2-tert-butyl-4-methoxyphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

In a Salmonella/microsome assay, the mutagenic activity of the test chemical was evaluated inSalmonella typhimuriumstrains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions from Aroclor-induced rats. At doses of 100 mg/plate, the phenolic antioxidant BHA exhibited toxic effects. However, a modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Therefore, exposure of the test chemical in Salmonella typhimurium, at concentrations below 500 mg/plate with or without metabolic activation by S9 liver fractions, is not regarded to be mutagenic.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was mixed with DMSO and used at dose level of 0, 0.000001, 0.00001, 0.000, or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours. The results showed evidence of cytotoxicity when treated with 2.5 or 5.0 mM for 24 and 48 hours. Independently of tested concentrations, the results showed no evidence of gene toxicity when exposed to < 2.5 mM. The number of cells and colonies at 2.5 and 5.0 mM were insufficient to say that these two concentration are non-genotoxic. Therefore, it is considered that The test chemical in the concentration of < 2.5 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

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:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Ames assay was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his+ gene

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA104

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fractions prepared from Aroclor-treated rats

Test concentrations with justification for top dose:

1, 10, 50, 100, 200, 500 or 1000 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: Without S9 mix Strain TA97: 4-nitro-o-phenylene-diamine

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Witout S9: Strain TA100: sodium azide Strain

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: TA102 tert.-butylhydroperoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: Strain TA104: methylglyoxal

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Mutagenicity test consisting of the combination of the test compound, the bacterial tester strain, and S9 mix in soft agar. Positive and negative controls are usually also included in each assay. However, negative control are not included in the current study. After incubation at 37°C for 48 h, revertant colonies are counted. A liquid preincubation procedure was also applied for some of the experiments to provide with a more sensitive assessment of mutagenic activity.

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Triplicate replications

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: N/A

OTHER EXAMINATIONS: N/A
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for no. of revertants/plate

Statistics:

No data

Species / strain:

S. typhimurium TA 97

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 102

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

Species / strain:

S. typhimurium, other: 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: Because BHA have antimicrobial properties a non-toxic dose range was established.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: No mutagenic potential

Mutagencity of the test chemical Salmonella Microsome Assay

                                            No. of his+ revertants per plate in strains

		TA97		TA100		TA102		TA104
Compound	Dose μg/plate	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
BHA	1	121 ±3	147± 9	83± 4	85± 8	243± 13	363 ±57	333 ±5	361 ±91
	10	117 2	146 6	76 12	81 14	224 24	387 2	371 21	409 50
	100	109 12	138 18	92 11	97 12	197 38	277 16	329 16	391 47
	200	136 9	174 19	102 8	76 12	171 11	381 9	32811	47254
	500	106 13	144 12	54 9	90 10	18 9	112 8	367 29	39268
	1000	0	0	0	0	15 1	182 67	0	17 10

 

Conclusions:

The test chemical dissolved in dimethylsulfoxide and given in the concentration of 1, 10, 100, 200, 500 or 1000 mg/plate, was not mutagenic in the Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions and hence it is not likely to be mutagenic as per the criteria mentioned in CLP regulation.

Executive summary:

In a Salmonella/microsome assay, the mutagenic activity of the test chemical was evaluated in Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions from Aroclor-induced rats. At doses of 100 mg/plate, the phenolic antioxidant BHA exhibited toxic effects. However, a modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Therefore, exposure of the test chemical in Salmonella typhimurium, at concentrations below 500 mg/plate with or without metabolic activation by S9 liver fractions, is not regarded to be mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosomal aberration test

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: A pseudo-diploid Chinese hamster cell line (Don)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimum essential medium supplemented with 10% fetal calf serum (pH 7.2) adjusted by HEPES6 buffer
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes, CHL cells had modal chromosome numbers of 21 and 25
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 induced with Aroclor 1254

Test concentrations with justification for top dose:

0, 0.000001, 0.00001, 0.0001 or 0.001 M

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Cells at the start of seeding: 1.0-1.2 X 106 cells per TD-40 culture bottle

DURATION
- Preincubation period: No data
- Exposure duration: 26 hrs
- Expression time (cells in growth medium): 26 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): aqueous solution of 33258 Hoechst and Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase plates for each dose

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

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations.

Rationale for test conditions:

No data

Evaluation criteria:

The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break.

