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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test

Under the conditions of this study, the test material was considered to be non-mutagenic.

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:
13 April 2017 to 24 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium
TA1537 (frame shift mutations)
TA98 (frame shift mutations)
TA1535 (base-pair mutations)
TA100 (base-pair mutations)

Escherichia coli
WP2 uvrA (base-pair substitution)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary test
1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate (with and without S9-mix)

Main test (1 and 2)
313, 625, 1250, 2500, 5000 µg/plate (with and without S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection
Untreated negative controls:
yes
Remarks:
(solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
0.01 µg/plate for TA100 and WP2uvrA, 0.1 µg/plate for TA98; without S9-mix
Untreated negative controls:
yes
Remarks:
(solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
0.5 µg/plate for TA1535; without S9-mix
Untreated negative controls:
yes
Remarks:
(solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl
Remarks:
1.0 µg/plate for TA1537; without S9-mix
Untreated negative controls:
yes
Remarks:
(solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5.0 µg/plate for TA100, TA98 and TA1537; with S9-mix
Untreated negative controls:
yes
Remarks:
(solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2.0 µg/plate for TA1535 and 10.0 µg/plate for WP2uvrA; with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C
- Selection time (if incubation with a selection agent): simultaneous with exposure

SELECTION AGENT (mutation assays): trace histidine or tryptophan supplemented

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertant colonies and reduction of bacterial background lawn

The test was performed by mixing 0.1 mL of bacterial culture, 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer. Five concentrations of the test material formulation and a vehicle control (sterile distilled water) were tested together with the positive controls (0.1 mL). In addition, three minimal agar plates were used for each dose level in the main tests which were performed at the same doses. For the sterility test, 0.1 mL of the test solution of the maximum concentration and 0.5 mL of the S9-mix wre put in each tube, 2.0 mL of top agar were then added to the tube, and the contents of the tube were poured over the surface of the minimal agar plate.
The operations were conducted under lamps with UV absorbent filter.
After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn with a stereomicroscope.

Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
Growth inhibition was not observed in any strain either with or without metabolic activation.

Mutation test:
In the two main tests, neither an increase in the number of revertant colonies (more tha twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type of frame-shit type, with or without metabolic activation.
The revertant colonies of the positive controls showed increases of more than twice that of the negative controls and they were within the limit of controls (mean ± 3SD) in historical data, indicating the study was performed correctly.
In the sterility test on the test solution and the S9-mix, no growth of bacteria was observed.
Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and in accordance with the Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals, under GLP conditions.

The test material was tested on the following strains of bacteria: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A both in the presence and absence of metabolic activation in the form of S9-mix.

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type of frame-shift type, with or without metabolic activation.

The revertant colonies of the positive controls showed increases of more than twice that of the negative controls and they were within the limit of controls (mean ± 3SD) in historical data, indicating the study was performed correctly.

In the sterility test on the test solution and the S9-mix, no growth of bacteria was observed.

Under the conditions of the study, the test material was considered to be non-mutagenic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and in accordance with the Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals, under GLP conditions.

The test material was tested on the following strains of bacteria: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A both in the presence and absence of metabolic activation in the form of S9-mix.

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type of frame-shift type, with or without metabolic activation.

The revertant colonies of the positive controls showed increases of more than twice that of the negative controls and they were within the limit of controls (mean ± 3SD) in historical data, indicating the study was performed correctly.

In the sterility test on the test solution and the S9-mix, no growth of bacteria was observed.

Under the conditions of the study, the test material was considered to be non-mutagenic.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.