2,6-di-tert-butyl-p-cresol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Weight of evidence: In vitro studies: BHT showed no potential to cause point mutations in bacterial and mammalian test systems. Overall, the available studies demonstrate that BHT has no overt clastogenic activity in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of the mutagenicity / genotoxicity

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Key result

Species / strain:

other: TA92, TA94, TA97, TA98, TA100, TA102, TA104, TA1530, TA1535, TA1537, TA1538, G46; Escherichia coli WP2 hrc trp, Sd-4 -73

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Without exception, all the studies demonstrated the lack of mutagenic potential of BHT in these test systems.

Conclusions:

Without exception, all the studies demonstrated the lack of mutagenic potential of BHT in these test systems.

Executive summary:

A host of bacterial point mutation studies with and without metabolic activation have been reported in this review, using the following strains: Salmonella typhimurium TA92, TA94, TA97, TA98, TA100, TA102, TA104, TA1530, TA1535, TA1537, TA1538, G46; Escherichia coli WP2 hrc trp, Sd-4 -73. Most studies were performed with standard methods using liver homogenates from rats as a metabolic activation system.Without exception, all the studies demonstrated the lack of mutagenic potential of BHT in these test systems.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of the mutagenicity / genotoxicity

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Key result

Remarks on result:

other: See any other information on results incl. tables

The majority of chromosome studies on BHT in mammalian cells in vitro indicate that BHT lacks clastogenic potential. Two studies with different results are briefly discussed below.

Newell and Maxwell (1972) report a sharp increase of chromosomal aberrations in anaphases of WI-38 cells obtained from human embryonic lung cells. This result was primarily due to a large increase in the number of acentric fragments at all dose levels. However, anaphase chromosomes were examined, an uncommon practice making it difficult to assess the relevance of the results.

In a study using Chinese hamster ovary (CHO) cells treatment with 1, 5 or 10 μg BHT/ml for 48 hours increased the number of cells with chromosomal abnormalities. The number of aberrant cells was nearly identical in 1.0 and 5.0 μg BHT/ml treated cells and was considerably lower (20%) at 10.0 μg BHT/ml (Patterson et al., 1987). Sufficient information on cytotoxicity and on sample size was not included for evaluation. The solvent (ethanol) used in this study already increased the percentage of abnormal cells from 4 -6% to 8 -12%.

On the other hand, CHO cells treated with BHT in the same concentration range (1.5 -16.0 μg/ml) did not significantly increase chromosome aberrations either in the presence or in the absence of S9 mix (Galloway et al., 1987).

Treatment of Chinese hamster fibroblast cells (CHL) with BHT concentrations up to 2.8 mg/ml dissolved in ethanol did not increase the number of aberrations (Ishidate and Odashima, 1977).

Under somewhat different experimental conditions up to 0.075 mg BHT/ml dissolved in DMSO did not increase the rate of polyploid cells or the number of structural aberrations (Ishidate et al., 1980, 1984).

In cultured Chinese hamster V79 cells concentrations of 50 μM BHT did not increase the incidence of chromosomal aberrations (Ochi and Ohsawa, 1985).

Conclusions:

Taking all the existing data into account, the weight of evidence suggests that BHT did not cause chromosomal aberrations in mammalian cells in vitro.

Executive summary:

The majority of chromosome studies on BHT in mammalian cells in vitro indicate that BHT lacks clastogenic potential. Two studies with different results are discussed. In one study (Newell and Maxwell, 1972), anaphase chromosomes were examined, an uncommon practice making it difficult to assess the relevance of the results. In the second study (Patterson et al., 1987), sufficient information on cytotoxicity and on sample size was not included for evaluation. The solvent (ethanol) used in this study already increased the percentage of abnormal cells from 4 -6% to 8 -12%. Taking all the existing data into account, the weight of evidence suggests that BHT did not cause chromosomal aberrations in mammalian cells in vitro.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of the mutagenicity / genotoxicity

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

other: Sister chromatid exchange and unscheduled DNA synthesis in mammalian cells in vitro

Key result

Remarks on result:

other: See any other information on results incl. tables

BHT inhibited DNA repair synthesis after UV irradiation of human lymphocytes. Semiconservative DNA synthesis was also inhibited in non-irradiated human lymphocytes at concentrations ranging from 10 to 30 μM (Daugherty et al., 1978). Both results were reproduced under very similar experimental conditions in a study reported by Daugherty and Franks (1986).

Excision repair and post-replication repair of DNA were not influenced in an aneuploid clonal cell line (V79 -4) as determined by [3H]thymidine incorporation (Goodman et al., 1976).

The formation of sister-chromatid exchanges (SCE) may reflect DNA damage and subsequent repair processes closely associated with mutational changes. BHT has been investigated in various SCE tests. Concentrations of 0.01 -1.0 M were used by Abe and Sasaki (1977) in the Chinese hamster derived cell line DON. Formation of SCEs was investigated in an unspecified cell line and in Chinese hamster lung fibroblast cells (CHL) (Shelby and Stasiewicz, 1984; Kawachi et al., 1980). No further details are available from these studies.

Concentrations of 1.6 -16 μg/ml were used in a study with cloned Chinese hamster ovary cells (CHO-W-B1). Tests were carried out with and without an in vitro metabolic activation system, S9 mix (Galloway et al., 1987).

The induction of SCE in Chinese hamster ovary cells were also studied in Williams et al., 1990.

None of the above assays revealed an induction of SCEs by BHT.

Hepatocytes, isolated from adult male F344 rats and exposed to 2.3 x 10 -5 M BHT simultaneously with [3H]thymidine did not show DNA repair (Williams et al., 1989). Concentrations ranging from 0.01 to 10.0 μg/ml BHT did not induce DNA repair under otherwise similar test conditions (Williams et al., 1990).

Conclusions:

None of the assays revealed an induction of SCEs by BHT.

Executive summary:

BHT inhibited DNA repair synthesis after UV irradiation of human lymphocytes. Semiconservative DNA synthesis was also inhibited in non-irradiated human lymphocytes at concentrations ranging from 10 to 30 μM (Daugherty et al., 1978). Both results were reproduced under very similar experimental conditions in a study reported by Daugherty and Franks (1986).

