Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria: Key study: Read-across from experimental results on the substance 6-tert-Butyl-2,4-xylenol. Test equivalent to OECD guideline 471. GLP study.

The substance is not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation.

Chromosomal aberrations in mammalian cells: Key study: Read-across from experimental results on the substance 6-tert-Butyl-2,4-xylenol. Test equivalent to OECD guideline 473. GLP study.

The substance did not produce chromosomal aberrations.

Gene mutation in mammalian cells: Key study: Experimental results. Test according to OECD guideline 476. GLP study.

The test substance is considered to be non-mutagenic in this HPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 mix

Test concentrations with justification for top dose:

0, 6.25, 12.5, 25, 50, 100 and 200 μg/plate (-S9 mix)
0, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/plate (+S9 mix)
0, 6.25, 12.5, 25, 50, 100 and 200 μg/plate (TA1537) (+S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

TA 1535: 0.5 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: AF2 (2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

TA 100, WP2: 0.01 µg/plate and TA 98: 0.1 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

TA 1537: 80 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 100: 1 µg/plate; TA 1535: 2 µg/plate; WP 2: 10 µg/plate; TA 98: 0.5 µg/plate; TA 1537: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method
NUMBER OF REPLICATIONS: 2
OTHER:
Number of plates/test: 3

Key result

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Toxicity (inhibition growth) was observed at concentrations of 200 µg/plate (100 µg/plate for TA100 and TA1537) (-)S9 Mix and 400 µg/plate (200 µg/plate for TA100 and TA1537) (+) S9 Mix.

 

Conclusions:

Not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation.

Executive summary:

Genotoxicity of 6-tert-butyl-2,4-xylenol was studied by the reverse mutation assay in bacteria, according to the JAPAN Guidelines for Screening Mutagenicity Testing Of Chemicals (equivalent to OECD Guideline 471). 6 -tert-Butyl-2,4-xylenol was found to be not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation.

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The substance CAS No. 1879-09-0 is one of the main components of the reaction mass of 2-tertbutyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol and it is present in a concentration of more than 70%. Therefore, the results obtained with the substance CAS No. 1879-09-0 can be used for the read-across approach.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: read-across from an analogue determined to be not mutagenic in the bacteria

Conclusions:

Based on read-across approach from experimental data on analogue substance 6-tert-butyl-2,4-xylenol (CAS 1879-09-0), the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol is considered not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation.

Executive summary:

Genotoxicity of 6-tert-butyl-2,4-xylenol was studied by the reverse mutation assay in bacteria, according to the JAPAN Guidelines for Screening Mutagenicity Testing Of Chemicals (equivalent to OECD Guideline 471). 6 -tert-Butyl-2,4-xylenol was found to be not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation. Based on these results, the read-across approach was applied and the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol is considered not mutagenic  in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Metabolic activation system:

S-9: Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 (continuous treatment): 0, 0.008, 0.017, 0.033 mg/ml
-S9 (short-term treatment): 0, 0.008, 0.017, 0.033 mg/ml
+S9 (short-term treatment): 0, 0.014, 0.028, 0.056 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.00005 mg/ml, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

0.005 mg/ml, with and without metabolic activation

Details on test system and experimental conditions:

DURATION
- Exposure duration: 24 and 48 h (without S9 mix) and 6 h (with and without S9 mix)

NUMBER OF CELLS EVALUATED: 200 (24 and 48 h) and 1, 10 and 200 (6 h)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: growth inhibition

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Other: structural aberrations, nº of cells with aberrations

OTHER:
Number of plates/test : 2

Key result

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

clastogenicity and polyploidy

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytogenetic effects were not seen under the conditions of this experiment.

Conclusions:

The test item 6-tert-Butyl-2,4-xylenol did not induce chromosomal aberrations.

Executive summary:

6-tert-Butyl-2,4-xylenol was studied in a chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells, according to the JAPAN Guidelines for Screening Mutagenicity Testing Of Chemicals (equivalent to OECD Guideline 473). Neither structural chromosomal aberrations nor polyploidy were induced in CHL/IU cells up to the concentration giving 50% cell growth inhibition, in the absence or presence of an exogenous metabolic activation system.           

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The substance CAS No. 1879-09-0 is one of the main components of the reaction mass of 2-tertbutyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol and it is present in a concentration of more than 70%. Therefore, the results obtained with the substance CAS No. 1879-09-0 can be used for the read-across approach.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

clastogenicity and polyploidy

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: read-across from an analogue determined to not induce chromosomal aberrations in a chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells.

Conclusions:

Based on read-across approach from experimental data on analogue substance 6-tert-butyl-2,4-xylenol (CAS 1879-09-0), the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol is considered to not produce chromosomal aberrations.

Executive summary:

6-tert-Butyl-2,4-xylenol was studied in a chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells, according to the JAPAN Guidelines for Screening Mutagenicity Testing Of Chemicals (equivalent to OECD Guideline 473). Neither structural chromosomal aberrations nor polyploidy were induced in CHL/IU cells up to the concentration giving 50% cell growth inhibition, in the absence or presence of an exogenous metabolic activation system. Based on these results, the read-across approach was applied and the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol is considered not mutagenic in a chromosomal aberration test, with and without metabolic activation. 