Statistics:

No data

Species / strain:

mammalian cell line, other: CHL

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.00001 and 0.0001 M

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Table: Frequencies of chromosome aberrations in Don cells after treatment with BUdR and solvent

Treatment	No. of cultures	No. of cultures observed for	Breaks/cell
			Mean±SE	Range
BUdR + DMSO	6	600	0.0666±0.0010	0.02- 0.11

 

TABLE 2. Chromosome aberrations in Don cells exposed to chemical

Chemical	Dose (M)	MI	Breaks/cell
Test chemical	0.000001	-	0.02
	0.00001	-	0.09
	0.0001	+	0.03
	0.001	++	-

Conclusions:

The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was mixed with DMSO and used at dose level of 0, 0.000001, 0.00001, 0.000, or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

In vitro mammalian cell gene mutation assay was performed for the test chemical

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Metabolic activation:

with

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)

Test concentrations with justification for top dose:

0, 0.5, 1.0, 2.5 or 5.0 mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: The test chemical was easily dissolved in ethanol.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT
6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable

STAIN (for cytogenetic assays):
Crystal violet

NUMBER OF REPLICATIONS:
A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED:
5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

Rationale for test conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical < 2.5 mM.

Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours. The results showed evidence of cytotoxicity when treated with 2.5 or 5.0 mM for 24 and 48 hours. Independently of tested concentrations, the results showed no evidence of gene toxicity when exposed to < 2.5 mM. The number of cells and colonies at 2.5 and 5.0 mM were insufficient to say that these two concentration are non-genotoxic. Therefore, it is considered that The test chemical in the concentration of < 2.5 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Drosophila melanogaster was tested in a test for sex-linked recessive lethals (SLRL) to detect induced mutations. Ten day germ cell-staged adult male flies were fed with a solvent containing the test chemical to detect if it induced any mutagenic responses. No mutagenic responses were detected when a SLRL test was carried out on Drosophila melanogaster.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: gene mutation

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:

data from handbook or collection of data

Principles of method if other than guideline:

The test for sex-linked recessive lethals (SLRL) in Drosophila melanogaster is used to detect induced mutations. The advantage of the test for both screening and hazard evaluation is its objectivity in testing for transmissible mutations in various chemicals. One of the chemicals studied include the test chemical of interest.

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Strain:

other: Balancer strains: Suitably marked inverted X chromosomes Tester strains: Males from wild type strains Oregon-K, Oregon-R, Canton-S, and Berlin-K.

Sex:

male

Route of administration:

oral: feed

Vehicle:

Vehicles
- Vehicle(s)/solvent(s) used: Test chemical is dissolved in either DMSO or ethanol

- Justification for choice of solvent/vehicle: DMSO is a commonly used solvent, while ethanol has an advantage since the flies have large quantities of the alcohol dehydrogenase (ADH) enzyme and can readily metabolize ethanol.

- Concentration of test material in vehicle: No data available

- Amount of vehicle (if gavage or dermal): No data available

- Type and concentration of dispersant aid (if powder): No data available

- Lot/batch no. (if required): No data available

- Purity: No data available

OTHER INFORMATION: In a test to screen for mutagenicity of a chemical, a single concentration of the chemical may be used to test for SLRLs. The selection of the final test concentration should be preceded by one or more exposure range-finding experiments in which two points are studied:
1. Toxicity to the males during treatment and during the breeding for the mating pattern analysis; and
2. Sterilizing effect due to lethal damage to one or several germ cell stages.

Based on the results obtained in such studies, a convenient exposure may be selected, usually at the LD50 level for treated males, provided sterility does not create problems. If sterility is induced, a lower exposure may have to be used. If no adverse effects are detected, the highest technically feasible exposure at which the flies will feed should be used.

Details on exposure:

For oral route
PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available

- Mixing appropriate amounts with (Type of food): No data available

- Storage temperature of food: No data available

Duration of treatment / exposure:

No data

Frequency of treatment:

No data

Post exposure period:

No data

Remarks:

No data

No. of animals per sex per dose:

No data

Control animals:

yes, historical

Positive control(s):

Yes, nonconcurrent positive control with ethylmethanesulphonate (EMS) or diethylnitrosamine (DEN)

Justification for choice of positive control(s): Nonconcurrent positive control are satisfactory for routine recessive lethal tests

- Route of administration: No data available

- Doses / concentrations: No data available

Tissues and cell types examined:

Eye shape and eye color

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No data available

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): No data available

DETAILS OF SLIDE PREPARATION: No data available

METHOD OF ANALYSIS:
Hazard evaluation has been made using dose to the germ cells of ionizing radiation, and similar use of this test system with chemical mutagens is possible with the development of dosimetry of chemical mutagens (Comment: I don’t think this statement is correct, however, I am not sure what to state here since there is no real directions on methods of analysis in the report text.)