Excision repair and post-replication repair of DNA were not influenced in an aneuploid clonal cell line (V79 -4) as determined by [3H]thymidine incorporation (Goodman et al., 1976).

The formation of sister-chromatid exchanges (SCE) may reflect DNA damage and subsequent repair processes closely associated with mutational changes. BHT has been investigated in various SCE tests. Concentrations of 0.01 -1.0 M were used by Abe and Sasaki (1977) in the Chinese hamster derived cell line DON. Formation of SCEs was investigated in an unspecified cell line and in Chinese hamster lung fibroblast cells (CHL) (Shelby and Stasiewicz, 1984; Kawachi et al., 1980). No further details are available from these studies.

Concentrations of 1.6 -16 μg/ml were used in a study with cloned Chinese hamster ovary cells (CHO-W-B1). Tests were carried out with and without an in vitro metabolic activation system, S9 mix (Galloway et al., 1987).

The induction of SCE in Chinese hamster ovary cells were also studied in Williams et al., 1990.

None of the above assays revealed an induction of SCEs by BHT.

Hepatocytes, isolated from adult male F344 rats and exposed to 2.3 x 10 -5 M BHT simultaneously with [3H]thymidine did not show DNA repair (Williams et al., 1989). Concentrations ranging from 0.01 to 10.0 μg/ml BHT did not induce DNA repair under otherwise similar test conditions (Williams et al., 1990).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of the mutagenicity / genotoxicity

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Key result

Remarks on result:

other: See executive summary

BHT did not cause point mutations at the HGPRT locus in a test ono adult rat liver epithelial cell line 18 in the concentration range of 50 -90 μg/ml used (Williams et al., 1990).

Significant increases in mutant frequencies were seen in the L5178Y TK+/- mouse lymphoma forward mutation assay, but only at cytotoxic concentrations in the presence of S9 mix (McGregor et al., 1988).

BHT concentrations below those affecting relative total growth of the mouse lymphoma cell populations did not cause an increase in mutation frequency either in the absence or in the presence of S9 mix. This test is known for its low specificity and high sensitivity to changes in the culture conditions, sometimes resulting in false positive results (Brusick, 1986).

In an experiment designed to investigate interaction with mutagenicity of benzo[a]pyrene by phenolic antioxidants in the Chinese hamster V79/HGPRT system in the presence of mouse liver S9, BHT alone showed mutagenic activity only at a level of low cell viability (Paschin and Bahitova, 1984). According to Fahrig et al (1991) evaluation of positive results is extremely difficult if they are only generated at highly toxic / cytotoxic doses / concentrations.

In a test using cultures of EUE cells, derived from human heteroploid epithelial-like cells, BHT did not cause point mutations, when using selection against diphtheria toxin as an endpoint (Ferreri et al., 1986). The concentration used was the highest that did not show cytotoxic effects.

Conclusions:

Taking all the existing data into account, the weight of evidence suggests that BHT did not cause point mutations in mammalian cells in vitro.

Executive summary:

BHT did not cause point mutations at the HGPRT locus in a test on adult rat liver epithelial cell line 18 in the concentration range of 50 -90 μg/ml used (Williams et al., 1990).

Significant increases in mutant frequencies were seen in the L5178Y TK+/- mouse lymphoma forward mutation assay, but only at cytotoxic concentrations in the presence of S9 mix (McGregor et al., 1988).

Taking all the existing data into account, the weight of evidence suggests that BHT did not cause point mutations in mammalian cells in vitro.

BHT concentrations below those affecting relative total growth of the mouse lymphoma cell populations did not cause an increase in mutation frequency either in the absence or in the presence of S9 mix. This test is known for its low specificity and high sensitivity to changes in the culture conditions, sometimes resulting in false positive results (Brusick, 1986).

In an experiment designed to investigate interaction with mutagenicity of benzo[a]pyrene by phenolic antioxidants in the Chinese hamster V79/HGPRT system in the presence of mouse liver S9, BHT alone showed mutagenic activity only at a level of low cell viability (Paschin and Bahitova, 1984). According to Fahrig et al (1991) evaluation of positive results is extremely difficult if they are only generated at highly toxic / cytotoxic doses / concentrations.

In a test using cultures of EUE cells, derived from human heteroploid epithelial-like cells, BHT did not cause point mutations, when using selection against diphtheria toxin as an endpoint (Ferreri et al., 1986). The concentration used was the highest that did not show cytotoxic effects.

Taking all the existing data into account, the weight of evidence suggests that BHT did not cause point mutations in mammalian cells in vitro.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Principles of method if other than guideline:

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in McCoy´s 5a medium with 10% fetal calf serum, L-glutamine and antibiotics. The sister chromatid exchange test was carried out with and without an in vitro metabolic activation system (S9 mix).

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

other: Chinese hamster ovary cells (CHO-W-B1)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Range: 1.6 - 16 μg/ml

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

(CHO-W-B1)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The sister chromatid exchange test with CHO cells was negative with and without metabolic activation.

Executive summary:

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in McCoy´s 5a medium with 10% fetal calf serum, L-glutamine and antibiotics. The sister chromatid exchange test was carried out with and without an in vitro metabolic activation system (S9 mix). The test with CHO cells was negative with and without metabolic activation.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

¡

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Acceptable to be used in a weight of evidence approach.

Principles of method if other than guideline:

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in McCoy´s 5a medium with 10% fetal calf serum, L-glutamine and antibiotics. The chromosome aberration test was carried out with and without an in vitro metabolic activation system (S9 mix).

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster ovary cells (CHO-W-B1)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Range: 1.6 - 16 μg/ml

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-W-B1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The chromosome aberration assay with CHO cells was negative without metabolic activation. With metabolic activation a slight increase and positive trend was observed, but the authors considered the overall result as negative.

