applied. A PNECwater of 0.0112 mg/L has been derived based on the available data.

Sediment

No data for effects on sediment organisms are available. According to BPR guidance Vol. IV Part B Section 3.5.2 an effect assessment for sediment organisms should be considered in cases where log KOW ≥ 3 applies. Out of the limonoids only salannin exceeds this trigger value (log KOW = 3.5 at pH 7). Therefore the equilibrium partition method (EPM) has been applied to identify a potential risk to sediment organisms as a screening approach. The input values for EPM calculation, Koc and Henry’s Law constant, are based on salannin which also shows the highest Koc of the limonoids. The calculated PNECsediment,EPM for margosa extract is 0.439 mg/kg wet weight sediment.

Inhibition of microbial activity

In a standard activated sludge respiration inhibition test with sludge from domestic sewage treatment plant no inhibitory effect of the test item up to and including a concentration of 100 mg a.s./L (nominal) was observed. Therefore the NOEC was ≥ 100 mg a.s./L and the EC50 > 100 mg a.s./L. The risk to the microorganism population of a sewage treatment plant can be characterised to be low regarding the results of these studies. From this data a PNECmicroorganisms of 10 mg a.s./L (nominal) was derived.

Terrestrial Compartment

Effect data for the terrestrial compartment were not provided and are only required if on basis of EPM an unacceptable risk for the terrestrial compartment has been indicated, or if direct or



17

long-term exposure represents a relevant exposure pathway. All of these conditions do not apply; therefore no effect data is required. Similar to sediment, the EPM has been applied as a screening approach, resulting in a PNECsoil,EPM of 0.350 mg/kg wet weight soil.

Atmosphere

Due to the very low vapour pressure of margosa extract (3.8 × 10-7 hPa at 20 °C) and the small Henry’s Law constants of the constituent limonoids (2.79 × 10-20 Pa m3/mol, 1.29 × 10-10 Pa m3/mol and 6.07 × 10-10 Pa m3/mol for azadirachtin, nimbin and salannin, respectively) only negligible volatilisation and transfer to the atmosphere is expected. Additionally, the tropospheric half-lives of the three limonoids were calculated to be in the range of 1.3 h to 1.7 h. According to these findings, accumulation and long-range transport of margosa extract or the constituent limonoids in the atmosphere followed by wet or dry deposition is not expected.

2.2.2.3. PBT Assessment and Exclusion Criteria

The PBT assessment was performed in line with the REACH legislation (Guidance on Information Requirements and Chemical Safety Assessment Part C: PBT/vPvB assessment, and Chapter R.11: PBT/vPvB assessment, Version 2.0), following the PBT and vPvB criteria laid down in Annex XIII of the REACH Regulation (EC) No 1907/2006. In addition, exclusion criteria according to Article 5(1) of the BPR were assessed according to ECHA Guidance on BPR Vol. IV Part B. The PBT assessment presented in Doc II-C 13.5 covers all constituents ≥1% (w/w), i.e. the lipid fraction, stigmasterol as well as individual limonoids salannin, nimbin and azadirachtin (A+B).

v(P) Assessment

P criterion: Half life > 40 d freshwater or >120 d in freshwater sediment or

> 120 d in soil

vP criterion: Half life > 60 d water or > 180 d in freshwater sediment or

>180 d in soil

According to the Closed Bottle test (OECD 301D), margosa extract is considered to be readily biodegradable fulfilling the 10-day window criterion. Generally, it is assumed that a chemical giving a positive result in a test of this type will rapidly biodegrade in the environment. However, it has to be noted that if a substance consists of more than one constituent PBT/vPvB assessment needs to be performed for all individual amounts ≥ 0.1 % (w/w). In case of margosa extract (CO2 extract from cold pressed Neem seed oil), this involves the individual limonoids (i.e. salannin, nimbin, and azadirachtin), stigmasterol as well as lipids (di- and triglycerides, free fatty acids).

In case of di- and triglycerides as well as free fatty acids it was considered scientifically unjustified to perform a PBT assessment, since they are ubiquitous and essential components of all living organisms.

In contrast to lipids, the substance class of triterpenoids, comprising substances such as the limonoids, (i.e. azadirachtin, nimbin, and salannin) is of higher complexity and structural diversity, although being of ubiquitous distribution, too. Triterpenoids are produced by many plants and animals as well. This supports the assumption that common pathways for the breakdown of limonoids exist in nature. Due to the lack of screening tests conducted with the individual limonoids, biodegradability was assessed using QSAR calculations in the first step.

Margosa Extract, cold-pressed oil Product-type 19 March 2017 of Azadirachta indica seeds without shells extracted with super-critical carbon dioxide

Furthermore, available information regard ing abiotic degradation (i.e. hydrolysis) as well as the degradation of azadirachtin A and B in soil was taken into account.

Based on this and due to the lack of further information about the degradation of azadirachtin, nimbin, and salann in in the aquatic and/or soil compartment as well as about the formation and degradation behaviour of relevant degradation products, it is concluded that all limonoids are considered potentially fulfilling the (v)P criterion.

Based on QSAR results and due to the lack of further information, it has to be concluded as well that stigmasterol potentially fulfils the (v)P-criterion.

B-Assessment

a-criterion: vB-criterion:

BCF > 2000 L/kg wet weight BCF > 5000 L/kg wet weight

The bioaccumulation for fish was calcu lated for all constituents exceeding 0.1 % (w/w) of Margosa extract. Based on the lack of measured bioaccumulation data, the assessment was performed following a screening approach on basis of the respective log Kow va lues. An octanol-water partitioning coefficient log Kow > 4.5 indicates that the substance is potentially (v)B. Out of the limonoids, sa lannin provided the highest octanol-water partitioning coefficient, but does not exceed the screening trigger value (salannin : Kow = 3.5; nimbin: Kow = 3.0; azadirachtin: Kow = 1.3), indicating a low potential for bioaccumulation for this group of constituents. Therefore the bioaccumulation ("B") criterion for the limonoid fraction of Margosa extract is not fulfilled. Based on physiological considerations, the bioaccumulation of glycerides and fatty acids as well as for stigmasterol conta ined in the a.s. can be considered as not relevant for the assessment of the B criterion. In conclusion the B criterion is not fulfilled for Margosa extract.

T-Assessment

T-criterion: Chronic NOEC < 0.01 mg/Lor CMR or endocrine disrupting effects

No chronic effects data is available for the Margosa extract, therefore screening on basis of acute effects has been performed. Rainbow trout (0. mykiss) represents the most sensitive aquatic organism in an acute test, with LC50 = 11.2 mg/L. Margosa extract cannot be considered as potentially toxic because all L(E)C50 to aquatic organisms are higher than 0.1 mg/L. Also considering that neither CMR properties are reported nor criteria for endocrinedisrupting effects (see next section) are met for the a.s., it can be concluded on the basis of the provided effect data for Margosa extract that the T criteria are not fu lfilled. However, based on available data for the limonoids as relevant constituents of Margosa extract the T criteria should be considered as potentially fulfilled.

Conclusion of the PBT assessment

The PST assessment for Margosa extract as a.s. and for the individual components ;;::: 0.1 % (w/w), i.e. the lipid fraction, stigmasterol, as well as the limonoids azadirachtin, nimbin and sa lannin, is summarised in the following table.

