Disodium 6-amino-5-[[4-[(2-bromo-1-oxoallyl)amino]-2-[(4-methyl-3-sulphonatophenyl)sulphonyl]phenyl]azo]naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies were taken into consideration. In Reverse Mutation Assay using bacteria (Salmonella Typhimurium) FAT 40066/B exerted a mutagenic effect in this test system. However, in another bacterial Reverse Mutation Assay using bacteria (Salmonella Typhimurium), FAT 40066/E revealed a non-mutagenic effect. Whereas it showed positive results in an in vitro chromosome aberration test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 April 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four tester strains tested; no tester strain to detect cross-linking mutagens was included

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from liver

Test concentrations with justification for top dose:

20 to 5120 µg per Petri dish

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive control substance:

other: 2-anthramine (6.25 µg))

Remarks:

For strains TA 1535, TA 1537, TA 98 and TA 100 - with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

for strain TA 1535 and TA 100 - without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA 1537 - without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Daunomycine

Remarks:

For strain TA 98 - without metabolic activation

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate accompanied by a dose-effect relationship.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

and TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

doubling of the spontaneous reversion was only noted at 5120 µg

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 1537 and TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

In the absence of S-9 mix a doubling of the spontaneous reversion was noted at 5120 µg with the strain TA 98. A mutagenic effect was observed with the strains TA 100 and TA 1537 over a concentration range of 1280 to 5120 µg of product per Petri Dish.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No mutagenic effect was observed with the strain TA 1535.
In the presence of S-9 mix a doubling of the spontaneous reversion was only noted at 5120 µg with the strains TA 100 and TA 1537.
In the absence of S-9 mix a doubling of the spontaneous reversion was noted at 5120 µg with the strain TA 98. A mutagenic effect was observed with the strains TA 100 and TA 1537 over a concentration range of 1280 to 5120 µg of product per Petri Dish.

Remarks on result:

other: four strains were used

Conclusions:

FAT 40066/B was found mutagenic for S. typhimurium strains TA 1537, TA 98 and TA 100.

Executive summary:

The substance FAT 40066/B was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 5120 µg per Petri dish both in the presence and absence of metabolic activation. Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 40066/B was found mutagenic for S. typhimurium strains TA 1537, TA 98 and TA 100. No mutagenic effect was observed with the strain TA 1535. In the presence of S-9 mix a doubling of the spontaneous reversion was only noted at 5120 µg with the strains TA 100 and TA 1537. In the absence of S-9 mix a doubling of the spontaneous reversion was noted at 5120 µg with the strain TA 98. A mutagenic effect was observed with the strains TA 100 and TA 1537 over a concentration range of 1280 to 5120 µg of product per Petri Dish. A mutagenic effect was observed with the strains TA 100 and TA 1537 both in the absence and the presence of S-9 mix, and with the strain TA 98 at 5120 µg only in the absence of S-9 mix. Based on the study results, FAT 40066/B was found mutagenic for S. typhimurium strains TA 1537, TA 98 and TA 100.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2016 to 6 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
SBL009/LR6G (India)
- Expiration date of the lot/batch:
18 December 2018
- Identification: FAT 40066/E

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
-10 °C to -30 °C, protected from light

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were: 0.2, 0.6, 2, 6, 20, 60, 200, 600, and 2000 µg/mL.
In the chromosome aberration assay, the concentrations tested were: 10, 25, 50, 100, 200, 300, 400, and 500 µg/mL in the non-activated 4-hour exposure group; 10, 20, 25, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, and 50 µg/mL in the S9-activated 4-hour exposure group; and 10, 25, 50, 75, 100, 125, 150, 175, 200, 300,and 400 µg/mL in the non-activated 20-hour exposure group.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed workable suspensions in water at concentrations ranging from approximately 15 to 25 mg/mL and was soluble in water at a concentration of approximately 10 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed. Cells were harvested 20 hours (±30 minutes) after initiation of treatment, which corresponds to 1.5 normal cell cycles.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assay

