Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 14 to August 18, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 05 July 2016 / signed on 28 October 2016
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name (as cited in study report): LABDANUM ABSOLUTE / F
- Storage condition of test material: closed containers, room temperature in the dark"

Method

Target gene:
Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction was pre-prepared using standardized in-house procedures (outside the confines of this study). The 10% S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
Test concentrations with justification for top dose:
Experiment 1 (Pre-incubation Method):
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix.
Experiment 2 (Pre-Incubation Method):
Salmonella strain TA100 (absence of S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strain TA98 (absence of S9): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
E.coli strain WP2uvrA and Salmonella strains TA1535 and TA1537 (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
All bacterial strains (presence of S9): 15, 50, 150, 500, 1500, 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 as immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL. Acetone was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in acetone by mixing on a vortex mixer. No correction was required for purity allowance. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations 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.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated: spontaneous mutation rates
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
Remarks:
untreated: spontaneous mutation rates
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: The bacteria used in the test were obtained from the University of California, Berkeley, and from the British Industrial Biological Research Association.

METHOD OF APPLICATION: preincubation (The test item was confirmed to either have unknown volatility or was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls).

DURATION
- Preincubation period: 20 minutes with shaking
- Exposure duration: approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level in experiment 1 and experiment 2.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - test item dose levels were selected based on Experiment 1 results. Up to seven test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.
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:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases 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.
Statistics:
NA

Results and discussion

Test results
Key result
Species / strain:
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
Remarks:
-S9: 150 µg/plate (TA1535, WP2uvrA and TA1537), 500 µg/plate (TA100) and 1500 µg/plate (TA98). +S9: at 5000 mg/plate ((TA100, TA1537 and TA1535)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: In solubility checks performed in house the test item was noted as immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL.
- Precipitation: A test item precipitate (light and globular in appearance) was noted at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
Not needed (no statistical significant increase were noted)
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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains from 150 µg/plate (TA1535, WP2uvrA and TA1537), 500 µg/plate (TA100) and 1500 µg/plate (TA98) in the absence of S9 mix and to three bacterial strains (TA100, TA1537 and TA1535) at 5000 µg/plate in the presence of S9-mix. Consequently, the toxic limit or the maximum recommended dose (5000 µg/plate) of the test item was employed as the maximum dose in the second mutation test, depending on bacterial tester strain type and presence or absence 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 150 µg/plate (TA1535, WP2uvrA and TA1537), 500 µg/plate (TA100) and 1500 µg/plate (TA98). In the presence of S9-mix weakened bacterial background lawns were noted to two of the bacterial tester strains at 5000 µg/plate (TA1535 and TA1537). 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 experiment.

CONCLUSION:
There were no biologically relevant 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. Similarly, no biologically relevant 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. Small increases in TA1535 revertant colony frequency were observed in the presence of S9 mix at 5000 µg/plate in both experiments. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the responses could be considered due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.
Remarks on result:
other:
Remarks:
Table of results are in "Attached background documents"

Any other information on results incl. tables

Cf Tables of results in attached background material

Applicant's summary and conclusion

Conclusions:
Under the test condition, test material 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 performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in acetone both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames pre‑incubation methods in Experiment 1 and 2.

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Up to seven test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the toxic limit of the test item.

 

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.

In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of thebacterial testerstrains from 150 µg/plate (TA1535, WP2uvrAand TA1537), 500 µg/plate (TA100) and 1500 µg/plate (TA98) in the absence of S9‑mix and to three bacterial strains (TA100, TA1537 and TA1535) at 5000 µg/plate in the presence of S9-mix. Consequently, the toxic limit or the maximum recommended dose (5000 µg/plate) of the test item was employed as the maximum dose in the second mutation test, depending on bacterial tester strain type and presence or absence 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 150 µg/plate (TA1535, WP2uvrAand TA1537), 500 µg/plate (TA100) and 1500 µg/plate (TA98). In the presence of S9-mix weakened bacterial background lawns were noted to two of the bacterial tester strains at 5000 µg/plate (TA1535 and TA1537). 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 experiment.

A test item precipitate (light and globular in appearance) was noted at 5000 mg/plate, this observation did not prevent the scoring of revertant colonies.

There were no biologically relevant 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. Similarly, no biologically relevant 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. Small increases in TA1535 revertant colony frequency were observed in the presence of S9‑mix at 5000 µg/plate in both experiments. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the responses could be considered due to additional histidine being available to His-bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

 

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.