Constituent (v)P (v)B T Conclusion

Lipids assessment scientifically unjustified not PST

Stigmasterol pot. vP scientifically not T pot. vP unjustified

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Margosa Extract, cold-pressed oil Product-type 19 of Azadirachta indica seeds without shells extracted with super-critical carbon dioxide

Salannin pot. (v)P not (v)B pot T

Nimbin pot. (v)P not (v)B pot T

Azadirachtin A + B pot. vP (aquatic) not (v)B pot T pot. P (soil)

March 2017

pot vP, pot T

pot vP, pot T

pot vP, pot T

Assessment of PBT criteria was performed on basis of data for the whole plant extract as well as for the relevant components of Margosa extract, consisting main ly of lipids and limonoids. For the group of lipids, including triglycerides, diglycerides - mainly based on stearic, oleic, pa lmitic and linoleic acid - as well as the corresponding free fatty acids, neither P, B nor T criteria are fulfilled. Therefore, this group of constituents neither meets PBT nor vPvB criteria. Stigmasterol potentially fulfils the (v)P-criterion but neither the B criterion nor the T-criterion. Therefore, t his constituent neither meets PBT nor vPvB criteria as well. The limonoids salann in, nimbin and azadirachtin had to be assessed separately. Azadirachtin consists of azadirachtin A and B, but no specific data was available for further PBT assessment. Azadirachtin potentially fulfils the (v)P criterion, does not fulfil the B criterion and potentially does fulfil the T criterion. Salannin and nimbin were potentially fu lfill ing (v)P as well, both were not fulfill ing the B criterion and both were potentially fulfill ing the T criterion based on a read-across approach.

In conclusion, none of the relevant compounds of Margosa extract fulfils the criteria of a PBT or a vPvB substance. Therefore, the whole Margosa extract does not represent a PBT/vPvB substance. Based on the P- and T-criterion, the limonoids are potentially fulfill ing two of the three PBT criteria. If PBT guidance and criteria are strictly applied, this conclusion has to be transferred to the active substance: Margosa extract potentially fulfils two out of three PBT criteria.

Based on the conta ined limonoids and both on the lack of information on breakdown/transformation products from hydrolysis and aerobic biodegradation in soil and -with regard to the T-criterion - on read-across with margosa extract PT18, the following stud ies for the limonoids and their transformation products could allow achieving a defin ite conclusion for the PBT assessment:

OECD 309 test (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) On the basis of the results of the OECD 309, if needed : a test OECD 307 (Aerobic and anaerobic transformation in soil including identification of transformation products) or a OECD 308 (water/sediment degradation study including identification of transformation products) shall be performed Long-term effect test with aquatic insects

The data are needed for the renewal process. A stepwise approach is conceivable, starting with investigating the T criterion . However, the applicant is advised to contact the eCA before performing the studies.

It should be considered that the limonoids represent only a small fraction of the whole extract ( < 2%), whereas azadirachtin A and B make up < 0.2%. In contrast, margosa extract approved in PT18 consists to a large share only of limonoids, mostly azadirachtin A/B. Therefore, a read-across from PT18 regarding the T criterion could be regarded as a worstcase approach (furthermore azadirachtin was regarded as being 'not P' for PT18). In addition, based on the data requ irements applying during submission of the dossier the submitted data set should be regarded as complete ("How to deal with extracts and oils of plant or animal origin?", endorsed at the 23rct CA-Meeting, and "Guidance to Member States and industry on the data requirements for naturally occurring substances used as attractants/repellents", endorsed at the 18th CA-Meeting). Due to this, further information should only be considered at the renewal stage of margosa extract in PT19. If P and T status could be confirmed by further

19



20

data, this could render the active substance margosa extract as a candidate for substitution, eventually triggering comparative assessment for biocidal products containing the active substance.

Endocrine-Disrupting Properties

ED-criteria: endocrine-disrupting properties that may cause adverse effects in humans, or endocrine-disrupting properties identified in accordance with Articles 57 (f) and 59(1) of Regulation (EC) No 1907/2006.

Until the Commission has specified scientific criteria, also transitional criteria apply:

carcinogen category 2 and toxic for reproduction category 2, or toxic for reproduction and having toxic effects on endocrine organs.

Margosa extract contains around 2% limonoids. These limonoids salannin, nimbin and azadirachtin are known to exhibit biological activity by inhibiting insect growth and development. Biological effects of azadirachtin include antifeedant activity, insecticidal activity and inhibition of hormone and enzyme activity, and similar effects are described in open literature for salannin and nimbin. The limonoids azadirachtin, salannin and nimbin are known to similar inhibit ecdysone 20-monoxygenase. This enzyme is responsible in insects for the conversion of the molting hormone ecdysone to its more physiologically active metabolite 20-hydroxyecdysone.

Extracts from Neem tree with higher content of limonoids, especially of azadirachtin, are used as insecticides (product type 18) and the effects on insects described above represent intended effects. Besides insects, no information is available on endocrine disrupting effects on further non-target organisms, especially considering vertebrates (including mammals and humans). Studies available in the human health risk assessment for margosa extract did not mention any indications for endocrine disrupting effects.

The BPR and supporting guidance does not suggest that endocrine effects on arthropod should be considered during assessment of endocrine-disrupting properties. Article 5(d) of the BPR only refers to “endocrine-disrupting properties that may cause adverse effects in humans or which are identified in accordance with Articles 57(f) and 59(1) of Regulation (EC) No 1907/2006 as having endocrine disrupting properties”. The active substance was not identified as SVHC under REACH and adverse effects in humans are presently not described in literature. In addition, the transitional criteria provided in the BPR are not met; the a.s. has neither been classified as carcinogen category 2 and toxic for reproduction category 2 nor as toxic for reproduction and have toxic effects on endocrine organs. The Commission still has to adopted definitive criteria for endocrine disruption. It should be noted that a neem extract with higher content of limonoids is authorised and used in line with the BPR as intended endocrine acting active substances. Therefore it can be concluded that in agreement with the BPR the present criteria for endocrine-disrupting properties are not fulfilled.

2.2.2.4. Exposure assessment and risk characterisation

Exposure assessment

For environmental exposure estimation data about one representative biocidal representative product is provided by the applicant. For the life cycle stage “production“, no exposure assessment has been performed as the production process takes places in a closed facility. As the representative product is technical identical to the a.s., no exposure assessment for the formulation has been performed.



21

According to the intended use emission to the environment are expected only for the life-cycle step “non-professional use”. Two different models were applied to estimate the emissions to the environment. First, a consumption based assessment was conductedincorporating specific information about the product and the intended use. The second model applied is a tonnage based model, which is more generic approach estimating emission from the produced and/or imported tonnage of the assessed active substance.

Consumption Scenario

The consumption based environmental exposure was assessed for the application and the cleaning step. The environmental exposure has been assessed applying the EU Technical Guidance Document (TGD) on Risk Assessment (2003), the OECD Emission Scenario Document Number 18 for Insecticides, Acaricides and Products to Control Other Arthropods (PT18) for Household and Professional Uses (July 2008) and the OECD Emission Scenario Document Number 22 (2009) for Coating Industry (Paints, Lacquers and Varnishes). The exposure of a.s. in the life cycle stage “non-professional use” of the biocidal representative product “Margosa extract barrier repellent” is estimated considering the application steps by non-professionals indoor and subsequent cleaning steps of the product. The application of the b.p. as a repellent barrier is envisaged to prevent garden ants Lasius niger from entering into domestic premises. Due to the proposed non-professional indoor use of the ready-to-use product the application mode can be described as:

− Targeted barrier surface application of a product in gel form with a brush

A detailed description of emission scenarios for gel application including the input and output values is given in chapter II-8.

Regarding the cleaning step, two general cleaning methods (wet and dry cleaning with emission to waste water or solid waste) are described in the OECD ESD No.18. Since the product is an oily liquid, it is not expected that residues of margosa extract can be removed by dry cleaning methods. Thus, the exposure pathway of solid waste to municipal landfill is negligible. However, the wet cleaning process is relevant for the environmental risk assessment of the margosa extract product. In addition, releases of margosa extract to the sewer due to wet cleaning of the brush were included in the exposure assessment. Assuming that residues of a.s. removed through wet cleaning may be emitted to waste water, the STP is considered as the primary receiving compartment for margosa extract. Subsequently, surface water, sediment, soil and groundwater represent secondary receiving compartments.

