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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation test:

The Substance was tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA 1535, TA 1537 and Escherichia Coli WP2 tester strain both in the presence and absence of liver S9 microsomal activation system. The test was performed according to OECD TG 471 and in compliance with GLP.

 

In vitro mammalian chromosome aberration assay:

An in vitro mammalian chromosome aberration test according to OECD TG 473 with the Substance is ongoing. Data will be added upon receiving the final study report.

 

In vitro mammalian cell gene mutation assay:

An in vitro gene mutation test in mammalian cells according to OECD TG 476 with the Substance will be conducted when negative results are obtained from OECD TG 471 and 473 studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 2002 - January 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
not specified
Principles of method if other than guideline:
To evaluate the mutagenic potential of test substance in bacteria by AMES assay.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus in the genome of Salmonella typhimurium and tryptophan locus of Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of either the uvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium) results in a deficient DNA excision repair system, which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin for growth.

Salmonella typhimurium tester strains also contain the rfa wall mutation, which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall.

Tester strains TA98 and TAlOO also contain the pKMlOl plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism by which this plasmid increases sensitivity to mutagens is by modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TAlOO, TA1535, and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 Homogenate (Aroclor) in S9Mix
Test concentrations with justification for top dose:
Salmonella tester strains (with S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Salmonella tester strains (without S9 mix): 3.33, 10.0, 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Escherichia coli tester strain (with and without S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate

Cytotoxicity was observed in the dose range finding study, and the highest dose level of test article used in the subsequent mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn.
Vehicle / solvent:
The test article was .observed to form a transparent, colorless solution at a concentration of 100 mg per mL in dimethylsulfoxide (DMSO). DMSO was selected as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution prepared for the mutagenicity assay, the test article was observed to form a transparent, non-viscous, colorless solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene - TA100, TA1535, TA1537, WP2uvrA with S9 Mix; ICR-191 - TA1537 without S9 Mix
Details on test system and experimental conditions:
Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls and positive controls were plated in triplicate.
Rationale for test conditions:
Experimental materials, methods and procedures are based on those described by Ames et al. (1975) and Green and Muriel (1976). The assay design is based on the OECD Guideline 471, updated and adopted 21 July 1997.
Evaluation criteria:
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.
Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.

Revertant colonies were counted by automated colony counter or by hand.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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 applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose Range Finding Assay
Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 ug per plate were tested.
Cytotoxicity was observed with tester strain TA100 at 333 ug per plate and above in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertants per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA in the presence or absence of S9 mix.

Mutagenicity Assay
In the initial mutagenicity assay, first trial (B1), all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.
In the confirmatory mutagenicity assay, second trial (C1), contamination was observed on many of the assay plates and several of the plates were observed to have reduced or absent bacterial background lawns. Due to the multiple technical problems observed the data generated were not used in the evaluation of the test article (the results have not been included).
The confirmatory assay was repeated in third trial (D1). In the repeat confirmatory mutagenicity assay, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In this trial, a 2.7-fold increase was observed with tester strain WP2uvrA in the presence of S9 mix, however, this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix in
fourth trial (D2). Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TAI100 at the same doses in the absence of S9 mix.
In the fourth trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.

All criteria for a valid study were met.
Conclusions:
The substance was tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA 1535, TA 1537 and Escherichia Coli WP2 tester strain both in the presence and absence of liver S9 microsomal activation system. The test was performed according to OECD TG 471 and in compliance with GLP.
Executive summary:

A bacterial reverse mutation assay was performed to assess the mutagenic activitythe test chemicalin in Salmonella typhimuriumTA 98, TA100, TA 1535, TA 1537 andEscherichia colistrain WP2 uvrA tester strains bytheplate incorporation method. The doses tested in the mutagenicity assay were selected based on the results of a dose-range finding assay using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 µg per plate, both in the presence and absence of mammalian liver microsomal enzymes (S9 mix).The growth inhibitory effect (cytotoxicity)induced by the test chemicalwas determinedas a decrease in thenumber of revertant colonies per plate and/or by a thinning or disappearance of the bacterialbackground lawn.Cytotoxicity induced by the test chemical was observed with tester strain TA100 at≥333 ug per plate in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertant per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA either in the presence or absence of S9 mix.The mutagenicity assay was performed using tester strains TA98, TA100, TA1535. TA1537, and WP2uvrA both in the presence and absence of S9 mix along with the appropriate vehicle (DMSO) and positive controls. The doses tested with the Salmonella tester strains were 0, 33.3, 100, 333, 1000, 3330, 5000 µg per plate in the presence of S9 mix and 0, 3.33, 10, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in the absence of S9 mix. The doses tested with tester strain WP2uvrA were 0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate both in the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in independentconfirmatoryexperiments. The number of revertant colonies per plate for the vehicle controls and all doses were counted manually.In the initial mutagenicity assay, no substantial increases in the mean number of revertantsper plate were observed with any of the tester strains either in the presence or absence of metabolic activation. In the confirmatory mutagenicity assay with Salmonella tester strains no substantial increase of His+colony numbers were seen at any doses tested either with or without metabolic activation. In E. coli WP2uvrA and in the presence of S9 mix, a 2.7-fold increase of revertant colony numbers was observed, but this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix. Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TA100 at the same doses without S9 mix. In the last trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.In conclusion,the test chemicaldid not induce gene mutations by base pair changes or frameshifts in the genome ofS. typhimurium and E. coli (WP2 uvrA) tester strainsneither in the presence nor in the absence of metabolic activation and consequently, it was considered non-mutagenic (negative) in bacterial reverse mutation assay, under the experimental conditions described.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
The study is ongoing and this information will be submitted later based on ECHA communication/decision number CCH-D-2114536388-40-01/F
Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
In vitro gene mutation study in mammalian cells according to OECD TG 476 with the registered substance will be performed if negative results are obtained from OECD 471 and OECD 473 studies and will be submitted later based on ECHA communication/decision number CCH-D-2114536388-40-01/F

Additional information

Bacterial reverse mutation test:

A bacterial reverse mutation assay was performed to assess the mutagenic activitythe test chemicalin in Salmonella typhimuriumTA 98, TA100, TA 1535, TA 1537 andEscherichia colistrain WP2 uvrA tester strains bytheplate incorporation method. The doses tested in the mutagenicity assay were selected based on the results of a dose-range finding assay using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 µg per plate, both in the presence and absence of mammalian liver microsomal enzymes (S9 mix).The growth inhibitory effect (cytotoxicity)induced by the test chemicalwas determinedas a decrease in thenumber of revertant colonies per plate and/or by a thinning or disappearance of the bacterialbackground lawn.Cytotoxicity induced by the test chemical was observed with tester strain TA100 at≥333 ug per plate in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertant per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA either in the presence or absence of S9 mix.The mutagenicity assay was performed using tester strains TA98, TA100, TA1535. TA1537, and WP2uvrA both in the presence and absence of S9 mix along with the appropriate vehicle (DMSO) and positive controls. The doses tested with the Salmonella tester strains were 0, 33.3, 100, 333, 1000, 3330, 5000 µg per plate in the presence of S9 mix and 0, 3.33, 10, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in the absence of S9 mix. The doses tested with tester strain WP2uvrA were 0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate both in the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in independentconfirmatoryexperiments. The number of revertant colonies per plate for the vehicle controls and all doses were counted manually.In the initial mutagenicity assay, no substantial increases in the mean number of revertantsper plate were observed with any of the tester strains either in the presence or absence of metabolic activation. In the confirmatory mutagenicity assay with Salmonella tester strains no substantial increase of His+colony numbers were seen at any doses tested either with or without metabolic activation. In E. coli WP2uvrA and in the presence of S9 mix, a 2.7-fold increase of revertant colony numbers was observed, but this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix. Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TA100 at the same doses without S9 mix. In the last trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.In conclusion,the test chemicaldid not induce gene mutations by base pair changes or frameshifts in the genome ofS. typhimurium and E. coli (WP2 uvrA) tester strainsneither in the presence nor in the absence of metabolic activation and consequently, it was considered non-mutagenic (negative) in bacterial reverse mutation assay, under the experimental conditions described.

Justification for classification or non-classification