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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

not mutagenic in bacteria (Ames Test)

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:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
mutant histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of Aroclor 1254-induced male Sprague Dawley rats. The S9 mix comprised 10 % S9 fraction.
Test concentrations with justification for top dose:
plate incorporation assay:
0, 50, 158, 500, 1581, 5000 µg/plate with and without S9 mix
preincubation assay:
0, 1.6, 5, 16, 50, 158, 500, 1581 µg/tube with and without S9 mix
The test was performed up to and including the limit dose of 5000 µg/plate and tube. Due to bacteriotoxic effects this dose could not be used for assessment. Bacteriotoxic effects were observed, starting at 158 µg/tube.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item formed a clear colorless solution in DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-nitro-o-phenylenediamine for TA 1537 and TA 98; 1-Anthracene-2-amine for TA 102 and S9-mix activity in all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar for the plate incubation assay; in medium for the preincubation assay
The amount of solvent for the test substance and control is usually 0.1 mL per plate. Due to the results of a stability study the test substance was solved in 0.01 mL per plate and the lacking volume of 0.09 mL was added directly to the tubes.

DURATION
- Preincubation period: 20 min treatment time
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: all plates were prepared in triplicate

DETERMINATION of Bacteriotoxicity
- reduction of background growth
- marked and dose-dependent reduction in the mutant count per plate, compared to negative controls
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100, and TA 98 this increase should be about twice that of negative controls. For TA 1537 this increase should be at least threefold. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
no statistics perfomed; evaluation based on criteria mentioned above
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate or tube with and without S9 mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Bacteriotoxic effects were observed, starting at 158 µg/tube. The dose of 5000 µg/plate and tube was thus not assessable.
No indication of mutagenic effects could be found for the test item at doses of up to and including 1581 µg per plate in any of the Salmonella typhimurium strains used, without and with metabolic activation, under the experimental conditions applied.
Conclusions:
no mutagenic effects observed
Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate/tube were tested. Strong bacteriotoxic effects occurred at 5000 µg per plate/tube. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.

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

Additional information

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate/tube were tested. Strong bacteriotoxic effects occurred at 5000 µg per plate/tube. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Justification for classification or non-classification

Based on the available data (Ames Test in bacteria) no classification is warranted for the test item. However, the registered substance ‘Reaction mass of phenol and tetraphenylphosphonium phenolate’ consists of ≥ 20 % phenol (CAS-No. 108-95-2). According to Annex VI to Regulation (EC) 1272/2008 phenol is classified with Muta 2 (H341). A mixture shall be classified as a mutagen when it contains an ingredient that is classified with Muta 2 in a concentration of ≥ 1 %. Thus, the classification with Muta 2 is applied to the registered substance as worst-case consideration.