Tonnage Scenario

The tonnage based environmental exposure has been assessed applying the EU Technical Guidance Document (TGD) on Risk Assessment (2003) and the ECHA ESD PT19 for repellents and attractants (2015). The applicant provided the production tonnage of the active substance margosa extract PT 19 for the years 2012 to 2016. This tonnage data represents a complete overview as the applicant is the only company producing and/or importing this active substance in/into the EU. The emission modelling contains a safety margin in order to cover a potential future increase of active substance production in the European Union.

As the dummy product is very specific with regard to the target organism, unequal spatial and temporal distribution of product use in the EU is predicted. In conclusion, the product is applied only in areas where the target organism Lasius niger is occurring in detrimental amounts during the seasons in which the occurrence of Lasius niger is so high to cause adverse effects. These temporal and spatial variations of product use were integrated into the parameterisation of the model.

Margosa Extract, cold-pressed oil Product-type 19 of Azadirachta indica seeds w ithout shells extracted w ith super-critical carbon dioxide

March 2017

Alike the Consumption Scenario, the STP is considered as the primary receiving compartment of margosa extract in the Tonnage Scenario. Subsequently, surface water, sediment, soil and groundwater represent secondary receiving compartments.

Summary of PECs

The PRCs for the Consumption Scenario and the Tonnage Scenario have been estimated for the aquat ic compartment including STP, surface water, and sediment, and for the terrestrial compartment including soil and groundwater . As the PEC9roundwater estimation accord ing to the pore water calcu lation model of the TOG resulted in unacceptable risks for the groundwater after sewage sludge application, the groundwater risk assessment was refined by using FOCUS model PEARL 4.4.4 (transport and fate simulation tool) .

Risk characterisation

Stages of the product 's life-cycle considered as relevant for the Consumption Scenario are targeted barrier surface application (including brush residues) and wet cleaning steps for nonprofessional use. Residues of b .p. removed through wet cleaning can be emitted to waste wat er, therefore STP is considered as the primary receiving compartment for the b.p., which is identical with the active substance. Subsequently, surface water, sediment, soi l and groundwater represent secondary receiving compartments. The same environmenta l emission pathway applies to the Tonnage Scenario.

Aquatic Compartment

STP represents the primary receiving compartment for the Consumption Scenario and the Tonnage Scenario. Secondarily affected compartments in the aquatic environment are surface water and sediment. PNECmicroorganism is based on the NOEC from an act ivated sludge respiration test and an AF of 10. PNECwater was derived on the basis of three acute effect studies with fish representing the most sensitive trophic level and an AF of 1000. For the sediment compartment no effect studies were avai lable and the equil ibrium partitioning method (EPM) was used therefore as a screening approach :

Table 2-3 PEC/PNEC ratios for different exposure situations concerning the

hydrosphere

Exposure Environmental PEC PNEC PEC I PNEC scenario comoartment

STP 299 ua/L 10 000 ua/L 0.03 Consumption Surface water 24.54 ua/L 11.2 µg/L 2.19

Scenario Sediment

961.33 µg/kg WW

439 µg/kg WW 2.19

STP 22.00 ua/L 10 000 ua/L 0.00 Tonnage Surface water 1.80 µq/L 11.2 µq/L 0.16 Scenario

Sediment 70.51 µg/kg

WW 439 µg/kg WW 0.16

Estimated PEC/PNEC ratios for surface water and sediment are exceeding the t rigger value of l , indicating an unacceptable r isk if the exposure estimation is performed without further refinement and not considering degradation in STP. Based on a tonnage scenario an acceptable risk, i.e. PEC/PNEC < 1, was found for all scenarios relevant for the aquatic compartment. It should be noted that the t onnage-based approach was considered as appropriate for demonstrating a safe use only for a.s. eva luation and that, as a general requirement, also the

22

Margosa Extract, cold-pressed oil Product-type 19 of Azadirachta indica seeds w ithout shells extracted w ith super-critical carbon dioxide

March 2017

consumption- based approach should indicate a safe use at the stage of product authorisation.

Terrestrial Compartment including Groundwater

One emission pathway was identified for the exposition of the terrestrial compartment with margosa extract, which applies to the Consumption Scenario and the Tonnage Scenario:

Emission via wastewater to STP leading to releases to soil via sewage sludge application and subsequently, leaching to groundwater

Based on a lack of effect data for the terrestrial compartment, PNEC50;1 was calculated as screening approach by applying the EPM. Groundwater assessment is based on the limit of 0. 1 µg/L set for biocides (active substances) in Council Directives 2006/118/EC and 98/83/EC. As the exposure estimation accord ing to the pore water calculation model of the TGD resulted in PEC9roundwater > 0.1 µg/L after sewage sludge application, the groundwater exposure assessment was refined by using FOCUS model PEARL 4.4.4 (transport and fate simulation tool). The groundwater assessment was refined for the two different emission models (Consumption Scenario or Tonnage scenario) and types or sewage sludge application (on arable land or grassland)

Table 2-4 PEC/PNEC ratios for different exposure situations concerning the

terrestrial compartment

Exposure Environmenta l PEC PNEC PEC I PNEC

scenario compartment

Soi l 2050.69 µg/kg 350 µg/kg WW 5.86

Consumption WW

Scenario Groundwater One safe (refined) scenario

0.1 µg/L acceptable

Soi l 150.18 µg/kg 350 µg/kg WW 0.43

Tonnage WW

Scenario Groundwater Six safe (refined) scenarios

0. 1 µg/L acceptable

The screening approach v ia EPM did not indicate an unacceptable r isk for soi l ( PEC/PNEC > 1) for the consumption -based scenario, which considers neither a refinement of the exposure estimation nor degradation in STP. The limit value for the groundwater of 0. 1 µg/L (Council Directives 2006/118/EC and 98/83/EC) is exceeded at none of the assessed locations (Tonnage Scenario, grassland) and at up to seven locations (Consumption Scenario, arable land) . However, at the 47th CA-Meeting held in July 2012 it was agreed that at the level of active substance assessment at least one of the nine locations needs to show a safe scenario. The modelling results comply with this requirement, as the location Sevilla is safe for all assessed scenarios. Consequently, the risks for the groundwater of the Consumption Scenario and the Tonnage Scenario can be considered as acceptable.

Considering tonnage data the risk assessment could demonstrate an acceptable risk for the terrestrial compartment. It should be noted that the tonnage-based approach was considered as appropriate for demonstrating a safe use only for a.s. evaluation and that, as a general requ irement, also the consumption- based approach should indicates a safe use at the stage of product authorisation.

Toxicity to honeybees and other beneficial arthropods 23



24

Based on the proposed use, direct exposure of bees and other beneficial arthropds to margosa extract from its application is highly improbable due to the limitation to indoor application. Merely individual bees or other arthropods might enter houses through open doors or windows and might be affected during an application process by accident, but such exposure by chance does not pose any hazard for natural communities.

Secondary Poisoning

Considering the complex composition of the a.s., the evaluation of the potential for bioaccumulation was done on basis of the limonoids azadirachtin, nimbin and salannin. Based on physiological considerations, the bioaccumulation of glycerides and fatty acids can be considered as not relevant for the assessment of bioaccumulation. Salannin provides the highest partition coefficient oil-water (log KOW = 3.5) out of the limonoids, resulting in a calculated BCF for fish of 95 L/kg wet weight.

