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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was shown to have no mutagenic potential in bacteria and mammalian cells in vitro.

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
mutant histidine gene
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of male Sprague Dawley rats, which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254, dissolved in corn oil, 5 days prior to sacrifice. The S9 mix comprised 10% S9 fraction.
Test concentrations with justification for top dose:
plate incorporation assay:
0, 50, 158, 500, 1581, 5000 µg/plate with and without S9 mix
preincubation assay:
0, 8, 16, 32, 64, 128, 256, 512 µg/tube with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Bisphenolharz spezial formed a clear colourless to yellowish solution in DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102 in plate incorporation assay), cumene hydroperoxide (only TA 102 in preincubation assay), 2-aminoanthracene
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
no statistics perfomed; evaluation based on criteria mentioned above
Species / strain:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Doses of >= 158 µg per plate had a strong, strain-specific bacteriotoxic effect. Therefore they could only partly be used for assessment purposes up to and including 256 µg per plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None of the five strains showed in the plate incorporation test a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Executive summary:

The test substance was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of phenobarital/beta-naphthoflavone induced male Wistar rats.
Test concentrations with justification for top dose:
In several experiments concentrations from 0.05 up to 2400 µg/ml were tested without S9 mix and from 0.4 to 2400 µg/ml with S9 mix; however, due to precipitation and test item induced cytotoxicity etc. the following concentrations were chosen for evaluation in the final experiments:
4 hours treatment without S9 mix (18 hour culture): 1.6, 3.1, and 6.3 µg/ml
4 hours treatment with S9 mix (18 hour culture): 6.3, 12.5, and 25 µg/ml
18 hours treatment without S9 mix (18 hour culture): 5, 10, and 20 µg/ml
4 hours treatment with S9 mix (28 hour culture): 6.3, 12.5, and 24 µg/ml
Evaluation of higher concentrations was impossible due to strong test item induced toxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality).
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO 0.5% (v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate, cyclophosphamide
Remarks:
ethylmethane sulfonate was used as positive control in cultures without S9 mix (1000 µg/ml for 4 hour treatment and 600 µg/ml for 18 hour treatment); cyclophosphamide was used as positive control in cultures with S9 mix (1.4 µg/ml).
Details on test system and experimental conditions:
Chromosomes were prepared 18 and 28 hours (for 4 hour treatment) or 18 hours (for 18 hour treatment) after start of treatment with test substances. The treatment interval was 4 hours with and without metabolic activation and 18 hours without metabolic activation. In each experimental group two paralell cultures were set up.

NUMBER OF CELLS EVALUATED: Chromosomes of approximately 200 metaphases per concentration, i.e. 100 metaphases from each of two parallel cultures, were examined.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid was added to the culture medium in a concentration of 0.2 µg/ml 2.5 hours prior to the end of the incubation period.

DETERMINATION OF CYTOTOXICITY:
- Cell survival and number of methaphases was determined in the presence and absence of S9 mix.

OTHER: Influence on pH and osmolality was assessed.
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data range, and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of indued structural chromosome aberrations is not in the range of the laboratory's historical control data range, and/or
- either a concentration-related or as significant increase of the number of strucutral chromosome aberrations is observed.
However, both biological and statistical significance shoud be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Statistics:
Fisher's exact test (p< 0.05); only cells with aberrations, excluding gaps, were included
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Strong test item induced toxic effects (reduced cell numbers and/or low metaphase numbers, partially praralleled by poor metaphase quality) were observed at 9.4 µg/ml and above in the 4 hour treatment cultures without S9 mix and at 40 µg/ml and above in the 18 hour treatment cultures without S9 mix. With S9 mix strong cytotoxic effects were observed at 50 µg/ml and above. Therefore, these and higher concentrations could not be evaluated.
Reference mutagens showed the expected results.
- Effects on pH: no effects
- Effects of osmolality: no effects
- Precipitation: at 150 µg/ml and above
None of the cultures treated with Bisphenolharz spezial in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Executive summary:

The clastogenic potential of the test substance was evaluated in a chromosome aberration test on Chinese hamster V79 cells according to OECD TG 473. Concentrations in the range of 0.05 to 2400 µg/ml were tested in pre-experiments with and without S9 mix. Due to precipitation, strong test item induced cytotoxicity (reduced cell numbers and/or low metaphase numbers, partially attended by poor metaphase quality etc.), only concentrations of 5 to 25 µg/ml were scorable.