Executive summary:

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in McCoy´s 5a medium with 10% fetal calf serum, L-glutamine and antibiotics. The chromosome aberration test was carried out with and without an in vitro metabolic activation system (S9 mix). The chromosome aberration assay with CHO cells was negative without metabolic activation. With metabolic activation a slight increase and positive trend was observed, but the authors considered the overall result as negative.

Reference 7

Endpoint:

genetic toxicity in vitro, other

Remarks:

anaphase-telophase test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Acceptable to be used in a weight of evidence approach.

Principles of method if other than guideline:

CHO cells were cultured as monolayer in cover glasses attached with a small drop of siliconised grease to the bottom of 90-mm Petri dishes. Three cover glasses were placed in each Petri dish. Each cover glass was seeded with 1.5 ml of culture medium containing about 50.000 cells. Cultures were incubated at 37 ºC and the set of cultures for each experiment was treated simultaneously for 8 hours before fixation to avoid the detachment of cells from cover slides. Each treatment was repeated 5 times. Regression analyses were performed to evaluate the mitotic index variations.

GLP compliance:

not specified

Type of assay:

other: anaphase-telophase test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Test concentrations with justification for top dose:

0.10, 0.25 and 0.5 μg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Remarks on result:

other: a significant increase of multipolar mitoses was found in anaphase-telophase cells after treatment with the highest dose

No significant increase of chromatin bridges, lagging chromosomes and lagging fragments was observed in CHO cells after treatment with BHT. Nevertheless, a significant increase of multipolar mitoses was found in anaphase-telophase cells after treatment with the highest dose. The cytotoxicity of BHT was established by scoring the mitotic index, which decreased with increasing doses of the chemical.

Conclusions:

No significant increase of chromatin bridges, lagging chromosomes and lagging fragments was observed in CHO cells after treatment with BHT. Nevertheless, a significant increase of multipolar mitoses was found in anaphase-telophase cells after treatment with the highest dose. The cytotoxicity of BHT was established by scoring the mitotic index, which decreased with increasing doses of the chemical.

Executive summary:

CHO cells were cultured as monolayer in cover glasses attached with a small drop of siliconised grease to the bottom of 90-mm Petri dishes. Three cover glasses were placed in each Petri dish. Each cover glass was seeded with 1.5 ml of culture medium containing about 50.000 cells. Cultures were incubated at 37 ºC and the set of cultures for each experiment was treated simultaneously for 8 hours before fixation to avoid the detachment of cells from cover slides. Each treatment was repeated 5 times. Regression analyses were performed to evaluate the mitotic index variations. No significant increase of chromatin bridges, lagging chromosomes and lagging fragments was observed in CHO cells after treatment with BHT. Nevertheless, a significant increase of multipolar mitoses was found in anaphase-telophase cells after treatment with the highest dose. The cytotoxicity of BHT was established by scoring the mitotic index, which decreased with increasing doses of the chemical.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to the recommended guidelines. Furthermore, a positive response is observed at cytotoxic doses.

Principles of method if other than guideline:

CHO cells were cultured for 15-16 hours in the presence of the different doses of BHT. Two hours before cell harvesting, cultures were added with colchicine. Each treatment was repeated 5 times and a total of 500 metaphases per treatment (100 per repetition) was scored in coded slides. Statistical analysis was performed using Chi-square test.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Test concentrations with justification for top dose:

0.10, 0.25 and 0.5 μg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

(genotoxic at cytotoxic doses)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(reduction in mitotic index)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Treatment with the three doses of BHT induced a significant increase of chromatid and isochromatid breaks with corresponding increase of abnormal metaphases. Treatment with 0.25 and 0.5 μg/ml of BHT induced a slight, not significant increase of chromatid exchange frequencies. The positive response is observed at cytotoxic doses.

Conclusions:

Treatment with the three doses of BHT induced a significant increase of chromatid and isochromatid breaks with corresponding increase of abnormal metaphases. Treatment with 0.25 and 0.5 μg/ml of BHT induced a slight, not significant increase of chromatid exchange frequencies. The positive response is observed at cytotoxic doses.

Executive summary:

CHO cells were cultured for 15-16 hours in the presence of the different doses of BHT. Two hours before cell harvesting, cultures were added with colchicine. Each treatment was repeated 5 times and a total of 500 metaphases per treatment (100 per repetition) was scored in coded slides. Statistical analysis was performed using Chi-square test. Treatment with the three doses of BHT induced a significant increase of chromatid and isochromatid breaks with corresponding increase of abnormal metaphases. Treatment with 0.25 and 0.5 μg/ml of BHT induced a slight, not significant increase of chromatid exchange frequencies. The positive response is observed at cytotoxic doses.

Reference 9

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Culture medium was added with 10 μg/ml of 5´-bromo-2´-deoxyuridine (BrdU) and the cells were incubated in complete darkness. Human lymphocytes were incubated for 72 hours and CHO cells for 30 hours. Two hours before fixation, cells were treated with colchicine. For each treatment 5 repetitions were made. Cytogenetic analysis was performed on coded slides. Statistical analysis was performed using multifactorial ANOVA.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Species / strain / cell type:

lymphocytes: human blood samples were obtained from newborn umbilical cord (two males and two females)

Metabolic activation:

without

Test concentrations with justification for top dose:

0.10, 0.25 and 0.5 μg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Key result

Species / strain:

primary culture, other: Lymphocytes of human umbilical cord

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

CHO cells: no differences were found between treatments with BHT and controls. However, BHT showed cytotoxic effects since only a few metaphases could be analysed in cells treated with 0.25 μg/ml and no cells at the second mitosis were found after treatment with 0.5 μg/ml of BHT. A significant decrease of cells in second division was observed in cultures treated with the 0.25 μg/ml dose of BHT.

Lymphocytes of human umbilical cord: treatment with BHT did not increase the SCE frequencies with respect to untreated and DMSO-treated controls. A cytotoxic effect similar to that observed in CHO cells was seen in human lymphocytes. A decrease of cells in second division with the three doses of BHT after continuous treatment for 72 hours and with the highest dose in treatments performed during the last 24 hours of incubation.

