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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

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:
27 October to 07 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
28 October 2016
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan for Salmonella typhimurium and Escherichia coli, respectively
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):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (10% v/v S9 fraction): S9 microsomal fraction was prepared from male rats induced with phenobarbital/β-naphthaflavone
Test concentrations with justification for top dose:
Experiment 1 – Plate Incorporation Method:
S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2 – Pre-Incubation Method:
S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 (with and without S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate. E.coli WP2uvrA (with and without S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In solubility checks performed in-house, the test item was noted to have formed an emulsion in sterile distilled water at 50 mg/mL and was only partially miscible in dimethyl sulphoxide at the same concentration but was fully miscible in acetone at 100 mg/mL. Therefore, acetone was selected as the vehicle.
- Preparation of test item formulation: The test item was accurately weighed and approximate half-log dilutions were prepared in acetone by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (10.2%) of the test item. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
with S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM
- Bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

METHOD OF APPLICATION: in agar (plate incorporation); pre-incubation

DURATION
- Preincubation period: 37 °C ± 3 °C for 20 minutes (with shaking)
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 h

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
All of the plates were incubated at 37 ± 3 °C for approximately 48 h and scored for the presence of revertant colonies using an automated colony counting system. Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
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.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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: A test item precipitate (globular in appearance) was observed under a low power microscope at 5000 μg/plate after the first mutation test (plate incorporation method). However, there was no evidence of a precipitate in the second mutation test after employing the pre-incubation modification.

MUTAGENICITY TEST
- In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains in both the absence and presence of S9-mix from 500 μg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA. Consequently, the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test depending on bacterial strain type. The test item induced a stronger toxic response in the second mutation test, after implementation of the pre-incubation method, with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (all Salmonella strains) and 150 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the Salmonella strains, initially from 150 μg/plate (TA1535 and TA100) and 1500 μg/plate (TA98 and TA1537). No toxic response was noted for Escherichia coli strain WP2uvrA dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was observed under a low power microscope at 5000 μg/plate after the first mutation test (plate incorporation method). However, there was no evidence of a precipitate in the second mutation test after employing the pre-incubation modification.
- 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 revertant colony frequency were observed in both the first and second mutation tests to TA1535, however these increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the responses are considered false and due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

OTHERS
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

None

Conclusions:
The test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to Guideline OECD 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test item diluted in acetone at the concentrations below. 

Experiment 1 – Plate Incorporation Method

S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537.

E. coli WP2uvrA (with and without S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Experiment 2 – Pre-Incubation Method

S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 (with and without S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate.

E.coli WP2uvrA (with and without S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Metabolic activation system used in this test S9 mix (10% v/v S9 fraction): S9 microsomal fraction was pre-prepared from male rats induced with phenobarbital/β-naphthaflavone. Negative control, vehicle and positive control groups were also included in mutagenicity tests. 

In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains in both the absence and presence of S9-mix from 500 μg/plate. No toxicity was noted to E. coli strain WP2uvrA. Consequently, the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test depending on bacterial strain type. The test item induced a stronger toxic response in the second mutation test, after implementation of the pre-incubation method, with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (all Salmonella strains) and 150 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the Salmonella strains, initially from 150 μg/plate (TA1535 and TA100) and 1500 μg/plate (TA98 and TA1537). No toxic response was noted for E. coli strain WP2uvrA dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was observed under a low power microscope at 5000 μg/plate after the first mutation test (plate incorporation method). However, there was no evidence of a precipitate in the second mutation test after employing the pre-incubation modification.

 

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 revertant colony frequency were observed in both the first and second mutation tests to TA1535, however these increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the responses are considered false and due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

The vehicle control plates (acetone) gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Therefore, the test item is not considered as mutagenic in these bacterial systems.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to Guideline OECD 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test item diluted in acetone. 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 revertant colony frequency were observed in both the first and second mutation tests to TA1535, however these increases were considered to have no biological relevance because weakened bacterial background lawns were also noted.

Therefore the responses are considered false and due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.

Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item is not considered as mutagenic in these bacterial systems.

Justification for classification or non-classification

As the registered substance was found negative in an Ames test, it has no mutagenic potential and is therefore not classified according to Regulation (EC) No. 1272/2008 (CLP).