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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative combining two separate tests

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
Species / strain / cell type:
other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat and hamster metabolic activation system.
Test concentrations with justification for top dose:
- Dose range finding test:
The test item was initially tested with strain TA100 in the presence and absence of metabolic activation over a wide dose range with an upper limit of 10 mg/plate. As a rule, at least one toxic dose was incorporated into the first mutagenicity test.

Five concentration were used in this study, ranging from 0.1 to 6.6 mg/plate.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO
Untreated negative controls:
yes
Remarks:
(untreated plates)
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
potassium chloride
Positive controls:
yes
Remarks:
2-aminoanthracene was used with all strains with metabolic activation
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Preincubation assay
Mutagenicity was tested in the preincubation assay. The mixture of S-9 mix or buffer, the overnight culture and the solvent of test chemical was allowed to incubate for 20 min at 37'C, at which time molten top agar was added, supplemented with L-histidine or D-biotin. The content of the tubes was mixed and poured onto minimal glucose bottom agar in petri dishes. When the top agar solidified, the plates were incubated at 37'C for 48 h.
Concurrent solvent and positive controls were tested with and without metabolic activation. Five dose levels of test item were tested, with three plates per dose level.

In this study E. coli was not used, neither TA102 strain. Therefore, this study is used in combination with another study in a WoA approach.
Evaluation criteria:
The criteria used for data evaluation, were as follows: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nonmutagenic response: when no increase in the number of revertants was elicited by the chemical.
Key result
Species / strain:
other: TA1535, TA1537, TA98 and TA100
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 precipitation was observed up to and including the top dose of 6.6 mg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In all strains toxicity was observed at the highest dose of 6.6 mg/plate.

5 concentrations were used for each strain, which resulted in at least 4 analyzable concentrations up to 3333 ug/plate (in some cases the highest dose of 6666 ug/plate was toxic).
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. The test was performed in a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose setting experiment. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100), neither in the absence nor in presence of S9-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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
Species / strain / cell type:
other: TA100, TA102 and TA104
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced liver S9 mix from F344 rats and B6C3F1 mice.
Test concentrations with justification for top dose:
Test item was tested at the concentration range of 33-3333 ug/plate. No number of doses applied in the test was mentioned in the publication. The highest dose was limited by toxicity, determined by thinning of the background lawn or a reduction in the number of colonies on the plates, or both.

Vehicle / solvent:
No information provided
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
mitomycin C
other: formaldehyde or crotonaldehyde for TA104; 2-amino anthracene was used for all strains with S9.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The test item was tested in a preincubation protocol. The highest dose was limited by toxicity, which was assesed based on below criteria.

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: determined by thinning of the background lawn or a reduction in the number of colonies on the plates, or both.


Numerous details of the study were not mentioned in the publication, like the number of applied doses, cytotoxicity presence and degree, solvent or negative controls. Therefore, this study is used in combination with another study in a WoE approach.
Evaluation criteria:
For the test substance to be considered mutagenic, positive responses had to be reproducible and dose-related.
Statistics:
The significance of mean revertant counts at individual dose levels was assessed using Dunnett's t-test and dose-response effects were analysed by two methods, Wahrendorf ranking and linear regression.
Key result
Species / strain:
other: TA100, TA102 and TA104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed similar to OECD 471.
Executive summary:

The mutagenic activity of the substance was evaluated in a study performed similar to OECD 471. The test was performed in a pre-incubation assay, both in the absence and presence of S9-mix. The highest dose was limited by toxicity. Adequate positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the three S. typhimurium tester strains (TA100, TA102 and TA104), neither in the absence nor in presence of S9-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.

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

Additional information

Two studies are used for this endpoint, because none of the studies covers the whole range of necessary bacterial strains. Two studies described below complement each other, as one included the following strains: TA1535, TA1537, TA98 and TA100 (not including the necessary E. coli WP2 or alternative TA102) and the other included the following ones: TA100, TA102 and TA104, therefore TA102 strain is covered.

Mortelmans:

The mutagenic activity of the substance was evaluated in accordance with OECD 471. The test was performed in a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose setting experiment. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100), neither in the absence nor in presence of S9-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.

Dillon:

The mutagenic activity of the substance was evaluated in a study performed similar to OECD 471. The test was performed in a pre-incubation assay, both in the absence and presence of S9-mix.The highest dose was limited by toxicity.Adequate positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the three S. typhimurium tester strains (TA100, TA102 and TA104), neither in the absence nor in presence of S9-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.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for mutagenicity in accordance with EU CLP and its amendments (1272/2008).