Conclusions:

CHO cells: no differences were found between treatments with BHT and controls. However, BHT showed cytotoxic effects since only a few metaphases could be analysed in cells treated with 0.25 μg/ml and no cells at the second mitosis were found after treatment with 0.5 μg/ml of BHT. A significant decrease of cells in second division was observed in cultures treated with the 0.25 μg/ml dose of BHT.

Lymphocytes of human umbilical cord: treatment with BHT did not increase the SCE frequencies with respect to untreated and DMSO-treated controls. A cytotoxic effect similar to that observed in CHO cells was seen in human lymphocytes. A decrease of cells in second division with the three doses of BHT after continuous treatment for 72 hours and with the highest dose in treatments performed during the last 24 hours of incubation.

Executive summary:

Culture medium was added with 10 μg/ml of 5´-bromo-2´-deoxyuridine (BrdU) and the cells were incubated in complete darkness. Human lymphocytes were incubated for 72 hours and CHO cells for 30 hours. Two hours before fixation, cells were treated with colchicine. For each treatment 5 repetitions were made. Cytogenetic analysis was performed on coded slides. Statistical analysis was performed using multifactorial ANOVA. In CHO cells no differences were found between treatments with BHT and controls. However, BHT showed cytotoxic effects since only a few metaphases could be analysed in cells treated with 0.25 μg/ml and no cells at the second mitosis were found after treatment with 0.5 μg/ml of BHT. A significant decrease of cells in second division was observed in cultures treated with the 0.25 μg/ml dose of BHT. In lymphocytes of human umbilical cord, treatment with BHT did not increase the SCE frequencies with respect to untreated and DMSO-treated controls. A cytotoxic effect similar to that observed in CHO cells was seen in human lymphocytes. A decrease of cells in second division with the three doses of BHT after continuous treatment for 72 hours and with the highest dose in treatments performed during the last 24 hours of incubation.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Only one concentration of BHT was tested and only 3 strains of Salmonella typhimurium were used. No data on replicates was provided. However, it provides scientific information to be taken into account for the weight of evidence analysis.

Principles of method if other than guideline:

The assay was carried out according to Ames et al. (1975) using Salmonella typhimurium TA98, TA100 and TA1537 with and without metabolic activation S9-mix. The test samples were dissolved in DMSO.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA98, TA100 and TA1537

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

10 μg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-quinoline-1-oxide, benzo[a]pyrene and 9-aminoacridine

Key result

Species / strain:

other: S. typhimurium TA98, TA100 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

BHT was negative in the Ames test when tested at a concentration of 10 μg/plate using Salmonella typhimurium TA98, TA100 and TA1537 with and without metabolic activation S9-mix.

Executive summary:

BHT was negative in the Ames test when tested at a concentration of 10 μg/plate using Salmonella typhimurium TA98, TA100 and TA1537 with and without metabolic activation S9-mix.

Reference 11

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Anaphase chromosomes were examined, an uncommon practice making it difficult to assess the relevance of the results.

Principles of method if other than guideline:

In vitro cytogenetic studies were performed by anaphase analysis of diploid human embryonic lung cells (WI-38). Dose levels used were the highest level that did not show cytotoxicity and two lower doses (10 and 100 times less). Duplicate slides were prepared for each treatment.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human embryonic lung cells (WI-38)

Key result

Species / strain:

other: Diploid human embryonic lung cells

Remarks:

WI-38

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

other:

Positive controls validity:

not specified

Newell and Maxwell (1972) report a sharp increase of chromosomal aberrations in anaphases of WI-38 cells obtained from human embryonic lung cells. This result was primarily due to a large increase in the number of acentric fragments at all dose levels. However, anaphase chromosomes were examined, an uncommon practice making it difficult to assess the relevance of the results.

Conclusions:

The authors considered the results observed as positive. Newell and Maxwell (1972) report a sharp increase of chromosomal aberrations in anaphases of WI-38 cells obtained from human embryonic lung cells. This result was primarily due to a large increase in the number of acentric fragments at all dose levels. However, anaphase chromosomes were examined, an uncommon practice making it difficult to assess the relevance of the results.

Executive summary:

In vitro cytogenetic studies were performed by anaphase analysis of diploid human embryonic lung cells (WI-38). Dose levels used were the highest level that did not show cytotoxicity and two lower doses (10 and 100 times less). Duplicate slides were prepared for each treatment. The authors considered the results observed as positive. Newell and Maxwell (1972) report a sharp increase of chromosomal aberrations in anaphases of WI-38 cells obtained from human embryonic lung cells. This result was primarily due to a large increase in the number of acentric fragments at all dose levels. However, anaphase chromosomes were examined, an uncommon practice making it difficult to assess the relevance of the results.

Reference 12

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This mouse lymphoma assay was reported as positive with metabolic activation and inconclusive without metabolic activation. However, small and large colonies were not distinguished in this test and the results of three independent trials are contradictory concerning effects; non-cytotoxic concentrations did not induce an increase in mutation frequency (with and without S9-mix).

Principles of method if other than guideline:

The objective of the study was to evaluate the ability of BHT to induce forward mutations at the thymidine kinase locus (tk) in a mammalian cell as assayed by colony growth of L5178Y clone 3.7.2C mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT).

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus (tk)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without metabolic activation: 1.25, 2.5, 5.0, 6.0, 10.0, 12.0, 15.0, 18.0, 20.0, 24.0, 25.0 and 40.0 μg/ml

With metabolic activation: 2.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 20.0, 24.0 and 28.0 μg/ml

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: with metabolic activation

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was a sharp decrease in the survival of cultures treated with more than about 20 μg BHT/ml in the absence of S9 mix. This effect made the choice of test concentrations difficult and was largely responsible for the overall inconclusive results from three trials in this activation condition. Although there was similar, or slightly greater, toxicity in the presence of S9 mix, there was greater success in demonstrating induced increases in the mutant fraction in three trials. It was concluded that butylated hydroxytoluene is mutagenic in this assay. However, small and large colonies were not distinguished in this test and the results of three independent trials are contradictory concerning effects; non-cytotoxic concentrations did not induce an increase in mutation frequency (with and without S9-mix).