With regard to the BCF estimations for the limonoids and the physiological role of triglycerides and fatty acids, the a.s. is not expected to bioconcentrate in aquatic and terrestrial organisms and to pose an unacceptable risk of biomagnification in the food chain of either compartment.

The representative biocidal product will only be used indoors as a barrier treatment and does not contain attractants. Therefore it can be assumed that insects will not feed the b.p. and insectivorous birds and small mammals should not be exposed indirectly to the a.s. via food and also to have no direct access to the applied b.p. Therefore, no assessment of secondary poisoning for insectivorous birds and mammals is required.

It can be concluded that the evaluated use of margosa extract in PT19 does not present a risk for secondary poisoning.

2.3. Overall conclusions

The outcome of the assessment for margosa extract in product-type 19 is specified in the BPC opinion following discussions at the 19th meeting of the Biocidal Products Committee (BPC). The BPC opinion is available from the ECHA web-site.

2.4. Requirement for further information related to the reference biocidal product

Missing information regarding:

▫ Acidity/alkalinity and if necessary pH value (1% in water)

▫ Storage stability — stability and shelf-life. Effects of light, temperature and humidity on technical characteristics of the biocidal product; reactivity towards container material (the possible increase of the aflatoxins content during storage should be addressed in this context)

▫ Technical characteristics of the biocidal product, e.g. wettability, persistent foaming, flowability, pourability and dustability

will have to be submitted within the process of biocidal product authorisation.

2.5. List of endpoints

The most important endpoints, as identified during the evaluation process, are listed in Appendix I.



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Appendix I: List of endpoints

Chapter 1: Identity, Physical and Chemical Properties, Classification and Labelling

Active substance (ISO Name) Margosa extract, cold-pressed oil of azadirachtia indica seeds without shells extracted with super-critical carbon dioxide

Product-type 19 (Repellents and attractants)

Identity

Chemical name (IUPAC) Not applicable since margosa extract is an UVCB substance

Chemical name (CA) Not applicable since margosa extract is an UVCB substance

CAS No 84696-25-3

EC No 283-644-7

Other substance No. Not allocated for margosa extract

Minimum purity of the active substance as manufactured (g/kg or g/l)

Margosa extract is an active substance with a specified purity of 100% (w/w); 1000g/kg

Identity of relevant impurities and additives (substances of concern) in the active substance as manufactured (g/kg)

As natural product margosa extract can contain aflatoxins as constituents: aflatoxine B1: < 2 µg/kg (0.0000002% w/w) aflatoxine B1, B2, G1, G2: < 4 µg/kg (0.0000004% w/w). Margosa extract can contain azadirachtin A, azadirachtin B, salannin and nimbin

Molecular formula Not applicable since margosa extract is an UVCB substance

Molecular mass Not applicable since margosa extract is an UVCB substance

Structural formula

Not applicable since margosa extract is an UVCB substance

Physical and chemical properties

Melting point (state purity) The melting range is -16 to + 20°C (100%) Since margosa extract is an UVCB substance no defined melting point can be expected

Boiling point (state purity) No boiling point at atmospheric pressure up to a temperature of 400 °C. Decomposition starts at 340 °C (100%)

Thermal stability / Temperature of decomposition

Exothermal effect in the temperature range 340-450 °C



26

Appearance (state purity) Yellow-brown liquid with onion/garlic like (100%)

Relative density (state purity) 0.92501 at 20 °C (100%).

Surface tension (state temperature and concentration of the test solution)

35.3 mN/m at 20 °C. (emulsion: c = 1 g/L)

Margosa extract is regarded as surface active

Vapour pressure (in Pa, state temperature)

3.8 x 10-7 hPa at 20 °C,

1.1 x 10-5 hPa at 50 °C vapour pressures for limonids and

azadirachtins:

Vap. Pr. Estimations (25°C)

estimated using MPBPWIN

v1.431 (US EPA) [Pa]

Azadirachtin 1.75E-018

Nimbin 7.03E-010

Salannin 1.16E-010

Linoleic acid 0.00765

α-Linolenic acid

0.00166

Oleic acid 0.00684

Palmitic acid 0.00736

Stearic acid 0.00111

Eicosanoic acid 0.0193

Henry’s law constant (Pa m3 mol -1) Calculation results at 25 °C:

Azadirachtin: 4.406x10-23 Pa m3 mol-1

Nimbin: 1.27x10-15 Pa m3 mol-1

Salannin: 5.99x10-15 Pa m3 mol-1

Linoleic acid: 5.617x10-4 Pa m3 mol-1

α-Linoleic acid: 3.705x10-5 Pa m3 mol-1

Oleic acid: 1.657x10-3 Pa m3 mol-1

Palmitic acid: 4.573x10-4 Pa m3 mol-1

Stearic acid: 8.857x10-3 Pa m3 mol-1

Eicosanoic acid: 1.9887x10-1 Pa m3 mol-1

Solubility in water (g/l or mg/l, state temperature)

pH__3____:



27

420 mg/kg at 10 °C

430 mg/kg at 20 °C

410 mg/kg at 30 °C

Measured as TOC, values > 9 days were

overlapped by hydrolysis. Samples were

measured at pH 3 in order to avoid

interference with atmospheric CO2; due to

hydrolysis samples could not be measured at

pH 7 and 9.

Calculation results at 25 °C:

Azadirachtin: 2.992 mg/L

Nimbin: 1.0051 mg/L

Salannin: 0.1477 – 3 mg/L

Linoleic acid: 0.03771 mg/L

α-Linolic acid: 0.1236 mg/L

Oleic acid: 0.011551 mg/L

Palmitic acid: 0.04073 mg/L

Stearic acid: 0.00512 mg/L

Eicosanoic acid: 0.00029 mg/L

Solubility in organic solvents (in g/l or mg/l, state temperature)

1,2 dichlorethane : > 250 g/l

octanol : > 250 g/l

aceton : 80 –100 g/l

i-propanol : 80 – 100 g/l

temperature: 20 °C

Stability in organic solvents used in biocidal products including relevant breakdown products

Not relevant

Partition coefficient (log POW) (state temperature)

pH___5___: at 10, 20 and 30°C



28

Azadirachtin A: 1.38, 1.37 and 1.35

Nimbin: 3.10, 3.10 and 3.09

Salannin: 3.51, 3.53 and 3.53 pH___7___:


Nimbin: 3.39, 3.15 and 3.13

Salannin: 3.83, 3.57 and 3.57 pH___9___:


Nimbin: 3.06, 3.06 and 3.23

Salannin: 3.49, 3.50 and 3.51 Calculation results:

Log Pow Linoleic acid: 7.51

Log Pow α-Linolic acid: 7.3

Log Pow Oleic acid: 7.73

Log Pow Palmitic acid: 6.96

Log Pow Stearic acid: 7.94

Log Pow Eicosanoic acid: 8.93



29

Hydrolytic stability (DT50) (state pH and temperature) (point VII.7.6.2.1)

pH 5, 35°C:

Azadirachtin: 275 h

Nimbin: 235.2 h

Salannin: 2632.8 h

pH 5, recalculated to 12°C:

Azadirachtin: 1731.5 h

Nimbin: 1480.9 h

Salannin: 16577.5 h pH 9, 35°C:

Nimbin: 316.8 h

Salannin: 1056.0 h


Nimbin: 1994.7 h

Salannin: 6649.1 h

Dissociation constant Azadirachtin A:

pKa= 9.78±0.70 (25°C)

Azadirachtin B:

pKa= 11.99±0.60 (25°C)

Nimbin: n.a.

Salannin: n.a.