NUMBER OF CELLS EVALUATED: a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture), whenever possible

DETERMINATION OF CYTOTOXICITY
- Method: cell growth index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Toxicity induced by treatment was based upon relative increase in cell counts and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

A test article was considered positive if it induced a statistically significant and dose dependent increase in the frequency of aberrant metaphases (p ≤0.05), and the results were outside the 95 % control limits of the historical negative control data. If only one criterion was met (statistically significant OR dose dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative. Other criteria also may be used in reaching a conclusion about the study results (e.g. comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

For the induction of structural and numerical chromosome aberrations in the non-activated test system and for the induction of numerical chromosome aberrations in the S9-activated test system

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At doses ≥300 µg/mL in the non activated 4 hour exposure group; at doses ≥ 32.5 µg/mL in the S9 activated 4-hour exposure group; and at doses ≥ 100 µg/mL in the non activated 20-hour exposure group.

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Preliminary Toxicity Assay

The test substance formed a workable suspension in water at a concentration of 20 mg/mL and was soluble in water at concentrations ranging from 0.002 to 6 mg/mL. No visible precipitate was observed in the treatment medium at any dose tested at the beginning and conclusion of the treatment period. The osmolality in treatment medium was measured as follows:

 

Dose tested	Doses (µg/mL)	Osmolality (mmol/kg)
Vehicle	0	266
Highest	2000	275

 

The osmolality of the test substance dose in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120 %. The pH of the highest dose of test substance in treatment medium was 7.32. Cytotoxicity (≥50 % reduction in cell growth index relative to the vehicle control) was observed at doses ≥600 µg/mL in the non-activated 4-hour exposure group; at doses ≥ 60 µg/mL in the S9-activated 4-hour exposure group; and at doses ≥200 µg/mL in the non-activated 20-hour exposure group. Based on the results of the preliminary toxicity test, the doses selected for testing in the chromosome aberration assay were as follows:

 

Treatment Condition	Treatment Time	Recovery Time	Doses (µg/mL)
Non-activated	4 hr	16 hr	10, 25, 50, 100, 200, 300, 400, 500
	20 hr	0 hr	10, 25, 50, 75, 100, 125, 150, 175, 200, 300, 400
S9-activated	4 hr	16 hr	10, 20, 25, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50

 

Chromosome Aberration Assay

The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period. The pH of the highest dose of test substance in treatment medium was 7.34. Cell growth and Mitotic Inhibition were observed as follows:

 

Treatment Condition	Treatment Time	Highest Evaluated Dose (µg/mL)	Cell Growth Inhibition (%)	Mitotic Inhibition (%)
Non-activated	4 hr	300	58	28
20 hr		100	57	21
S9-activated	4 hr	32.5	59	28

 

In the non-activated 4-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (21.3 %) were observed at 300 µg/mL (p ≤0.01; Fisher’s Exact test and p ≤0.05; Cochran-Armitage test). In order to confirm dose-responsiveness, a lower dose of 200 µg/mL was included in the evaluation. A statistically significant and dose-dependent increases in structural aberrations (13.3 %) were observed at 200 µg/mL (p ≤0.01; Fisher’s Exact test and p ≤0.05; Cochran-Armitage test). In addition, a statistically significant and dose-dependent increase in numerical (polyploid or endoreduplicated cells) aberrations (6.3%) was observed at 200 µg/mL (p ≤0.05; Fisher’s Exact and Cochran-Armitage tests). In the S9-activated 4-hour exposure group, no significant and dose-dependent increases in structural aberrations were observed at any dose (p >0.05; Fisher’s Exact and Cochran-Armitage tests). A statistically significant increase in numerical aberrations (8.0 %) was observed at 10 µg/mL (p ≤0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p >0.05).

