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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 04, 2017 to August 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: See remarks
Remarks:
rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galacto se metabolism; chl: mutation in nitrate reductase; bio defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced Aroclor 1254
Test concentrations with justification for top dose:
Treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. The highest concentration of the test substance used in the mutation assays was 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines).
Vehicle / solvent:
Purified water for the test substance
DMSO or purified water for positive controls
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO or saline
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene (AAN)
Details on test system and experimental conditions:
-The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix.

The test substance was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments without and with S-9 for test substance, vehicle and positive controls. These plating’s were achieved by the following sequence of additions to molten agar at 45±1°C:

• 0.1 mL bacterial culture
• 0.1 mL of test substance solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution

Followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 2 to 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertants colonies were counted.

As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test substance, vehicle control solution or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Rationale for test conditions:
- Based on the most recent OECD and EC guidelines.
- First mutation experiment
Evaluation criteria:
For valid data, the test substance was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. The positive trend/effects described above were reproducible.

The test substance was considered positive in this assay if both of the above criteria were met.
The test substance was considered negative in this assay if neither of the above criteria were met.
Statistics:
No formal hypothesis testing was done.
- Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria were counted.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 , TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
- The test substance was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.
- All test substance treatments in this study were performed using formulations prepared in water for irrigation (purified water), and all concentrations are expressed in terms of pure compound.
- Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed in all strains from 50 or 160 μg/plate in the absence of S-9 and from 500 μg/plate in the presence of S-9.
- Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For all strains, the maximum test concentration was reduced based on strain specific toxicity observed in Mutation Experiment 1. Narrowed concentration intervals were employed covering the ranges 2.5-160 μg/plate or 10-500 μg/plate, in order to examine more closely those concentrations of the test substance approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed in all strains at concentrations of 80 and 160 μg/plate in the absence of S-9 and additionally in TA98 at 320 and 500 μg/plate and in TA102 at 40 μg/plate. In the presence of S-9, evidence of toxicity was noted in all strains at concentrations of 320 and 500 μg/plate and additionally in TA102 at 160 μg/plate.
- No precipitation was observed on the test plates following incubation. Vehicle and positive control treatments were included for all strains in both experiments.
- The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following test substance treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control.
- This study was considered therefore to have provided no evidence of any test substance mutagenic activity in this assay system it was concluded that the test substance did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium bacterial reverse mutation assay.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471, in compliance with GLP. The test substance was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9 mix), in two separate experiments. All test substance treatments were performed in purified water. In Experiment 1 treatments of all the tester strains were performed in +/- of S9 mix, using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following treatment, evidence of toxicity was observed in all strains from 50 or 160 μg/plate in the absence of S9 and from 500 μg/plate in the presence of S9. In Experiment 2 treatments of all the tester strains were performed in the presenec and absence of S9. For all strains, the maximum test concentration was reduced based on strain-specific toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 2.5 -160 μg/plate or 10 -500 μg/plate, in order to examine more closely those concentrations of the test substance approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S9 were further modified by the inclusion of a pre-incubation step. Following treatment, evidence of toxicity was observed in all strains at 80 and 160 μg/plate in the absence of S9 mix and additionally in TA98 at 320 and 500 μg/plate and in TA102 at 40 μg/plate. In the presence of S9, evidence of toxicity was noted in all strains at 320 and 500 μg/plate and additionally in TA102 at 160 μg/plate. No precipitation was observed on the test plates following incubation. Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following test substance treatment in the presence and absence of S9, no increase in revertant numbers was observed that was greater than 1.5 -fold (in strain TA102), ≥2 -fold (in strains TA98 or TA100) or ≥3 -fold (in strains TA1535 or TA1537) of the concurrent vehicle control. Therefore, no evidence of test substance-induced mutagenic activity in this assay system was observed. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Dreher, 2017).