Conclusions:

This mouse lymphoma assay was reported as positive with metabolic activation and inconclusive without metabolic activation. However, small and large colonies were not distinguished in this test and the results of three independent trials are contradictory concerning effects; non-cytotoxic concentrations did not induce an increase in mutation frequency (with and without S9-mix).

Executive summary:

The objective of the study was to evaluate the ability of BHT to induce forward mutations at the thymidine kinase locus (tk) in a mammalian cell as assayed by colony growth of L5178Y clone 3.7.2C mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT). This mouse lymphoma assay was reported as positive with metabolic activation and inconclusive without metabolic activation. However, small and large colonies were not distinguished in this test and the results of three independent trials are contradictory concerning effects; non-cytotoxic concentrations did not induce an increase in mutation frequency (with and without S9-mix).

Reference 13

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Mouse lymphoma L5178Y TK+/- cells were maintained at 37° C as suspension cultures in Fischer's medium. Treated cultures contained 6 x 10exp6 cells in 10 mL of medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with the test chemical continued for 4 hours. Cells were resuspended in 20 mL of fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 10exp6 cells were plated in medium and soft agar supplemented with TFT for selection of TFT resistant cells (TK-/-) and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 ºC in 5% CO2 for 10 to 12 days.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

L5178Y/tk+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 from the livers of either Aroclor 1254-induced or noninduced male Fischer 344 rats.

Test concentrations with justification for top dose:

Non-activation trial 1: 1.25, 2.5, 5, 10, 20 and 40 µg/ml
Non-activation trial 2: 6, 12, 18 and 24 µg/ml
Non-activation trial 3: 14, 18, 22, 26 and 30 µg/ml
Non-activation trial 4: 5, 10, 15, 20 and 25 µg/ml
Induced S9 trial 1: 8, 12, 16, 20, 24 and 28 µg/ml
Induced S9 trial 2: 2, 8, 14 and 20 µg/ml
Induced S9 trial 3: 2, 6, 10 and 14 µg/ml

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation Migrated to IUCLID6: (MCA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: The highest dose of test chemical was determined by solubility or toxicity, and did not exceed 5 mg/kg in the absence of dose-limiting toxicity. Mouse lymphoma L5178Y TK+/- cells were maintained at 37° C as suspension cultures in Fischer's medium supplemented with 2 mM l-glutamine, 110 µg/mL sodium pyruvate, 0.05% luronic F68, antibiotics, and heat-inactivated horse serum. Normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine (TFT) resistant cells, subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for one day and to normal medium for 3 to 5 days. For cloning, horse serum content was increased and Noble agar was added.

DURATION
- Exposure duration: Treated cultures contained 6 x 10exp6 cells in 10 mL of medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with the test chemical continued for 4 hours.
- Expression time (cells in growth medium): Cells were resuspended in 20 mL of fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained.
- Selection time (if incubation with a selection agent): After the 48-hour expression period, 3 x 10exp6 cells were plated in medium and soft agar supplemented with TFT for selection of TFTresistant cells (TK-/-) and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 ºC. in 5% CO2 for 10 to 12 days.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Quality control factors (minimum criteria for accepting an experiment as valid) included such factors as cloning efficiencies within acceptable parameters for control and treated cultures, relative total growth of treated cultures above 1%, absence of test chemical precipitate, and two or more acceptable cultures per dose set.

Statistics:

All data were evaluated statistically for trend and peak responses. Both responses had to be significant (P < 0.05) for a chemical to be considered capable of inducing TFT resistance; a single significant response led to a "questionable" conclusion, and the absence of both a trend and a peak response resulted in a "negative" call.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The mouse lymphoma assay was reported as positive with metabolic activation and inconclusive without metabolic activation.

Executive summary:

Mouse lymphoma L5178Y TK+/- cells were maintained at 37° C as suspension cultures in Fischer's medium. Treated cultures contained 6 x 10exp6 cells in 10 mL of medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with the test chemical continued for 4 hours. Cells were resuspended in 20 mL of fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 10exp6 cells were plated in medium and soft agar supplemented with TFT for selection of TFTresistant cells (TK-/-) and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 ºC in 5% CO2 for 10 to 12 days. The mouse lymphoma assay was reported as positive with metabolic activation and inconclusive without metabolic activation.

 

Reference 14

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Test tubes containing a suspension of one strain of Salmonella typhimurium plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of petri dishes containing standard bacterial culture medium. The number of colonies is usually counted after 2 days.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

RLI = induced male Sprague Dawley rat liver S9 and HLI = induced male Syrian hamster liver S9

Test concentrations with justification for top dose:

TA1535 and TA100: 0.1, 0.3, 1, 3.3, 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate
TA98 and TA1537: 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation for TA100 and TA1535

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation for TA98

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation for TA1537

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation. In the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of petri dishes containing standard bacterial culture medium. The number of colonies is usually counted after 2 days.

DURATION
- Preincubation period: 20 minutes at 37º C

NUMBER OF REPLICATIONS: Duplicates and triplicates

Evaluation criteria:

If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be
significantly greater than the corresponding control plates for that strain of bacteria.

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

Remarks:

(slight toxicity)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed in some cases (showed in the tables as "p")

Conclusions:

The test substance showed no potential to cause point mutations in the bacterial test systems used in this assay with or without metabolic activation.

Executive summary:

In a bacterial reverse mutation test, tubes containing a suspension of one strain of Salmonella typhimurium plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical at concentrations in the range of 0.1 to 10000 µg/plate. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of petri dishes containing standard bacterial culture medium. The number of colonies is usually counted after 2 days. The test substance showed no potential to cause point mutations in the bacterial test systems used in this assay with or without metabolic activation.