In the non-activated 20-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (2.7 % and 3.7 %) were observed at doses 75 and 100 µg/mL, respectively (p ≤0.05 or p ≤0.01; Fisher’s Exact test and p ≤0.05; Cochran-Armitage test). A statistically significant increase in numerical aberrations (5.7 %) was observed at 75 µg/mL (p ≤0.01; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p >0.05). The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. Under the conditions of the assay described in this report, FAT 40066/E was positive for the induction of structural and numerical chromosome aberrations in the absence of the exogenous metabolic activation system in thein vitromammalian chromosome aberration test using CHO cells. FAT 40066/E was negative for the induction of structural chromosome aberrations and positive for the induction of numerical chromosome aberrations in the presence of the exogenous metabolic activation system in the in vitro mammalian chromosome aberration test using CHO cells.

Conclusions:

FAT 40066/E was positive for the induction of structural and numerical chromosome aberrations in the absence of the exogenous metabolic activation system while it was negative for the induction of structural chromosome aberrations and positive for the induction of numerical chromosome aberrations in the presence of the exogenous metabolic activation system.

Executive summary:

FAT 40066/E was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. The study was carried out according to OECD guideline 473. Water was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were: 0.2, 0.6, 2, 6, 20, 60, 200, 600, and 2000 µg/ml. In the chromosome aberration assay, the concentrations tested were: 10, 25, 50, 100, 200, 300, 400, and 500 µg/mL in the non-activated 4-hour exposure group; 10, 20, 25, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, and 50 µg/mL in the S9-activated 4-hour exposure group; and 10, 25, 50, 75, 100, 125, 150, 175, 200, 300,and 400 µg/mL in the non-activated 20-hour exposure group. In the chromosome aberration assay, cytotoxicity (55±5 % reduction in cell growth index relative to the vehicle control) was observed at doses≥300 µg/mL in the non activated 4 hour exposure group; at doses≥32.5 µg/mL in the S9 activated 4 -hour exposure group; and at doses≥100 µg/mL in the non activated 20-hour exposure group. The doses initially selected for evaluation of chromosome aberrations were 25, 100, and 300 µg/mL for the non activated 4 hour exposure group; 10, 20, and 32.5 µg/mL for the S9 activated 4-hour exposure group; and 50, 75, and 100 µg/mL for the non activated 20-hour exposure group. In the S9-activated 4-hour exposure group, no significant and dose‑dependent increases in structural aberrations were observed at any dose (p >0.05; Fisher’s Exact and Cochran-Armitage tests). A statistically significant increase in numerical aberrations (8.0 %) was observed at 10 µg/mL (p ≤ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p >0.05). The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. These results indicate FAT 40066/E was positive for the induction of structural and numerical chromosome aberrations in the absence of the exogenous metabolic activation system while it was negative for the induction of structural chromosome aberrations and positive for the induction of numerical chromosome aberrations in the presence of the exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Testing proposal for in-vivo Comet assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study planned

Study period:

Subject to ECHA approval

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
Hazard endpoint for which vertebrate testing was proposed: Genetic toxicity in vivo with the registered substance.
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Reactive Red 084 (EC# 286-122-7).
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no available GLP studies on the substance or on read-across analogues suitable to fill the endpoint.
- Available non-GLP studies: There are no available non-GLP studies on the substance or on read-across analogues suitable to fill the endpoint.
- Historical human data: There is no historical human data on the substance or on read-across analogues suitable to fill the endpoint.
- (Q)SAR: (Q)SAR analysis is not sufficient to fill the endpoint. There are no adequate models to address this end point.
- In vitro methods: Already available, but further in vivo data needed. Available studies: 1. Study with methodology equivalent to OECD Guideline 471 (Bacterial Reverse Mutation Assay); 2. In vitro Mammalian Chromosomal Aberration Test conducted according to OECD Guideline 473
- Weight of evidence: There is not sufficient data on the substance or read across analogues to be able to establish a weight of evidence argument.
- Grouping and read-across: There is not sufficient data on the substance or read-across analogues to be able to group or propose read-across.
- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: Not applicable
- Other reasons [if applicable]: Not applicable
CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Test proposal is fully in line with ECHA guidance document* and can neither be replaced by in vitro testing nor by using other data from other substances.
* Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: OECD 489: In Vivo Mammalian Alkaline Comet Assay.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