Eicosanoic acid (Arachidic acid), Palmitoleic

acid, Oleic acid, Linoleic acid, Stearic acid,

Palmitic acid:

pKa= 4.78±0.10 (25°C) UV/VIS absorption (max.) (if absorption > 290 nm state ε at wavelength)

503.0 mg margosa extract /L methanol: λmax

= 207.5 nm

113.2 mg margosa extract /L methanol: λmax = 204 nm It is not possible to calculate extinction coefficients as margosa extract is a mixture of different organic compounds. No significant absorption above 290 nm of azadirachtin, nimbin and salannin

Flammability or flash point The self-ignition temperature is 395°C

Explosive properties Margosa extract has no explosive properties



30

Oxidising properties No oxidising properties

Auto-ignition or relative self ignition temperature

The self-ignition temperature is 395 °C.

Classification and proposed labelling

with regard to physical hazards None

with regard to human health hazards None

with regard to environmental hazards none

Chapter 2: Methods of Analysis

Analytical methods for the active substance

Technical active substance (principle of method)

Limonoids: HPLC-UV Free fatty acids, mono-, di- and triglycerides: quantitative C-NMR Sterols: quantitative H-NMR

Impurities in technical active substance (principle of method)

Impurities: Aflatoxin: HPLC-fluorescence detection

Analytical methods for residues

Soil (principle of method and LOQ) Primary and confirmatory method missing residue definition: lead compounds nimbin and salannin

Air (principle of method and LOQ) HPLC-DAD, Nucleosil C18 column, 215 nm

LOQ: 2.0 µg/m³ nimbin, 5 µg/m³ salannin; confirmation for salannin by LC-MS/MS, C18 column, ESI+, m/z 597→419+245+391 ; LOQ: 11.7 µg/L

Water (principle of method and LOQ) Drinking water: primary and confirmatory method missing Surface water: primary and confirmatory method missing residue definition: lead compounds nimbin and salannin

Body fluids and tissues (principle of method and LOQ)

Not required, not classified as toxic or very toxic

Food/feed of plant origin (principle of method and LOQ for methods for monitoring purposes)

No relevant residues expected

Food/feed of animal origin (principle of method and LOQ for methods for monitoring purposes)

No relevant residues expected



31

Chapter 3: Impact on Human Health

Absorption, distribution, metabolism and excretion in mammals

Rate and extent of oral absorption: No data, technically not feasible

100 % oral absorption by default

Rate and extent of dermal absorption*: No data, technically not feasible

75/25 % dermal absorption by default, depending on concentration in the product (EFSA 2012).

Distribution: No data, technically not feasible

Potential for accumulation: No data, technically not feasible

Rate and extent of excretion: No data, technically not feasible

Toxicologically significant metabolite(s) No data, technically not feasible * the dermal absorption value is applicable for the active substance and might not be usable in product authorization

Acute toxicity

Rat LD50 oral 2000 mg/kg bw

Rat LD50 dermal 2000 mg/kg bw

Rat LC50 inhalation 82 mg/L (4-h exposure, nose-only, max. attainable conc.)

Skin corrosion/irritation Not irritating

Eye irritation Not irritating

Respiratory tract irritation Not irritating

Skin sensitisation (test method used and result)

Not sensitising (M & K)

Respiratory sensitisation (test method used and result)

Not sensitising

Repeated dose toxicity

Short term

Species / target / critical effect Rat: liver weight increases



32

Relevant oral NOAEL / LOAEL Rat: 450 mg/kg bw/d

Relevant dermal NOAEL / LOAEL Rat: 1000 mg/kg bw/d (for systemic

effects)

100 mg/kg bw/d (for local

effects)

Rabbit*: 800 mg/kg bw/d (for systemic

effects)

50 mg/kg bw/d (for local effects)

Relevant inhalation NOAEL / LOAEL No data available, not required * Data from prenatal toxicity study

Genotoxicity No evidence for a genotoxic potential in humans

Carcinogenicity

Species/type of tumour No data available, accepted

Relevant NOAEL/LOAEL No data available, accepted

Reproductive toxicity Developmental toxicity

Species/ Developmental target / critical effect

Rabbit: no effects on development up to the highest dose tested, maternal: reduced bw, not considered adverse

Relevant maternal NOAEL 800 mg/kg bw/d

Relevant developmental NOAEL 800 mg/kg bw/d

Fertility

Species/critical effect No data available, accepted

Relevant parental NOAEL No data available, accepted

Relevant offspring NOAEL No data available, accepted

Relevant fertility NOAEL No data available, accepted

Neurotoxicity

Species/ target/critical effect No data available, not required

Developmental Neurotoxicity

Species/ target/critical effect No data available, not required

Other toxicological studies



33

No data available, not required

Medical data

Open literature and medical surveillance report in production of margosa extract

Intoxications reported with various neem oil preparations not relevant for margosa extract. No data available for margosa extract. No effects in manufacturing staff and users of margosa extract containing products reported.

Summary

Value Study Safety factor

AELacute 5.0 mg/kg bw/d 28-d rat, oral 200

AELmedium-term 2.3 mg/kg bw/d 90-d rat, oral 200

AELlong-term 1.1 mg/kg bw/d 90-d tar, oral 400

ADI2 Not allocated, no residues in food or feed

ARfD2 Not allocated, no residues in food or feed

Chapter 4: Fate and Behaviour in the Environment

Route and rate of degradation in water

Hydrolysis of active substance and relevant metabolites (DT50) (state pH and temperature)

pH 5 pH 5, 35°C:

Azadirachtin: 275 h

Nimbin: 235.2 h

Salannin: 2632.8 h



Nimbin: 1480.9 h

Salannin: 16577.5

2 If residues in food or feed.



34

pH7 pH 7, 35°C:


Nimbin: 283.2 h

Salannin: 3504.0 h



Nimbin: 1783.2 h

Salannin: 22063.1 h

pH8 pH 8, 35°C:




pH 9 pH 9, 35°C:

Nimbin: 316.8 h

Salannin: 1056.0 h


Nimbin: 1994.7 h

Salannin: 6649.1 h

Photolytic / photo-oxidative degradation of active substance and resulting relevant metabolites

Photolysis in water is not relevant. Indirect phototrasformation in air was modelled (AOPWIN, Version 1.91): Half-lives of 1.696 h (azadirachtin), 1.258 h (nimbin) and 1.325 h (salannin)

Readily biodegradable (yes/no) Yes, fulfilling the 10-d window criterion but rate constant of k = 0 h-1 for the elimination in sewage treatment plant based on QSAR calculations for limonids

Biodegradation in seawater No data

Non-extractable residues No data

Distribution in water / sediment systems (active substance)

No data (supportive information for unlabelled azadirachtin A and B available)

Distribution in water / sediment systems (metabolites)

No data

Route and rate of degradation in soil

Mineralization (aerobic) No data

Laboratory studies (range or median, with number of measurements, with regression coefficient)

Supportive information available for unlabelled azadirachtin A and B



35

DT50lab (20°C, aerobic): No data



DT50lab (20°C, anaerobic): No data

degradation in the saturated zone: No data

Field studies (state location, range or median with number of measurements)

DT50f: No data

DT90f: No data

Anaerobic degradation No data

Soil photolysis Not required

Non-extractable residues Not applicable

Relevant metabolites - name and/or code, % of applied a.i. (range and maximum)

Not applicable

Soil accumulation and plateau concentration

Not required

Adsorption/desorption

Ka , Kd Kaoc , Kdoc

pH dependence (yes / no) (if yes type of dependence)

Determined with HPLC-method according to

OECD Guideline 121

KOC = 144 L/kg (azadirachtin)

KOC = 809 L/kg (nimbin)

KOC = 1766 L/kg (salannin)

Fate and behaviour in air

Direct photolysis in air Not relevant

Quantum yield of direct photolysis

Photo-oxidative degradation in air Tropospheric half-lives of 1.696 h (azadirachtin), 1.258 h (nimbin) and 1.325 h (salannin) were determined with AOPWIN, Version 1.91, (according to Atkinson, reaction with OH radicals, concentration: 5.105 OH/cm3)

