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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two reverse mutation assays were run with the test substance, one with a 62% pure sample and the other with an 84.9% pure sample. The first assay was positive in Escherichia coli WP2 uvrA, but at the highest concentrations only. These effects may have resulted from impurities or by-products of the test substance. When the study was re-run with the higher purity sample, the outcome was negative. Based on these results, the test substance is not considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 04 November, 1997 to 02 April, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No deviations had a detrimental impact on the outcome of the study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
No deviations had a detrimental impact on the outcome of the study
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix fom induced rat liver and uninduced hamster liver
Test concentrations with justification for top dose:
Based on toxicity tests, the concentration range included two logarithmic decades. In this study 6 dose levels with adequately spaced intervals were tested in experiments I and II, and 4 dose levels were tested in experiment III, The maximum dose level was 5000 µg/plate (active ingredient) in experiments I and II, and 8000 µg/plate (active ingredient) in experiment III
Exp I and II: 33; 100; 333; 1000; 2500; and 5000 µg/plate (active ingredient)
Exp III (strain WP2 uvrA only)- 2000; 4000; 6000; and 8000 µg/plate (active ingredient)
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
congo red
methylmethanesulfonate
other: 2-aminoanthracene; 4-Nitro-o-phenylene-diamine
Details on test system and experimental conditions:
Ames plate incorporation test (experiment I) and the Prival preincubation test (experiment II and III)

For each strain and dose level, including the controls three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or 59 mix substitution buffer (for test without metabolic activation),
100 µI Bacteria suspension (cf test system, pre-culture of the strains)

According to the pre-incubation method 100 µl test solution, 500 pl S9 mix / 59 mix substitution buffer, and 100 µI bacteria suspension were mixed in a test tube and incubated at
30°C for 30 minutes. After pre-incubation 2.0 ml overlay agar (45°C) was added to each tube The mixture was poured on minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Acceptability of the Assay
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria.:
- normal background growth in the negative and solvent control
- normal range of spontaneous reversion rates in the negative and solvent control
- the positive control substances should produce a significant increase in mutant colony frequencies


Evaluation criteria:
A test article is considered positive if either a dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced..
A test article producing neither a dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A biologically relevant response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.

Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
NA
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No relevant increase in the number of revertant colonies was observed in the plate incorporation assay (experiment I) in any of the strains used, neither in the presence nor absence of metabolic activation. In experiment II (pre-incubation test), a dose dependent increase was observed in strain WP2 uvrA with and without metabolic activation. The threshold of twice the number of the spontaneous revertant colonies of the corresponding solvent control was reached at 5000 µg/plate without 59 mix and 2500 µg/plate with S9 mix. To verify these results, an additional pre-incubation experiment was performed using strain WP2 uvrA with and without metabolic activation. In this third experiment, in the presence and absence of Metabolic activation, the threshold was reached at 2000 µg/plate and clearly exceeded in the concentrations above The diverging results of the plate incorporation method versus the preincubation
procedure are based upon the different test design and the increased sensitivity of the pre-incubation assay, not upon the application of rat S9 in the first experiment and hamster
S9 in the other experiments since the mutagenic effect also occurred in the absence of metabolic activation. However, since mutagenicity was observed at high concentrations only,
these effects may also result from impurities or by-products of the test article

Solubility. No precipitation of the test article occurred up to the highest investigated concentration.
Toxicity. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation up to the highest investigated dose.
Conclusions:
Under the study conditions, the test substance was considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays. Since mutagenicity was observed at high concentrations only, these effects may result from impurities or by-products of the test article.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 401 and EU Method B13/B14, in compliance with GLP. Both an Ames incorporation test (Experiment I) and the Prival pre-incubation test (Experiments II and III) were run. The strains used were Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA. The dose ranges corresponded to 33, 100, 333, 1000, 2500 and 5000 µg active ingredient (a.i.)/plate in Experiments I and II and to 2000, 4000, 6000 and 8000 µg a.i./plate in Experiment III (E. coli WP2 uvrA only). The Ames plate incorporation test was run with and without induced rat liver S9 mix. The Prival pre-incubation test was run with and without uninduced hamster liver S9 mix. The test substance did not precipitate up to the highest investigated concentration. There were no toxic effects, evident as a reduction in the number of revertants, up to the highest dose.