In vitro Bacterial Reverse Mutation Assay:

FAT 40066/B was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 5120 µg per Petri dish both in the presence and absence of metabolic activation. Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 40066/B was found mutagenic for S. typhimurium strains TA 1537, TA 98 and TA 100. No mutagenic effect was observed with the strain TA 1535. In the presence of S-9 mix a doubling of the spontaneous reversion was only noted at 5120 µg with the strains TA 100 and TA 1537. In the absence of S-9 mix a doubling of the spontaneous reversion was noted at 5120 µg with the strain TA 98. A mutagenic effect was observed with the strains TA 100 and TA 1537 over a concentration range of 1280 to 5120 µg of product per Petri Dish. A mutagenic effect was observed with the strains TA 100 and TA 1537 both in the absence and the presence of S-9 mix, and with the strain TA 98 at 5120 µg only in the absence of S-9 mix.

 

In another study, FAT 40066/C was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in the range of 61.7 to 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 96.5 to 7812.5 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 40066/C led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Based on the study results, FAT 40066/C is considered as non-mutagenic.

 

In-vitro Chromosomal Aberration test:

FAT 40066/E was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese Hamster Ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. The study was carried out according to OECD guideline 473. Water was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were: 0.2, 0.6, 2, 6, 20, 60, 200, 600, and 2000 µg/ml. In the chromosome aberration assay, the concentrations tested were: 10, 25, 50, 100, 200, 300, 400, and 500 µg/mL in the non-activated 4-hour exposure group; 10, 20, 25, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, and 50 µg/mL in the S9-activated 4-hour exposure group; and 10, 25, 50, 75, 100, 125, 150, 175, 200, 300,and 400 µg/mL in the non-activated 20-hour exposure group. In the chromosome aberration assay, cytotoxicity (55±5 % reduction in cell growth index relative to the vehicle control) was observed at doses≥300 µg/mL in the non activated 4 hour exposure group; at doses≥32.5 µg/mL in the S9 activated 4 -hour exposure group; and at doses≥100 µg/mL in the non activated 20-hour exposure group. The doses initially selected for evaluation of chromosome aberrations were 25, 100, and 300 µg/mL for the non activated 4 hour exposure group; 10, 20, and 32.5 µg/mL for the S9 activated 4-hour exposure group; and 50, 75, and 100 µg/mL for the non activated 20-hour exposure group.

In the S9-activated 4-hour exposure group, no significant and dose‑dependent increases in structural aberrations were observed at any dose (p >0.05; Fisher’s Exact and Cochran-Armitage tests). A statistically significant increase in numerical aberrations (8.0 %) was observed at 10 µg/mL (p≤ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p >0.05). The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. These results indicate FAT 40066/E was positive for the induction of structural and numerical chromosome aberrations in the absence of the exogenous metabolic activation system. FAT 40066/E was negative for the induction of structural chromosome aberrations and positive for the induction of numerical chromosome aberrations in the presence of the exogenous metabolic activation system. Under the conditions of the assay described in this report, FAT 40066 was concluded to be positive for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant. The information on this endpoint is not available for the registered substance but needs to be present in the technical dossier to meet the data requirements. Consequently, there is a data gap and it is necessary to generate the data for this endpoint. Therefore, a proposal to conduct the In vivo Comet Assay according to OECD guideline 489 has been made to ECHA.

Justification for classification or non-classification

Based on the above mentioned results a judgement on the mutagenic and clastogenic activity of the test item can't be performed without the generation of additional in vivo data. No classification regarding genotoxicity in accordance to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 will be applied. The final classification for the test item will be proposed after completion of proposed test. According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant. Therefore, a proposal to conduct an in vivo Comet Assay according to OECD guideline 489 has been made to ECHA.