Reference 15

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

A chromosome aberration assay with CHO cells (concentrations tested: 1.6 - 16 μg/ml) was conducted with and without metabolic activation (S9). In the test without S9, cells were incubated for 8- 12 hours with the test chemical. Colcemid was added and incubation continued for 2 hours. The cells were harvested by mitotic shake-off, fixed, and stained with Giemsa. For the test with S9, cells were treated with the test chemical and S9 for 2 h, after which the treatment medium was removed and the cells incubated for 10 hours in fresh medium, with Colcemid present for the final 2 h. Cells were harvested in the same manner as for the treatment without S9.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Induced Rat liver S9

Test concentrations with justification for top dose:

1.6, 5 and 16 µg/ml

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the test without S9, cells were incubated for 8- 12 hours with the test chemical in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics; Colcemid was added and incubation continued for 2 hours. The incubation time and the dose levels selected were determined from the information on cell cycling and toxicity obtained from the Sister Chromatid Exchanges test; if cell cycle delay was anticipated in the Chromosomic aberrations test, the incubation period was extended to permit accumulation of sufficient cells in the first metaphase for analysis. The cells were harvested by mitotic shake-off, fixed, and stained with Giemsa.

For the test with S9, cells were treated with the test chemical and S9 for 2 h, after which the treatment medium was removed and the cells incubated for 10 hours in fresh medium, with Colcemid present for the final 2 h. Cells were harvested in the same manner as for the treatment without S9.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). One hundred or two hundred first-division metaphase cells were scored at each dose level. The classes of aberrations that were recorded included "simple" (breaks and
terminal deletions), "complex" (rearrangements and translocations), and "other" (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Evaluation criteria:

For a single trial, a statistically significant (P<0.05) difference for one dose point and a significant trend (P<0.015) was considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A strong trend (P < 0.003) with a single significant dose level was designated weak positive, to indicate a high level of induced aberrations. A strongly positive trend (P < 0.003), in the absence of a statistically-significant increase at any one dose point, led to an equivocal call. Ultimately, the trial calls were based on a consideration of the statistical
analyses as well as the biological information. Trials that gave a weak positive or positive result were repeated.

Statistics:

To arrive at a statistical call for a trial, analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control (Galloway et al., 1987).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

A chromosome aberration assay with CHO cells (concentrations tested: 1.6 - 16 μg/ml) was negative without metabolic activation. With metabolic activation a slight increase and positive trend was observed, but the authors considered the overall result as negative.

Executive summary:

A chromosome aberration assay with CHO cells (concentrations tested: 1.6 - 16μg/ml) was conducted with and without metabolic activation (S9). In the test without S9, cells were incubated for 8- 12 hours with the test chemical. Colcemid was added and incubation continued for 2 hours. The cells were harvested by mitotic shake-off, fixed, and stained with Giemsa. For the test with S9, cells were treated with the test chemical and S9 for 2 h, after which the treatment medium was removed and the cells incubated for 10 hours in fresh medium, with Colcemid present for the final 2 h. Cells were harvested in the same manner as for the treatment without S9. The assay was negative without metabolic activation. With metabolic activation a slight increase and positive trend was observed, but the authors considered the overall result as negative.

Reference 16

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

In the SCE test without S9, CHO cells were incubated with the test chemical for 26 hours in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics. 5-Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium with test chemical was removed and replaced with fresh medium plus BrdU and Colcemid, without test chemical. Incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no test chemical. Incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. Fifty second-division metaphase cells were scored to determine the frequency of SCE/cell for each dose level.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix.

Test concentrations with justification for top dose:

1.6, 5 and 16 µg/ml

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the SCE test without S9, CHO cells were incubated with the test chemical for 26 hours in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics. 5-Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium with test chemical was removed and replaced with fresh medium plus BrdU and Colcemid, without test chemical. Incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no test chemical. Incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Hoechst 33258 and Giemsa

NUMBER OF CELLS EVALUATED: Fifty second-division metaphase cells were scored to determine the frequency of SCE/cell for each dose level. If significant chemical-induced cell cycle delay was seen in treated cultures, the incubation time was lengthened to ensure the accumulation of a sufficient number of scorable (second-division metaphase) cells.

Evaluation criteria:

An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response (Galloway et al., 1985). The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. An increase of 20%, or greater, at any single dose, was considered weak evidence of activity (weak positive); increases at two or more doses resulted in a determination that the trial was positive. A trend P-value of less than 0.005, in the absence of any responses reaching 20% above background, led to a call of equivocal for the trial.

Statistics:

Statistical analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control (Galloway et al., 1987).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Conclusions:

Under the conditions of this sister chromatid exchange study, the test substance was negative.

Executive summary:

In the SCE test without S9, CHO cells were incubated with the test chemical for 26 hours in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics. 5-Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium with test chemical was removed and replaced with fresh medium plus BrdU and Colcemid, without test chemical. Incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no test chemical. Incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. Fifty second-division metaphase cells were scored to determine the frequency of SCE/cell for each dose level. Under the conditions of this sister chromatid exchange study, the test substance was negative.

 

Reference 17

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although the study was not conducted according to the recommended guidelines (no data on positive controls, number of replicates, etc.) it provides some scientific valid information to assess the in vitro mutagenicity of the substance (weight of evidence).

Principles of method if other than guideline:

In the presence of metabolic activation the mutagenicity of BHT in Chinese hamster V79 cells was studied.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Metabolic activation system:

Mixture prepared from mouse liver

Test concentrations with justification for top dose:

0-10 μg/ml

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive control substance:

not specified

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only at cytotoxic concentrations)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

not specified

Conclusions:

The HGPRT assay with Chinese hamster V79 cells and metabolic activation was positive only at cytotoxic concentrations.

Executive summary:

In the presence of metabolic activation the mutagenicity of BHT in Chinese hamster V79 cells was studied at concentrations between 0 and 10 μg/ml. Under the conditions of this study, positive results were only observed at cytotoxic concentrations.

Reference 18

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the in vitro mutagenicity of the substance.

Principles of method if other than guideline:

BHT was tested using the plate incorporation method with metabolic activation using four bacterial strains: S. typhimurium TA102 and TA2638 and E.coli WP2/pkM101 and WP2 uvrA/pKM101

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA102 and TA2638

Species / strain / cell type:

E. coli, other: WP2/pKM101 and WP2 uvrA/pKM101

Metabolic activation:

with

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 313, 625, 1250, 2500 and 5000 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


NUMBER OF REPLICATIONS: BHT was tested in at least two replicate experiments using three plates per dose. The chemical was tested in two laboratories to assess reproducibility.