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 16th to June 18th, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus in V79 cells of the Chinese hamster

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: the V79 cell line are stored in liquid nitrogen in the cell bank of Harlan CCR
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 0.6, 1.3, 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL
with S9 mix: 3.8, 7.5, 15.0, 30.0, 40.0, 50.0 and 60.0 µg/mL
Experiment II:
without S9 mix: 2.5, 5.0, 10.0, 20.0, 40.0, 50.0 and 60.0 µg/mL
with S9 mix: 2.5, 5.0, 10.0, 20.0, 40.0, 50.0 and 60.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: according to its solubility properties and its relative non-toxicity to the cells.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium MEM (minimal essential medium)

DURATION
- Preincubation period: first experiment: 2 days; second experiment: 3 days
- Exposure duration: first experiment: 4 h and second experiment: 4 h and 24 h
- Expression time (cells in growth medium): 7 days

SELECTION AGENT (mutation assays): for the selection of mutant cells the medium is supplemented with 11 µg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Approximately 1500000 (single culture) and 500 cells (in duplicate) were seeded in MEM with 10 % FBS (complete medium) for the determination of mutation rate and toxicity, respectively.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item was observed up to the maximum concentration in all experiments.

RANGE-FINDING/SCREENING STUDIES: In the range finding pre-experiment test item concentrations between 13.9 and 1783.0 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Relevant toxic effects were observed at 13.9 µg/mL and above in the absence of metabolic activation and at 55.7 µg/mL and above with metabolic activation (4 h treatment). Following continuous treatment (24 hours) strong toxic effects occurred at 55.7 µg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: the numbers of mutant colonies per 1000000 cells found in the solvent controls fall within the laboratory historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the experimental part without S9 mix the relative cloning efficiency I was approximately 90.0 % in cultures I and II (10 μg/mL). In the presence of S9 mix the relative cloning efficiency I was reduced to 58.0 % in culture I (40.0 μg/mL) and 31.0 % in culture II (50.0 μg/mL). However, due to strong test item induced cytotoxicity higher test item concentrations than the evaluated were not evaluable for mutagenicity. In the main experiment II the cytotoxicity criteria, given as relative cloning efficiency I reduced to 10 – 20 %, were fulfilled in both experimental parts. Following 24 hours treatment in the absence of S9 mix the relative cloning efficiency I was reduced to 6.6 % in culture I and to 11.6 % in culture II after treatment with a test item concentration of 40.0 μg/mL. After 4 hours treatment with 50.0 μg/mL in the presence of S9 mix the relative cloning efficiency I was reduced to 6.9 % and 12.1 % (cultures I and II).

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. Nearly all mutant frequencies remained well within the historical range of solvent controls. The two values of mutant colony numbers/10⁶cells (34.7 and 35.9) slightly exceeded the historical range of solvent controls of (3.0 – 33.2 mutant colony numbers/106 cells). This effect however was judged to be not biologically relevant for the outcome of the study. The induction factor did not exceed the threshold of three times the corresponding solvent control in any test item dose group in any culture. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

 

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. The test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration in the pre-experiment (1783.0 µg/mL) was equal to a molar concentration of about10 mM. The dose range of the main experiments was limited by toxicity of the test item.No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system. In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria: Key study: Genotoxicity of 6-tert-butyl-2,4-xylenol was studied by the reverse mutation assay in bacteria, according to the JAPAN Guidelines for Screening Mutagenicity Testing Of Chemicals (equivalent to OECD Guideline 471). 6 -tert-Butyl-2,4-xylenol was found to be not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation.Based on these results, the read-across approach was applied and the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol is considered not mutagenic  in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, with and without metabolic activation.

 

Chromosomal aberrations in mammalian cells: Key study: 6-tert-Butyl-2,4-xylenol was studied in a chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells, according to the JAPAN Guidelines for Screening Mutagenicity Testing Of Chemicals (equivalent to OECD Guideline 473). Neither structural chromosomal aberrations nor polyploidy were induced in CHL/IU cells up to the concentration giving 50% cell growth inhibition, in the absence or presence of an exogenous metabolic activation system. Based on these results, the read-across approach was applied and the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol is considered not mutagenic inachromosomal aberration test, with and without metabolic activation.

 

Gene mutation in mammalian cells: Key study: The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration in the pre-experiment (1783.0 µg/mL) was equal to a molar concentration of about10 mM. The dose range of the main experiments was limited by toxicity of the test item.No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system. In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Justification for selection of genetic toxicity endpoint

No study was selected, since the results obtained in the in vitro studies (Ames test, Chromosome aberrations test and Mammalian cell gene mutation assay) were negative.

Justification for classification or non-classification

Based on the results obtained in the in vitro genetic toxicity studies, the substance is considered as negative in all of them. Therefore, the substance is not classified.