Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).

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:
10 October 2018 - 31 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOX
Test concentrations with justification for top dose:
Test for Mutagenicity (Experiment 1) – Direct Plate Incorporation Method:
Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 1000, 300, 100, 30 and 10 μg/plate

Test for Mutagenicity (Experiment 2) - Pre-incubation Test:
Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 1000, 300, 100, 30 and 10 μg/plate
Vehicle / solvent:
Aboslute ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cis-Platinum (II) Diammine Dichloride (1 µg/plate E. coli - S9), 2-Nitrofluorene (2 µg/plate, TA98 - S9) and 2-Anthramine (1 or 2 µg/plate, TA strains + S9)
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM:
Strains of Salmonella typhimurium and Escherichia coli are purchased from MOLTOX

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period:
37 °C for 30 minutes (with shaking)
- Exposure duration:
Plates were incubated at 37 °C ± 3 °C for approximately 48-72 hours


NUMBER OF REPLICATIONS:
Triplicate plates per dose level.

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

Rationale for test conditions:
The highest dose tested was 1000 μg/plate due to over toxicity from 1500 µg/plate
Evaluation criteria:
The following validity criteria were checked to validate each experiment:
 the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %.
 The spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
 the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
 the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
 Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
STERILITY CONTROLS:
absence of any bacterial growth in the presence of the various concentrations of the test item.
absence of any bacterial growth in the presence of "S9-mix".

BACTERIOSTATIC ACTIVITY CONTROL:
Results showed a high toxicity from 1500 to 5000 μg/plate in TA 100. Therefore the test item was tested at these doses (1000, 300, 100, 30 and 10 μg/plate).

MUTATION ASSAY
- There was no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
- There was no evidence of any increase in the number of revertant colonies in the presence and absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and in E. coli WP2(uvrA-) (pKM101) at any dose tested with the test item up to cytotoxic concentrations.
- thinning of the bacterial lawn and a high decrease of the number of revertant colonies with metabolic activation and pre-incubation for all strains according to the toxicity measured can be observed at the highest doses tested.
- Results were confirmed in an independent experiment.
Conclusions:
There is no evidence of any increase in the number of revertant colonies in the presence and absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and in E. coli WP2(uvrA-) (pKM101) at any dose tested with the test item up to cytotoxic concentrations.
Executive summary:

The test item was tested for its capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvr A¯) (pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out.

For assay n° 1, various concentrations (from 10 to 1000 µg/plate) of test item in absolute ethanol were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2, various concentrations (from 10 to 1000 µg/plate) of test item in absolute ethanol were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There was no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory.

There was no evidence of any increase in the number of revertant colonies in the presence and absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and in E. coli WP2(uvrA-) (pKM101) at any dose tested with the test item up to cytotoxic concentrations.

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 test

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

Savineau, 2019

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100,

WP2 uvrA-

-S9

+S9

Up to cytotoxic or highest recommended concentration

-S9 : negative

+S9 : negative

 

 

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no classification is proposed.