Volatilization Not relevant

Reference value for groundwater

According to BPR Annex VI, point 68 0.1 µg/L



36

Monitoring data, if available

Soil (indicate location and type of study) No data

Surface water (indicate location and type of study)

No data

Ground water (indicate location and type of study)

No data

Air (indicate location and type of study) No data

Chapter 5: Effects on Non-target Species

Toxicity data for aquatic species (most sensitive species of each group)

Species Time-scale

Endpoint Toxicity(mg a.s./L)-

1

Fish

Oncorhynchus mykiss 96h LC50 11.2 (mm)

Invertebrates

Daphnia magna 48h EC50 > 128 (mm)

Algae

Desmodesmus subspicatus

72h ErC50

NOErC > 237 (mm) 1.05 (mm)

Microorganisms

Activated sludge predominately from municipal wastewater treatment plant

3 hours; static

Respiration inhibition (oxygen consumption), NOEC, EC50

≥ 100

> 100

Effects on earthworms or other soil non-target organisms

Acute toxicity to …………………………………..

No data available

Reproductive toxicity to …………………………

No data available

Effects on soil micro-organisms

Nitrogen mineralization No data available

Carbon mineralization No data available

Effects on terrestrial vertebrates



37

Acute toxicity to mammals No data available

Acute toxicity to birds No data available

Dietary toxicity to birds No data available

Reproductive toxicity to birds No data available

Effects on honeybees

Acute oral toxicity No data available

Acute contact toxicity No data available

Effects on other beneficial arthropods

Acute oral toxicity No data available

Acute contact toxicity No data available

Acute toxicity to ………………………………….. No data available

Bioconcentration

Bioconcentration factor (BCF) Azadirachtin: 3.35 L/kg ww (estimated)

Nimbin: 44.3 L/kg ww (estimated)

Salannin: 94.69 L/kg (estimated)

Depration time (DT50) No data available

Depration time (DT90) No data available

Level of metabolites (%) in organisms accounting for > 10 % of residues

No data available

Chapter 6: Other End Points



38

Appendix II: List of Intended Uses

Object and/or situation

Product Name

Organisms controlled Formulation Application Applied amount per

treatment Re

marks:

Type

Conc. of a.s.

method kind

number min max

interval between applications (min)

g a.s./L min max

water L/m2 min max

g a.s./m2

min max

repellent to deter ants from entering buildings (non-professional user)

Margosa extract

Black Garden Ant (Lasius niger)

100% Application by brushing

1.6g – 4g Margosa extract per linear meter, in a brushed line of 0.3 cm or 2 cm

The formulation used was 100% Margosa extract. At the stage of product authorisation, the mortality of the target organisms has to be recorded in the efficacy tests. Acceptable mortality values should be set at product authorisation stage depending on the target organisms. Products that show undesirable effect onto target organisms at the claimed dose rates should be rejected.



39

Appendix III: List of studies

Data protection is claimed by the applicant in accordance with Article 60 of Regulation (EU) No 528/2012.

Author(s)

Annex point/ reference number

Year Title

Source (where different from company)

Company name, Report No., Date,

GLP status (where relevant), published or notFehler! Unbekannter Name für Dokument-Eigenschaft.

Data protecti-on claimed

Owner

Ben-Shabat S, Baruch N, Sintov AC

IIA 3.8.1 2007 Conjugates of unsaturated fatty acids with propylene glycol as potentially less-irritant skin penetration enhancers

Drug Development and Industrial Pharmacy 33, 1169-1175

Published

No -

Bhaskar M.V. et al.

IIA 3.10 2010 MR IMAGING FINDINGS OF NEEM OIL POISONING

AJNR, 31, pp. E60 – E61

No -

Boeke, S.J. et al. IIA 3.10 2004 Safety evaluation of neem (Azadirachta indica) derived pesticides. J Ethnopharmacol. 94, pp. 25-41 Report no.: not applicable GLP: unknown Published: yes

No -

Bol, R. et al. IIC 13.5 1996 The 14C age and residence time of organic matter and its lipid constituents in a stagnohumic gley soil. European Journal of Soil Science 47, 215–222. GLP: unknown Published: yes

N Published

Child, R., Ramanathan, S.

IIC 13.5 1936 The fatty acids of margosa oil. J. Soc. Chem. Ind., London 55:124T GLP: no Published: yes

N Published

Christianson-Heiska, I. et al.

IIA 4.2.5 2007 Endocrine modulating actions of a phytosterol mixture and its

N Publish



40

oxidation products in zebrafish (Danio rerio). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 145(4), 518-527. GLP: unknown Published: yes

ed

Chróst, R.J., Gajewski, A.J.

IIC 13.5 1995 Microbial utilization of lipids in lake water. FEMS microbiology ecology 18.1 (1995): 45-50. GLP: unknown Published: yes

N Published

Dhongade R.K. et al.

IIA 3.10 2008 nEEM OIL POISONING. Indian Pediatrics, 45, pp. 56-57

No -

EC 2000 FOCUS groundwater scenarios in the EU review of active substances. Report of the FOCUS Groundwater Scenarios Workgroup, EC Document Reference SANCO/321/2000 Rev.2

N Published

ECB 2003 Technical Guidance Document in support of Commission Directive 93/67/EEC on Risk Assessment for new notified substances, Part II; Commission Regulation (EC) No 1488/94 on Risk Assessment for existing substances and Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market. EUR 20418 EN/2

N Published

ECHA 2014 Guidance on the Biocidal Products Regulation Volume IV: Environment Part A: Information Requirements Version 1.1

N Published

ECHA 2015 Guidance on the Biocidal Products Regulation Volume IV: Environment Part B: Risk Assessment (active substances) Version 1.0

N Published

ECHA 2015 Emission Scenario Document for Product Type 19 Repellents and attractants

No Published

Hilditch, T. P., Murti, K. S.

IIC 13.5 1939 The fatty acids glycerides of neem (margosa oil). J. Soc. Chem. Ind., London, 58, 310-312. GLP: no Published: yes

N Published



41

Howell, W.M., Denton, T.E.

IIA 4.2.5 1989 Gonopodial morphogenesis in female mosquitofish, Gambusia affinis affinis, masculinized by exposure to degradation products from plant sterols. Environmental Biology of Fishes, 24(1), 43-51. GLP: unknown Published: yes

N Published

Iyyadurai R. et. al.

IIA 3.10 2010 AZADIRACHTIN POISONING: A CASE REPORT. Clinical Toxicology, 48/8, pp. 857-858

No -

James A, Meikandan D, Shankar SK, Mahadevan A, Behl HM, Meenakshi-Sundaram S

IIA 3.10 2006 Neurotoxic effects of neem oil poisoning Annals of Neurology 60, Suppl. 10, S32 Published

No -

Kaushik, N., Vir, S.

IIC 13.5 2000 Variations in fatty acid composition of neem seeds collected from the Rajasthan state of India. Biochemical Society Transactions, 28(6), 880-882. GLP: unknown Published: yes

N Published

Lai S.M. et al. IIA 3.10 1990 MARGOSA OIL POISONING AS A CAUSE OF TOXIC ENCEPHALOPATHY. Singapore Med J., 31; pp. 463-465

No -

MacLatchy, D.L., Van der Kraak, G.J.

IIA 4.2.5 1995 The phytoestrogen β-sitosterol alters the reproductive endocrine status of goldfish. Toxicology and applied pharmacology, 134(2), 305-312. GLP: unknown Published: yes

N Published

MacLatchy, D.L., Van der Kraak, G.J.

