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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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, the Registrant proposes to conduct the following tests using the indicated test method:

- EU Method B.12 Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus test or the equivalent OECD Guideline 474 – Mammalian Erythrocyte Micronucleus test (see Genetic toxicity in vivo Testing proposal).

- OECD Guideline 486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo (see Genetic toxicity in vivo Testing proposal).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
conducted according to previous version available in 1981.
Deviations:
yes
Remarks:
Only four tester strains were used for the experiment and no tester strain to detect cross-linking mutagens was included.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
The mutation in the histidine operon.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-liver extract from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
Five dose levels: 0.2, 2, 20, 200 and 2000 µg per petri dish.
Vehicle / solvent:
DMSO: Dimethylsulfoxide.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: MNNG= N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
A sample of FAT 36052/D (red dyestuff) was tested in Salmonella typhimurium strains TA 1535, 1537, TA 98 and TA 100 at five dose levels: 0.2, 2, 20, 200 and 2000 µg per petri dish.
The substance was dissolved in DMSO and every concentration was tested in triplicate. The tubes were agitated using a vortex mixed and poured onto the minimal medium base. The plates were incubated for 48 hours at 37 °C in the dark.

The colonies, the revertants to the wild type, were counted manually or electronically using a Fisher Colony counter.
Evaluation criteria:
The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-response relationship.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 98 and TA 100
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
Additional information on results:
The evidence for mutagenicity existed only in the presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 20 to 2000 µg of product per petri dish.

None

Conclusions:
FAT 36052/D is not mutagenic for S. typhimurium strains TA 1535, TA 98 and TA 100 in the presence and absence of metabolic activation, but was mutagenic for S. typhimurium strain TA 1537 in presence of S-9 mix.
Executive summary:

The substance FAT 36052/D was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at five dose levels:0.2, 2, 20, 200 and 200 microgramme (or nl) per petri dish. both in presence and absence of metabolic activation. The test substance was dissolved in DMSO and every concentration was tested in triplicate.

Under the experimental conditions, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 36052/D was not mutagenic for S. typhimurium strains TA 1535, TA 98 and TA 100 in the presence and absence of metabolic activation, but was mutagenic for S. typhimurium strain TA 1537 in presence of S-9 mix.

The evidence for mutagenicity existed only in the presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 20 to 2000 microgramme of product per petri dish.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Genotoxicity of the test item was assessed in three in vitro systems.

In Reverse Mutation Assay using Bacteria (Salmonella Typhimurium) FAT 36052/D exerted a very weak mutagenic effect in this test system.

 

In vitro Mammalian Chromosome Aberration Test, the test substance FAT 93504/B TE induced structural chromosome aberrations in the V79 Chinese hamster cell line in the absence and the presence of metabolic activation.

In vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells, FAT 93504/B TE and its metabolites did not show any mutagenic activity in this forward mutation system.

 

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 (see genetic toxicity in vivo testing proposal).

Justification for classification or non-classification

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, the registrant proposes to conduct the following test by oral route using the indicated test method: EU Method B.12 Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus test or the equivalent OECD Guideline 474 – Mammalian Erythrocyte Micronucleus test.

Hence, the genotoxicity data is considered to be inconclusive for classification of the substance as per the Regulation (EC) No. 1272/2008 (CLP) criteria.