Statistics:

Linear regression test

Key result

Species / strain:

other: S. typhimurium TA102 and TA2638 and E. coli WP2/pKM101 and WP2 uvrA/pKM101

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In the bacterial gene mutation assay with S. typhimurium strains TA102 and TA2638, E. coli WP2/pKM101 and WP2 uvrA/pKM101 with metabolic activation BHT was negative

Executive summary:

BHT was tested using the plate incorporation method with metabolic activation using four bacterial strains: S. typhimurium TA102 and TA2638 and E.coli WP2/pkM101 and WP2 uvrA/pKM101. Under the conditions of this study, negative results were observed.

Reference 19

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the in vitro mutagenicity of the substance. No data were given on cytotoxicity, but the highest concentration tested was 10000 µg/plate.

Principles of method if other than guideline:

Salmonella typhimurium mutagenesis assay. The Salmonella/microsome assay (Ames et al., 1975) was conducted using the pre-incubation method described by Sugimura and Nagao (1980). Strains TA98, TA100, TA1535, TA1537 and TA1538 were used. S-9 was prepared from Aroclor-induced Sprague-Dawley rats. Revertant colonies were counted after 72 hr of exposure, and the mean of triplicate plates was calculated. A three-fold increase in revertants was accepted as a positive result.

GLP compliance:

not specified

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:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 was prepared from Aroclor-induced Sprague-Dawley rats.

Test concentrations with justification for top dose:

100, 1000 and 10000 µg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 and TA1538 Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-anthramine

Remarks:

TA98, TA100, TA1535, TA1537 and TA1538

Details on test system and experimental conditions:

METHOD OF APPLICATION: The pre-incubation method described by Sugimura and Nagao (1980).

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: Triplicate plates

Evaluation criteria:

A three-fold increase in revertants was accepted as a positive result.

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:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 7.6.1 -1: Results of Salmonella/microsome mutagenicity test for BHT

Compound	Concn (µg/plate)	No. of revertants/plate
		S-9	TA98	TAl00	TA1535	TA1537	TA1538
BHT	10,000	-	38	110	26	5	33
	1000	-	34	152	20	7	29
	100	-	47	130	26	7	23
	10,000	+	59	174	28	10	26
	1000	+	66	176	23	6	24
	100	+	48	130	26	6	30
DMSO		- +	45 45	114 133	27 21	5 4	25 32
Sodium azide	5	-		1401	1087
9-Aminoacridine	100	-				375
2-Nitrofluorene	5	-	1257				1145
2-Anthramine	5	+	1252	1218	1213	741	1440

Conclusions:

No increase in revertants was observed in bacteria exposed to BHT. All positive control compounds elicited the expected responses. The test substance showed no potential to cause point mutations in bacterial test systems.

Executive summary:

The Salmonella/microsome assay was conducted using the pre-incubation method described by Sugimura and Nagao (1980). Strains TA98, TA100, TA1535, TA1537 and TA1538 were used. S-9 was prepared from Aroclor-induced Sprague-Dawley rats. Revertant colonies were counted after 72 hr of exposure, and the mean of triplicate plates was calculated. A three-fold increase in revertants was accepted as a positive result. No increase in revertants was observed in bacteria exposed to BHT. All positive control compounds elicited the expected responses. The test substance showed no potential to cause point mutations in bacterial test systems.

Reference 20

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Adult rat liver epithelial cell HGPRT mutagenesis assay. Mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was
assayed according to previously described procedures (Tong et al., 1984; Tong and Williams, 1980). Cells from adult rat liver (ARL) line 18, at passage 25, were used. An aliquot of cryopreserved cells was cultured in WME containing 10% calf serum (WMES). Log phase cultures containing 0.25-0.5 x 10exp5 cells/cm2 were exposed to the test compound for 3 days, then washed. The cultures were then maintained with intermittent subculturing for a minimum of 21-23 days for mutant expression, before they were seeded for selection for HGPRT-deficient mutants. For mutant selection, 10exp4 cells/cm2 were seeded in WMES in 25-cm2 flasks. Twenty-four h later, WMES containing 20 µg/ml 6-thioguanine (TG) was added. The cultures were then re-fed every 4 days. After 14 days, TG' colonies were fixed with formalin and stained with Giesma for counting. The colony-forming efficiency
in non-selective media was determined by seeding 20 cells/cm2 in WMES in 25-cm2 culture flasks. At the end of 7-9 days, colonies were fixed and stained as above. The data were analysed using Student's t-test.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

other: Cells from adult rat liver (ARL) line 18, at passage 25

Details on mammalian cell type (if applicable):

An aliquot of cryopreserved cells was cultured in WME containing 10% calf serum (WMES)

Metabolic activation:

with

Metabolic activation system:

Cells from adult rat liver

Test concentrations with justification for top dose:

50, 60, 70, 80 and 90 µg/ml

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

DURATION
- Exposure duration: Log phase cultures containing 0.25-0.5 x 10exp5 cells/cm2 were exposed to the test compound for 3 days, then washed.
- Expression time (cells in growth medium): The cultures were then maintained with intermittent subculturing for a minimum of 21-23 days for mutant expression, before they were seeded for selection for HGPRT-deficient mutants.
- Selection time (if incubation with a selection agent): For mutant selection, 10exp4 cells/cm2 were seeded in WMES in 25-cm2 flasks. Twenty-four h later, WMES containing 20 µg/ml 6-thioguanine (TG) was added. The cultures were then re-fed every 4 days.
- Fixation time (start of exposure up to fixation or harvest of cells): After 14 days, TG' colonies were fixed with formalin and stained with Giesma for counting.

The colony-forming efficiency in non-selective media was determined by seeding 20 cells/cm2 in WMES in 25-cm2 culture flasks. At the end of 7-9 days, colonies were fixed and stained as above.