IIA 4.2.5 1995 The phytoestrogen β-sitosterol alters the reproductive endocrine status of goldfish. Toxicology and applied pharmacology, 134(2), 305-312. GLP: unknown Published: yes

N Published

Miskelly, A. IIA 4.2.5 2009 Effects of pulp mill wastewater treatment on phytosterol biotransformation and genomic response in rainbow trout Doctoral dissertation, University of British Columbia. GLP: unknown Published: yes

N Published

Nakari, T., Erkomaa, K.

IIA 4.2.5 2003 Effects of phytosterols on zebrafish reproduction in

N Publish



42

multigeneration test. Environmental Pollution, 123(2), 267-273. GLP: unknown Published: yes

ed

Niemann, L., Stinchcombe, S., Hilbig, B.,

IIA 3.10 2002 TOXICITY OF NEEM TO VERTEBRATES AND SIDE EFFECTS ON BENEFICIAL AND OTHER ECOLOGICALLY IMPORTANT NON-TARGETED ORGANISMS. TOXICITY TO MAMMALS INCLUDING HUMANS The Neem Tree (Azadirachta indica A Juss.) and Other Meliaceous Plants. (Ed. Schmutterer), Weinheim, Germany: VCH, 607-623 Report-no. GLP/GEP: no Published: yes

No -

OECD 2008

OECD Series on Emission Scenario Documents (Number 18) Emission Scenario Document for Insecticides, Acaricides and Products to control other Arthropods for Household and Professional Uses

N Published

OECD 2009 OECD Series on Emission Scenario Documents (Number 22) Emission Scenario Documents on Coating Industry (Paint, Laquers and Varnishes)

N Published

Orrego, R. et al. IIA 4.2.5 2009 Pulp and paper mill effluent treatments have differential endocrine‐disrupting effects on rainbow trout. Environmental Toxicology and Chemistry, 28(1), 181-188. GLP: unknown Published: yes

N Published

Schmutterer, H. (ed.)

IIA 3.10 2002 The Neem tree. Neem foundation, Mumbai/India Report no.: not applicable GLP: unknown Published: yes

No -

Senanayake M.P. et al.

IIA 3.10 2009 MARGOSA (KOHOMBA) OIL INDUCED TOXIC ENCEPHALOPATHY FOLLOWING HOME REMEDY FOR INTESTINAL WORMS. Ceylon Med. J., 54/4: p. 140

No -

Sinniah, D. & Baskaran, G.

IIA 3.10 1981 MARGOSA OIL POISONING AS A CAUSE OF REYE’S SYNDROME. The Lancet, 1/8218, pp. 487-489

No -

Sinniah, D. et al. IIA 3.10 1981 MARGOSA OIL POISONING IN INDIA AND MALAYSIA

Transactions of the Royal Society of Tropical Medicine and

No -



43

Hygiene. 75/6; pp 903-904

Sivashanmugham, R. et al.

IIA 3.10 1984 VENTRICULAR FIBRILLATION AND CARDIAC ARREST DUE TO NEEM LEAF POISONING

J. Ass. Phys. India, 32; pp. 610-611

No -

Skellon, J. H. et al.

IIC 13.5 1962 The fatty acids of neem oil and their reduction products. Journal of the Science of Food and Agriculture, 13(12), 639-643. GLP: no Published: yes

N Published

Sri Ranganathan, S. et al.

IIA 3.10 2005 Kohomba oil induced encephalopathy : lessons in prescribing traditional medicines.

Sri Lanka Journal of Child Health, 34, Pp. 94-95

No -

Sundarvalli, N. et al.

IIA 3.10 1982 NEEM OIL POISONING. The Indian J Pediat. 49/3; pp. 357-359

No -

Tremblay, L. Van der Kraak, G.

IIA 4.2.5 1998 Use of a series of homologous in vitro and in vivo assays to evaluate the endocrine modulating actions of β-sitosterol in rainbow trout. Aquatic toxicology, 43(2), 149-162. GLP: unknown Published: yes

N Published

Tremblay, L. Van der Kraak, G.

IIA 4.2.5 1999 Comparison between the effects of the phytosterol β‐Sitosterol and pulp and paper mill effluents on sexually immature rainbow trout. Environmental Toxicology and Chemistry, 18(2), 329-336. GLP: unknown Published: yes

N Published

Venkataram, T.V. IIA 3.10 2002 Employees Health Record 2001 not applicable Trifolio-M GmbH Report-no. not applicable GLP/GEP: no Published: no

yes TRF

Ventakaram, T.V. IIA 3.10 2003 Employess Health Record 2002 not applicable Trifolio-M GmbH Report-no. not applicable GLP/GEP: no Published: no

yes TRF



44

Doc IIIA

Author(s)


Year Title Source (where different from company) Company name, Report No., Date, GLP status (where relevant), published or notFehler! Unbekannter Name für Dokument-Eigenschaft.

Data protect. claimed

Owner

Anonymous A9_01 2005 EU Safety Data Sheet according to 91/155/EEC, Terra Nostra, No, Unpublished

Yes Terra Nostra GmbH

Bär, Christian A3.4 2005 UV/VIS Absorption Spectrum, Infareed Absorption-Spectrum and 1H-NMR Spectrum, of Margosa Extract: CO2-Extract from cold pressed Oil form Neem Seed without Shell, GAB GmbH, 20051094/01-PCSD, Yes, Unpublished


Blackwell,A. A5.3_05 2008 Assessment of Repellent Activity of Oil Extracts of the Kernels of the Neem Tree ( Azadirachtata indica ) against the Scottish Highland Midge Culicoides impunctatus

Y Terra Nostra Registration Service SRL

Bockholt, K. A7.1.1.1.1.03 2006 Hydrolysis as a Function of pH, UCL GmbH, PR05/028, Yes, Unpublished


Bockholt, K. A7.1.3 2005 Estimation of the Adsorption Coefficient (Koc) on Soil and Sewage Sludge using High Performance Liquid Chormatography (HPLC),UCL GmbH, PR05/013, Yes, Unpublished


Bockholt, K. A3.3 A3.3.8.01

2005 Accelerated Storage Stability Test at 54°C, UCL GmbH, PR05/017, Yes, Unpublished


Bockholt, K. A3.5.01 2006 Margosa Extract (CO2-Extract from cold pressed Oil from Neem Seed without shells), UCL GmbH, Study no. PR06/001, March 2006


Ventakaram, T.V. IIA 3.10 2004 Employees Health Record 2003 not applicable Trifolio-M GmbH Report-no. 4826, not applicable GLP/GEP: no Published: no

yes TRF



45

Author(s)




Owner

Bockholt, K. A3.9.01 2006 Partition Coefficient (n-Octanol/Water) by High Performance Liquid Chromatography (HPLC) pH 5,7,9, UCL GmbH, PR05/021, Yes, Unpublished


Cawelius,A. A3.3.7.02 2006 Solubility of Limonoids in 2-Propanol at 10 and 30°C, Flavex Naturextrakte GmbH, No, Unpublished


Cawelius,A. A3.3.8.02 2006 Storage stability control of Limonoids in Milbiol reference samples, Terra Nostra GmbH,No, Unpublished


A6.1.1 2003 Acute Toxicity Study of Neemoil by Oral Administraion to Rats,

16315/02, Yes, Unpublished


A6.1.2 2003 Acute Toxicity Study of Neemoil in CD Rats by Dermal Administration,

, 16316/02 Yes, Unpublished


A6.1.3 2003 Aucte Inhalation Toxicity Study of Neemoil in CD Rats, 16317/02, Yes, Unpublished


A6.3.1 2006 Margosa extract (CO2 extract from pressed Neem Seed oil 4 Week Dietary Toxicity Study in Rats Followed by a 2 Week Recovery Period, 43990, Yes, Unpublished