None of the cultures assessed showed biologically relevant or statistically significant increased numbers of aberrant metaphases. Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to the highest evaluable concentration.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of phenobarital/beta-naphthoflavone induced male Wistar rats.
Test concentrations with justification for top dose:
Based on the results of the pre-experiment the following concentration range of the main experiments was selected:
Exp. I, 4 hour treatment without S9 mix: 0.5, 1.0, 2.0, 4.0, 6.0, (8.0, 10.0) µg/ml
Exp. I, 4 hour treatment with S9 mix: 1.1, 3.8, 7.5, 15,0, 30.0, 45.0, (60.0, 90.0) µg/ml
Exp. II, 4 hour treatment without S9 mix: (0.5, 1.0), 2.0, 4.0, 5.0, 6.0, 7.0 µg/ml
Exp. II, 4 hour treatment with S9 mix: 1.1, 10.0, 20.0, 40.0, 45.0, 50.0, (55.0, 60.0) µg/ml
In both main experiments the individual concentrations were spaced by a factor of 2. Closer spacing was used at high concentrations to cover the toxic range more closely. In the first main experiment with and without metabolic activation and in the second experiment with metabolic activation the cultures at the two highest concentrations were not continued due to exceedingly severe cytotoxic effects. In the second main experiment without metabolic activation the cultures of the two lowest concentrations were not continued.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulphonate; 7,12-dimethylbenz(a)anthracene
Remarks:
ethylmethane sulfonate used as positive control in cultures without S9-mix (150 µg/mL); dimethylbenzanthracene used as positive control in cultures with S9-mix (1.1 µg/mL).
Details on test system and experimental conditions:
Influence of test item in a concentration of 240 µg/mL on pH and osmolality was assessed.
Four days (experiment I) and three days (experiment II) after treatment 1.5x10 E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Fowowing the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 5x10 E5 cells each in medium containing 6-Thioguanine (11 µg/ml). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A positive response is described as folIows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (3.3 -33.2 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Ganal Boulevard, Suite G, Richmond, GA 94804, USA) statistics software. The number of mutant colonies obtained for the cultures treated with the test item were compared to the solvent controls. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Additional information on results:
No reproducible increase in mutant fequencies was observed in the main experiments up to acceptable levels of cytotoxicity. The induction factor of three times the corresponding solvent control was exceeded at 3.8 and 15.0 µg/mL in the first experiment with metabolic activation (culture II). In the second experiment the induction factor was exceeded at 4.0 µg/mL in the absence of metabolic activation in culture II and at 20 µg/mL and 40 µg/mL in the second culture with metabolic activation. However, these effects were judged as biologically irrelevant since the absolute values of the mutation frequency remained well within the historical control range and none of these increases was reproduced in the parallel cultures. Furthermore, the increased values of the mutation frequency described above were not dose dependent as indicated by the lacking statistical significance.
The relatively large induction factors are based on the low solvent control counts of 2.8 to 5.4 colonies per 10 E6 cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency below 50% of the solvent control occurred in the first experiment at 6.0 µg/mL and above without metabolie activation and at 60.0 µg/mL and above with metabolie activation. In the second experiment relevant cytotoxicity was observed at 7.0 µg/mL without metabolic activation and at 50.0 µg/mL and above with metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: no relevant changes
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 2000µg/ml Polyether P 293.
- Precipitation or turbidity: at 120 to 240 µg/mL with S9 mix (determined in the pre-test)
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Executive summary:

The test substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476 in concentrations of up to and including 7 µg/mL without S9 mix 50 µg/ml with S9 mix. The highest concentrations induced acceptable levels of cytotoxicity. Cultures with higher concentrations were not continued due to exceedingly strong toxic effects. Precipitation or turbidity was observed at 120 µg/mL and above in the cultures with metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the cultures with and without S9 mix. Based on these results the test substance is considered to be non-mutagenic in this V79/HPRT test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The test substance was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

The clastogenic potential of the test substance was evaluated in a chromosome aberration test on Chinese hamster V79 cells according to OECD TG 473. Concentrations in the range of 0.05 to 2400 µg/ml were tested in pre-experiments with and without S9 mix. Due to precipitation, strong test item induced cytotoxicity (reduced cell numbers and/or low metaphase numbers, partially attended by poor metaphase quality etc.), only concentrations of 5 to 25 µg/ml were scorable.

None of the cultures assessed showed biologically relevant or statistically significant increased numbers of aberrant metaphases. Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to the highest evaluable concentration.

The test substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476 in concentrations of up to and including 7 µg/mL without S9 mix and 50 µg/ml with S9 mix. The highest concentrations induced acceptable levels of cytotoxicity. Cultures with higher concentrations were not continued due to exceedingly strong toxic effects. Precipitation or turbidity was observed at 120 µg/mL and above in the cultures with metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the cultures with and without S9 mix. Based on these results the test substance is considered to be non-mutagenic in this V79/HPRT test.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In several in vitro mutagenicity studies the substance showed no mutagenic properties. Therefore no classification is required.