OTHER: No data available

Evaluation criteria:

Statistical evaluation

Statistics:

1. Comparison of control and treated frequencies
The most common problem is a comparison of the mutation frequencies obtained in a control and in a treated group. As long as the total number of mutations recovered (control and treated) is below 100, the Kastenbaum-Bowman test should be used. If more than 100 mutations are obtained, the chi square test is appropriate.

2. Analysis of the pattern of successive matings
The pooling of data from submatings is one by adding the number of chromosomes tested and the number of mutations found. In this way a weighted mean is obtained. For each of the samples, the statistical significance of a difference between the mutation frequency found in the control and in the treated groups should be checked as described above.
In a second step, the same procedure is repeated to obtain the analysis of the data pooled from the two samples.

3. Dose-response data
Dose-response data can be analyzed using standard regression techniques when germ cell dosimetry is available.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

other: Yes, solvent (If used) without the test chemical (administered in the same way as the treatment) is used for the negative control.

Positive controls validity:

other: Nonconcurrent positive control

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No data available
- Solubility: No data available
- Clinical signs of toxicity in test animals: No data available
- Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: No data available
- High dose with and without activation: No data available
- Other: No data available

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data available
- Induction of micronuclei (for Micronucleus assay): No data available
- Ratio of PCE/NCE (for Micronucleus assay): No data available
- Appropriateness of dose levels and route: No data available
- Statistical evaluation: No data available

Conclusions:

The test chemical is regarded to be negative for any mutagenic response when a SLRL test carried out on male Drosophila melanogaster.

Executive summary:

Drosophila melanogaster was tested in a test for sex-linked recessive lethals (SLRL) to detect induced mutations. Ten day germ cell-staged adult male flies were fed with a solvent containing the test chemical to detect if it induced any mutagenic responses. No mutagenic responses were detected when a SLRL test was carried out on Drosophila melanogaster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Gene mutation in vitro:

In a Salmonella/microsome assay, the mutagenic activity of the test chemical was evaluated in Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions from Aroclor-induced rats. At doses of 100 mg/plate, the phenolic antioxidant BHA exhibited toxic effects. However, a modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Therefore, exposure of the test chemical in Salmonella typhimurium, at concentrations below 500 mg/plate with or without metabolic activation by S9 liver fractions, is not regarded to be mutagenic.

This is further supported by data another publication. Bacterial reverse mutation assay was performed for the test chemical. The study was performed using E.coli WP2 (PKM101) with metabolic activation. The test chemical gives negative results for gene mutation conducted on E.coli WP2 (PKM101) with metabolic activation.

In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was mixed with DMSO and used at dose level of 0, 0.000001, 0.00001, 0.000, or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and with and without S9-induced metabolic activation for 3 hours. The results showed evidence of cytotoxicity when treated with 2.5 or 5.0 mM for 24 and 48 hours with S9 and 1.0, 2.5 or 5.0 mM for 24 and 48 hours without S9. Independently of tested concentrations, the results showed no evidence of gene toxicity when exposed to < 2.5 mM with S9 and < 1.0 mM without S9. The number of cells and colonies at 2.5 and 5.0 mM were insufficient to say that these two concentration are non-genotoxic. Therefore, it is considered that the test chemical in the concentration of < 2.5 mM with S9 and < 1.0 mM without S9 does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Sister chromatid exchange assay was also performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in HBSS and used at dose levels of 0, 0.000001, 0.00001, 0.0001 or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 10⁶ cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). Sister chromatids were differentiated by the fluorescence or Giemsa staining techniques. The acridine orange technique was used for fluorescence, and a modified FPG technique was used for Giemsa staining. Taking the existence of a dosage effect greater than two fold background as a criterion, the frequency of SCE remained at about twice the background value for all concentrations shown. Based on these considerations, the test chemical did not induce SCEs in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

In an evaluation of acute cytotoxicity of metabolic endpoints in V79 fibroblasts, the cells was exposed to the test chemical in the concentrations of 50 or 100mM. The results show that the test chemical was lethal and may also induce a 50% loss of cellular energy charge when V79 cells are exposed to the test chemical. It may also act as an inhibitor of oxygen consumption. It however did not induce DNA damage in the V79 fibroblast cell line and hence it is not likely to be mutagenic in vitro.

Gene mutation in vivo:

Drosophila melanogasterwas tested in a test for sex-linked recessive lethals (SLRL) to detect induced mutations. Ten day germ cell-staged adult male flies were fed with a solvent containing the test chemical to detect if it induced any mutagenic responses. No mutagenic responses were detected when a SLRL test was carried out onDrosophila melanogaster.

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.