4-tert-butylbenzonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

During the described mutagenicity test and under the experimental conditions reported, 4-tert-Butylbenzonitrile did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, 4-tert-Butylbenzonitrile is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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:

18.06.2014 - 07.07.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Gene involved in histidine synthesis.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

n. a.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II
without S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
with S9 mix: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

The test item 4-tert-Butylbenzonitrile was dissolved in DMSO (purity > 99 %, 50 mg/mL in both experiments). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium 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

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, 4-tert-Butylbenzonitrile did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, 4-tert-Butylbenzonitrile is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of 4 -tert-Butylbenzonitrile to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation according to the OECD Guideline 471 ("Bacterial Reverse Mutation Test"). Each concentration, including the positive and negative controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II
without S9 mix:                              3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
with S9 mix:                                   1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes with S9 mix from 1000 to 5000 µg/plate in experiment I and from 333 up to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was not observed. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced back­ground growth in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with 4-tert-Butylbenzonitrile at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutage­nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, 4-tert-Butylbenzonitrile is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A study was performed to investigate the potential of 4 -tert-Butylbenzonitrile to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation according to the OECD Guideline 471 ("Bacterial Reverse Mutation Test"). Each concentration, including the positive and negative controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II
without S9 mix:                              3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
with S9 mix:                                   1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes with S9 mix from 1000 to 5000 µg/plate in experiment I and from 333 up to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was not observed. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced back­ground growth in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with 4-tert-Butylbenzonitrile at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutage­nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, 4-tert-Butylbenzonitrile is considered to benon-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Justification for classification or non-classification

4 -tert-Butylbenzonitrile was tested negative for genotoxic effects in a GLP Guideline study according to OECD 471 ("Bacterial Reverse Mutation Test"). Therefore no classification is necessary for this endpoint.