Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 December 2016 - 6 February 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:
(1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
Species / strain
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:
Six concentrations of the test item (15, 50, 150, 500, 1500 and 5000 µg/plate) were initially assayed in triplicate against each tester strain, however, results from the second mutation test (pre-incubation method) showed significant toxicity of the test item to several of the bacterial tester strains and consequently an insufficient number of non-toxic dose levels were initially attained for TA100, TA1535, TA98 and TA1537 dosed in absence of S9-mix. Therefore, these strains were repeated employing an amended test item dose range of:
Salmonella strains TA100, TA98 and TA1537 (without S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strain TA1535 (without 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
Controls
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.

Results and discussion

Test results
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 any plate up to and including the top dose of 5000 µg/plate

EXPERIMENTS 1 AND 2:
- There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of metabolic activation (S9-mix) in the first mutation test (plate incorporation method).
- Excessive toxicity was noted in the second mutation test, resulting in four bacterial strains (TA100, TA1535, TA98 and TA1537 in the absence of S9-mix) not achieving four non-toxic dose levels. These strains were, therefore, repeated at a later date employing amended dose ranges with the toxic limit of the test item utilised as the maximum dose. The test item induced a toxic response in the second mutation test following the utilisation of the pre-incubation modification, with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA100 and TA1535), 500 µg/plate (TA98 and TA1537) and 1500 µg/plate (WP2uvrA). In the presence of S9 mix, weakened bacterial background lawns were noted to all of the tester strains at and above 1500 µg/plate.

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. Positive control counts for Salmonella strain TA98 dosed in the presence of S9-mix in the second mutation test were slightly below the 2016 minimum level but were within the 2015 range. The response also achieved a 3.8 fold increase over the concurrent vehicle control and was, therefore, considered acceptable.

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 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 TA100 revertant colony frequency were observed in the first mutation test at 15 µg/plate in the presence of S9-mix. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 15 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay 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. 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 tothe 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.coliWP2uvrA) 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.