4-tert-butyl-3-hydroxy-2,6-xylylacetonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, restriction in design (only one positive control used to test efficacy of the S9-mix), but adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

, only one positive control used to test efficacy of the S9-mix

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

, only one positive control used to test efficacy of the S9-mix

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

, only one positive control used to test efficacy of the S9-mix

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF Aktiengesellschaft, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butyl-3-hydroxy-2,6-xylylacetonitrile

EC Number:

259-766-1

EC Name:

4-tert-butyl-3-hydroxy-2,6-xylylacetonitrile

Cas Number:

55699-10-0

Molecular formula:

C14H19NO

IUPAC Name:

2-(4-tert-butyl-3-hydroxy-2,6-dimethylphenyl)acetonitrile

Details on test material:

- Name of the test substance used in the study report: 4-tert-Butyl-3-hydroxy-2,6-xylylacetonitrile
- Physical state: solid, white to yellowish
- Storage: room temperature

Method

Target gene:

- S. typhimurium: his-locus
- E. coli: trp-locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1: 20, 100, 500, 2500, 5000 µg/plate
Experiment 2 (TA 1525 without S9 mix): 125, 250, 500, 1000, 2000 µg/plate
Experiment 2 (TA 1535 with S9 mix, all other tester strains ± S9 mix): 31.3, 62.5, 125, 250, 500 µg/plate
Experiment 3 (TA 1535, E.coli): 125, 250, 500, 1000, 2000 µg/plate
Experiment 3 (TA 100, TA 98): 31.3, 62.5, 125, 250, 500 µg/plate
Experiment 3 (TA 1537): 62.5, 125, 250, 5000, 1000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

TA: 2.5 µg/plate, E.coli: 60 µg/plate

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains tested with metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

TA 1535, TA 100, without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

TA 98, without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

100 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

TA 1537; without metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

E.coli WP2 uvrA; without metabolic activation

Details on test system and experimental conditions:

--> EXPERIMENT 1 and 2

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (reduced his- or trp- background growth), reduction in the titer


--> EXPERIMENT 3

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72h

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (reduced his- or trp- background growth), reduction in the titer

Evaluation criteria:

- The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
- A test substance is generally considered non-mutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

standard plate test and preincubation test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

standard plate test and preincubation test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- precipitation: test substance precipitation was found from about 2000 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test from about 500 μg/plate Salmonella strains) and from about 2500 μg/plate (E. coli) onward.
- In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 250 μg – 500 μg/plate onward with the Salmonella strains and from about 1000 μg/plate onward using E. coli.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, it is concluded that the test substance is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The substance was tested for its mutagenic potential in an OECD 471 guideline study (compliant with GLP) based on the ability to induce point mutation in selected loci of several bacterial strains, i.e. Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA98) and Escherichia coli (WP2 uvrA) in a reverse mutation assay. A standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix) were used. Bacteria were exposed to 20 - 5000 μg/plate (SPT; all strains), 125 - 2000 μg/plate (PIT; TA 1535, E. coli), 31.3 - 500 μg/plate (PIT; TA 100, TA 98), 62.5 - 1000 μg/plate (PIT; TA 1537). Precipitation of the test substance was found from about 2 000 μg/plate onward with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 250 - 500 μg/plate onward. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. It was therefore concluded that the test substance is not mutagenic under these experimental conditions.