No relevant increase in the number of revertant colonies was observed in the plate incorporation experiment (Experiment I). In Experiment II, a dose-dependent increase was observed in strain WP2 uvrA with and without metabolic activation. The threshold of twice the number of spontaneous revertant colonies of the corresponding solvent control was reached at 5000 µg a.i./plate without S9 mix and 2500 µg a.i./plate with S9 mix. To verify these results, an additional experiment was performed with strain WP2 uvrA with and without metabolic activation using concentrations up to 8000 µg a.i./plate. In this third experiment, in the presence and absence of metabolic activation, the threshold was reached at 2000 µg a.i./plate and clearly exceeded in the concentrations above. Since mutagenicity was observed at the highest concentrations only, these effects may result from impurities or by-products of the test substance.

Under the study conditions, the test substance was considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays (Wollny, 1998).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Study 1, test substance at 62% purity

A study was conducted to determine the mutagenic potential of the test substance (62% purity) according to OECD Guideline 401 and EU Method B13/B14, in compliance with GLP. Both an Ames incorporation test (Experiment I) and the Prival pre-incubation test (Experiments II and III) were run. The strains used were Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA. The dose ranges corresponded to 33, 100, 333, 1000, 2500 and 5000 µg active ingredient (a.i.)/plate in Experiments I and II and to 2000, 4000, 6000 and 8000 µg a.i./plate in Experiment III (E. coli WP2 uvrA only). The Ames plate incorporation test was run with and without induced rat liver S9 mix. The Prival pre-incubation test was run with and without uninduced hamster liver S9 mix. The test substance did not precipitate up to the highest investigated concentration. There were no toxic effects, evident as a reduction in the number of revertants, up to the highest dose.

No relevant increase in the number of revertant colonies was observed in the plate incorporation experiment (Experiment I). In Experiment II, a dose-dependent increase was observed in strain WP2 uvrA with and without metabolic activation. The threshold of twice the number of spontaneous revertant colonies of the corresponding solvent control was reached at 5000 µg a.i./plate without S9 mix and 2500 µg a.i./plate with S9 mix. To verify these results, an additional experiment was performed with strain WP2 uvrA with and without metabolic activation using concentrations up to 8000 µg a.i./plate. In this third experiment, in the presence and absence of metabolic activation, the threshold was reached at 2000 µg a.i./plate and clearly exceeded in the concentrations above. Since mutagenicity was observed at the highest concentrations only, these effects may result from impurities or by-products of the test substance.

Under the study conditions, the test substance was considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays (Wollny, 1998).

Study 2, test substance at 84.9% purity

A study was conducted to determine the mutagenic potential of the test substance (84.9% purity) according to OECD Guideline 401 and EU Method B13/B14, in compliance with GLP. Both an Ames incorporation test and the Prival pre-incubation test were run. The strains used were Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA. The doses ranged from 24 to g000 µg active ingredient/plate in both experiments. The Ames plate incorporation test was run with and without induced rat liver S9 mix. The Prival pre-incubation test was run with and without uninduced hamster liver S9 mix. The test substance did not precipitate up to the highest investigated concentration. A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 3000 µg/plate onwards.

No relevant increase in the number of his’ or trp’ revertant colonies was observed either in the Ames standard plate incorporation test or in the Prival pre-incubation test, with or without S9 mix.

Under the study conditions, the test substance was not considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays (Engelhardt, 1999).

Justification for classification or non-classification

Insufficient information is available to determine classification for genotoxicity according to CLP (EC 1272/2008) criteria.