A6.3.2 2005 Margosa extract (CO2 extract from pressed Neem Seed oil 4 Week Dermal Toxicity Study in Rats , 44070 Yes, Unpublished


A6.8.1/02 2006 Margosa Extract (CO2 Extract from cold pressed Neem Seed Oil) Prenatal Developmental Toxicity Study in Rabbits by Dermal Application, 44800, Yes, Unpublished



46

Author(s)




Owner

Dengler, Detlef A7.1.1.2.1 2005 Assessment of the Ready Biodegradability of Margsoa Extract: CO2-Extract from Cold Pressed Oil from Neem Seed without Shell, GAB GmbH, 20051094/01-AACB, Yes, Unpublished


Dengler, Detlef A7.4.1.3 2005 Testing of Toxic Effexts of Margosa Extract: CO2-Extract from Cold Pressed Oil from Neem Seed without Shell to the Single Cell Green Alga Desmodesumus subspicatus, GAB GmbH, 20051094/01-AADs, Yes Unpublished


Dengler, Detlef A7.4.1.4 2005 Acute Toxicity Testing of Margosa Extract: CO2-Extract from Cold Pressed Oil from Neem Seed without Shell, GAB GmbH, 20051094/01-AAHT, Yes, Unpublished


Fábregas, Eulàlia A7.3.1 2005 Calculation of the Indirect Phototransformation Dr. Knoell Consultant, KC-PD-02/05, No, Unpublished


Fàbregas, Eulàlia A7.4.2-BCF 2006 Calculation of the Bioconcentration Factor (BCF), Dr. Knoell GmbH, No, Unpublished


Franke, J. A3.2 2005 Vapour Pressure, Siemens AG, 20050729.02 Yes, Unpublished


Franke, J. A3.16 2005 Oxidizing Properties of Liquids A.21, Siemens 20050729.01, Yes, Unpublished


A6.6.2 2003 Gene Mutation Test in Chinese Hamster V79 Cells in vitrowith Neemoil,

T, Yes,

Unpublished


A6.6.3 2003 Chromosome Aberration Test in Chinese Hamster V79 Cells in vitro with Neemoil,

, Yes,

Unpublished




47

Author(s)




Owner

Jarvis, Andrew P., Johnson, Shaun, Morgan, David

A7.1.1.1.1.02 1998 Stability of the Natural Insecticide Azadirachtin in Aqueous and Organic Solvents, Literature Pesticide Science 1998, 53, 217-222, No, Published

No

Jung, R. A5.3.04 2010 Repellent effect of margosa extract formulation against Red poultry mite

Y Terra Nostra Registration Service SRL

A6.1.4.eye 2003 Acute Eye Irritaion Study of Neemoil by Instillation into the Conjunctival Sac of Rabbits, , 16319/02, Yes, Un- published


A6.1.4.skin 2003 Acute Skin Irritaion Test (Patch Test) of Neemoil in Rabbits,


Lüpkes, K.-H. A5.3.03 2006 Repellent effect of Margosa extract formulation against various crawling and flying test insects

Y Terra Nostra GmbH

Lüpkes, K.-H. A5.3.06 2014 Simulated-Use Test showing Repellent Efficacy against Black Ants: Repellent efficacy of Margosa extract, applied on terrace board made of acacia wood and plywood in test arenas against black ants Lasius niger

Y Terra Nostra GmbH

Müller, Jacki A3.2.1 A3.5.02 A3.9.02

2006 Calculation of Henry's Law constant, Water solubility and Partition coefficient


Oetzel, Holger A5_3_1 2006 Effectiveness against target organisms, 01TN2403/06, Ingenieurbüro Oetzel, No, Unpublished


A6.4.1 2009 Margosa Extract (CO2 Extract from Cold Pressed Neem Seed Oil) 90-Day Dietary Toxicity Study in Rats Followed by a 4 Week Recovery Period.

GLP Unpublished




48

Author(s)




Owner

A6.1.5 2006 Margosa extract (CO2 extract from pressed Neem Seed oil) Delayed Dermal Sensitisation Study in Guinea Pigs (Magnusson and Kligmann Test),

., 49060, Yes, Unpublished


Schmidt, Franz

A3.17 2006 Reactivity towards container material Margosa Extract, Terra Nostra, No, Unpublished


Smeykal, H. A3.1.1 A3.1.2

2003 Melting Point/Melting Range Boiling Point/Boiling Range, Siemens Axiva GmbH&Co. KG, 20021483.01, Yes, Unpublished


Smeykal, H. A3.10 A 3.11 A3.15

2003 Auto-Flammability (Determination of the Temperature of Self-Igniton of Volatile Liquids and of Gases, Siemens Axiva GmbH&Co. KG, 2002143.02, Yes, Unpublished


A7.4.1.1 2005 Acute Toxicity Testing of Margosa Extract: CO2-Extract from Cold Pressed Oil from Neem Seed without Shell in Rainbow Trout (Oncorhynchus mykiss) (Teleostei, Salmonella) ,

20051094/01-AAOm, Yes, Unpublished


Stäbler, Dietmar A7.4.1.2 2005 Assessment of Toxic Effects of Margosa Extract: CO2-Extract from Cold Pressed Oil from Neem Seed without Shell on Daphnia magna using the 48h Acute Immobilisation Test, GAB GmbH, 20051094/01-AADm, Yes, Unpublished


Stark J.D., Walter J.F.

A7.2.1.01 1995 Persistence of Azadirachtin A and B in soil: Effects of temperature and microbial activity, Literature Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 30:5, 685-698

No

Szeto, Sunny Y., Wan, Michael T.

A7.1.1.1.1.01 1996 Hydrolysis of Azadirachtin in Buffered and Natural Waters, Literature J. Agric. Food Chem 1996, 44 Page 1160-1163, No, Published

No



49

Author(s)




Owner

Thompson, D.G. Chartrand, D.T. Kreutzweiser, D.P.

A7.1.2.2.2 2004 Fate and effects of Azadirachtin in aquatic mesocosms -1: Fate in water and bottom sediments, Literature Ecotoxicology and Environmental Safety 59, (2004) 186-193, No, Published

No

Uhde, H. A6.6.1 2003 Mutagenicity Study of Neemoil in the Salmonella Thyphimurium Reverse Mutation Assay (In Vitro), LPT KG, 16320/02, Yes Unpublished


Uhde, H. A6.6.4 2003 Micronucleus Test of Neemoil in Bone Marrow Cells of te NMRI Mouse by Oral Administration, LPT KG, 16321/02, Yes Unpublished


Wilfinger, Wolfgang

A3.1.3 2003 Relative Density of Neemoil, GAB GmbH&IFU GmbH, 20021424/01-PCRD, Yes, Unpublished


Wilfinger, Wolfgang

A3.3.7.01 2003 Solubility of Neemoil in Organic Solvents, GAB GmbH&IFU GmbH, 2002142/01-PSBO, Yes, Unpublished


Wilfinger, Wolfgang

A3.12 2005 Flash Point of Margosa: Co2-Extract from cold pressed Oil from Neem Seed without Shell, GAB GmbH, 20021424/01-PCFB, Yes, Unpublished


Wilfinger, Wolfgang

A.13 2003 Surface Tension of Neemoil, GAB GmbH, 20021424/01-PCST, Yes, Unpublished


Wilfinger, Wolfgang

A.14 2003 Viscosity of Neemoil, GAB GmbH, 20021424/01-PCVC, Yes, Unpublished


Xie, Y.S., Fields, P.G., Isman,M.B.

A5_3_02 1995 Repellency and Toxicity of Azadirachtin and Neem Concentrates to Three Stored-Product Beetles

No Literature

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