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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 November - 8 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
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:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
- Experiment 1:
TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA (without and with S9): doses of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 2:
Salmonella strains TA1535 (presence of S9) and TA1537 (absence and presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
Salmonella strain TA1535 (absence of S9) and Salmonella strains TA100 and TA98 (absence and presence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
E.coli strain WP2uvrA (absence and presence of S9): 15, 50, 150, 500, 1500 and 5000 µg/plate.

However, results from the second mutation test (pre-incubation method) showed that the toxicity of the test item yielded results that differed significantly from Experiment 1 and consequently an insufficient number of non-toxic dose levels were initially attained for WP2uvrA in the absence of S9-mix. Therefore, this strain was repeated employing an amended test item dose range as follows:
WP2uvrA (absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 5000 µg/plate
Untreated negative controls:
yes
Remarks:
(untreated plates)
Negative solvent / vehicle controls:
yes
Remarks:
(50 or 100 µL/plate DMSO)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: plate incorporation method (0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. In case of treatment with metabolic activation, 0.5 mL of S9 mix was added to the molten instead of phosphate buffer.)
- Experiment 2: pre-incubation method (0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. In case of treatment with metabolic activation, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer.)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain, in all experiments

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation
Evaluation criteria:
For the test substance to be considered mutagenic:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitation was observed on the plates up to and including the top dose of 5000 µg/plate

EXPERIMENTS 1 AND 2:
- In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except WP2uvrA dosed in the presence of S9-mix), initially from 150 µg/plate in both the absence and presence of S9-mix.
- In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA100, TA1535, WP2uvrA and TA98) and 150 µg/plate (TA1537). In the presence of S9-mix, weakened bacterial background lawns were noted from 500 µg/plate (TA100, TA1535, TA98 and TA1537) and 1500 µg/plate (WP2uvrA).

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test from 150 to 5000 µg/plate in the absence of S9-mix and from 500 to 5000 µg/plate in the presence of S9-mix. Statistically significant increases in TA1535 revertant colony frequency were again observed in the second mutation test at one dose only (500 µg/plate) in the absence and presence of S9-mix. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same test item dose levels. Therefore the ‘response’ observed would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and present as non-revertant colonies.
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay (Ames test) performed according to OECD 471.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles, in the Ames test. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on the dose finder study, up to the maximum recommended dose level of 5000 µg/plate. Adequate negative and positive controls were included. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains (TA1535, TA1537, TA98, TA100 and E.coli WP2uvrA), with any dose of the test item, either with or without metabolic activation. Based on the results of this study, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

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

Additional information

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles, in the Ames test. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on the dose finder study, up to the maximum recommended dose level of 5000 µg/plate. Adequate negative and positive controls were included. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains (TA1535, TA1537, TA98, TA100 and E.coli WP2uvrA), with any dose of the test item, either with or without metabolic activation. Based on the results of this study, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for mutagenicity in accordance with EU CLP and its amendments ( EC/1272/2008).