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Administrative data

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 limit or 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:
18 October to 17 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline 471 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on July 18-20, 2017/ signed on November 28, 2017)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
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:
10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Test for Mutagenicity (Experiment 1) – Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Justification: The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in DMSO at 50 mg/mL in solubility checks performed in-house. DMSO was therefore selected as the vehicle.
- Preparation of test formulation: The test item was accurately weighed and, on the day of each experiment, appropriate dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer. The test item was confirmed as a UVCB product, therefore no correction was required for purity. 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:
DMSO
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:
DMSO
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.

METHOD OF APPLICATION:
Experiment 1 and 2 were performed using the preincubation method.
Negative (untreated) controls were performed on the same day as the mutation test employing the plate incorporation method.

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

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

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

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 μg/plate because of a test item film. Several manual counts were required due to revertant colonies on the very edge of plates and base agar interference, thus distorting the actual plate count.
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Up to six 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:
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. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Key result
Species / strain:
S. typhimurium, other: 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
- Water solubility: No
- Precipitation: None

HISTORICAL CONTROL DATA
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2015 and 2016 of the corresponding Testing Laboratory.
See the "Attached background material" section for Historical control data

CYTOTOXICITY AND MUTAGENICITY:
In Experiment 1, the maximum dose level of the test item was the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), although sporadic strains (particularly TA100 and TA1535) exhibited slight decreases in colony frequency at 5000 μg/plate. Additionally, there were no 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 2, the same maximum dose level was used as the maximum dose in the first mutation test (5000 μg/plate). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). As in Experiment 1, there were no 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).
A test item film (creamy in appearance) was observed at and above 1500 μg/plate in both the presence and absence of S9-mix in both experiments. This observation did not prevent the scoring of revertant colonies and, therefore, has no impact on the interpretation of results and consequently, the conclusion of the study.

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.
Remarks on result:
other: Table of results are in "Attached background documents"

Cf Tables of results in attached background material

Conclusions:
Under the test conditions, test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA strains.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2uvrA were exposed to the test substance diluted in DMSO at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

 

Test for Mutagenicity (Experiment 1) – Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

 

Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

The vehicle (dimethyl sulphoxide (DMSO)) 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 Experiment 1, the maximum dose level of the test item was the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), although sporadic strains (particularly TA100 and TA1535) exhibited slight decreases in colony frequency at 5000 μg/plate. Additionally, there were no 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 2, the same maximum dose level was used as the maximum dose in the first mutation test (5000 μg/plate). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). As in Experiment 1, there were no 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).

 

A test item film (creamy in appearance) was observed at and above 1500 μg/plate in both the presence and absence of S9-mix in both experiments. This observation did not prevent the scoring of revertant colonies and, therefore, has no impact on the interpretation of results and consequently, the conclusion of the study.

 

Under the test conditions, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coliWP2uvrA.

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

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

 Test Guideline / reliability

Focus

Strains
tested

Metabolic
activation

Test
concentration

Statement

1
Envigo, 2017

Ames Test (OECD 471)
K, rel. 1

Gene mutation

TA 1535, TA 1537, TA 98, TA 100, E. coli WP2uvrA

-S9
+S9

Up to limit concentration

-S9 : non mutagenic
+S9
 : non mutagenic

Gene mutation Assays (Tests n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (Test n°1, see Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonised System of classification and labelling of chemicals (GHS).