Statistics:

The data were analysed using Student's t-test.

Key result

Species / strain:

other: Cells from adult rat liver (ARL) line 18, at passage 25

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 7.6.1 -2: Results of ARL cell/HGPRT mutagenicity test for BHT

Compound	Concn (µg/ml)	TG^rmutants per l0^6CFC	Evaluation
BHT	90 80 70 60 50	67 ± 20 51 ± 31 77 ± 23 66 ± 19 45 ± 20	Negative Negative Negative Negative Negative
Benzo[a]pyrene	1 x 10^-5M	334 ± 69	Positive
DMSO	1 %	67 ± 25	Negative
None		53 ± 26	Negative
CFC =colony-forming cells

Conclusions:

In ARL cells exposed to BHT, no statistically significant increase in TGr mutants was observed. The test substance showed no potential to cause point mutations in the selected mammalian test system.

Executive summary:

Mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was assayed. Cells from adult rat liver (ARL) line 18, at passage 25, were used. An aliquot of cryopreserved cells was cultured in WME containing 10% calf serum (WMES). Log phase cultures containing 0.25-0.5 x 10exp5 cells/cm2 were exposed to the test compound for 3 days, then washed. The cultures were then maintained with intermittent subculturing for a minimum of 21-23 days for mutant expression, before they were seeded for selection for HGPRT-deficient mutants. For mutant selection, 10exp4 cells/cm2 were seeded in WMES in 25-cm2 flasks. Twenty-four h later, WMES containing 20 µg/ml 6-thioguanine (TG) was added. The cultures were then re-fed every 4 days. After 14 days, TG' colonies were fixed with formalin and stained with Giesma for counting. The colony-forming efficiency in non-selective media was determined by seeding 20 cells/cm2 in WMES in 25-cm2 culture flasks. At the end of 7-9 days, colonies were fixed and stained as above. The data were analysed using Student's t-test. In ARL cells exposed to BHT, no statistically significant increase in TGr mutants was observed. The test substance showed no potential to cause point mutations in the selected mammalian test system.

Reference 21

Endpoint:

genetic toxicity in vitro, other

Remarks:

Type of genotoxicity: other: reverse mutation, gene mutation, chromosomal aberrations, DNA repair and DNA damage

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of genotoxicity studies

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

other: review of different studies

Key result

Genotoxicity:

negative

Remarks on result:

other: See any other information on results incl. tables

BHT did not cause DNA damage in Bacillus subtilis (Kinae et al., 1981) or mutation in Salmonella typhimurium (Ben-Hur et al., 1981; Brusick, 1993; McKee and Tometsko, 1979; Shelef and Chin, 1980; Williams et al., 1990). In one study, it was reported to be mutagenic to culture Chinese hamster V79 cells in the presence of an exogenous metabolic system (Paschin and Bahitova, 1984). BHT was negative for DNA repair in isolated hepatocytes (Williams et al., 1990). BHT did not induce micronuclei in bone marrow or dominant lethal mutations in mice (Bruce and Heddle, 1979; Epstein et al., 1977). The weight of evidence, therefore, supports the conclusion that BHT is not genotoxic.

Conclusions:

The weight of evidence, therefore, supports the conclusion that BHT is not genotoxic.

Executive summary:

BHT did not cause DNA damage in Bacillus subtilis (Kinae et al., 1981) or mutation in Salmonella typhimurium (Ben-Hur et al., 1981; Brusick, 1993; McKee and Tometsko, 1979; Shelef and Chin, 1980; Williams et al., 1990). In one study, it was reported to be mutagenic to culture Chinese hamster V79 cells in the presence of an exogenous metabolic system (Paschin and Bahitova, 1984). BHT was negative for DNA repair in isolated hepatocytes (Williams et al., 1990). BHT did not induce micronuclei in bone marrow or dominant lethal mutations in mice (Bruce and Heddle, 1979; Epstein et al., 1977). The weight of evidence, therefore, supports the conclusion that BHT is not genotoxic.

Reference 22

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

The mutagenicity in strains TA98 and TA100 of Salmonella typhimurium was investigated in the preincubation plate incorporation method. The S9 mix contained 10% rat liver S9.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

100, 330 and 1000 μg/plate

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

BHT did not show mutagenic activity on the preincubation modified Ames assay using the strains TA98 and TA100 of S. typhimurium under the conditions of this study.

Conclusions:

BHT did not show mutagenic activity on the preincubation modified Ames assay using the strains TA98 and TA100 of S. typhimurium under the conditions of this study.

Executive summary:

The mutagenicity of BHT was investigated in strains TA98 and TA100 of Salmonella typhimurium in the preincubation plate incorporation method in the presence and absence of metabolic activation. BHT did not show mutagenic activity on the preincubation modified Ames assay using the strains TA98 and TA100 of S. typhimurium under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Weight of evidence: In vivo: BHT showed no potential to cause point mutations in in vivo test systems. Overall, cytogenicity studies demonstrate that BHT has no clastogenic activity in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

Review

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of the mutagenicity / genotoxicity

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

other: dominant-lethal tests and mouse heritable translocation assay

Key result

Sex:

male/female

Remarks on result:

other: In the view of the general lack of clastogenic potential of BHT in the established in vivo systems at the high dose levels described, the sperm-head abnormalities cannot be considered to be a consequence of genotoxicity.

Single intraperitoneal administration of 0, 250, 500, 1000 or 2000 mg/kg of BHT to groups of 7 or 9 male ICR/Ha Swiss mice and subsequent mating with 3 untreated virgin female mice, did not cause any significant effects on the number of implantations or fetal deaths (Epstein et al., 1972; Epstein and Shafner, 1968).

In a preliminary range finding study 11 male (101 x C3H)F1 mice were given 0 or 1% BHT in their diets for periods of 8 weeks (ca. 1500 mg BHT/kg per day). After this period they were allowed to mate with (SEC x C57BL)F1 females. The same dose regimen was used in the main study using 36 male mice and mating them after the 8 -week treatment period with 66 -76 (SEC x C57BL)F1 hybrid female mice. In both studies there was no evidence of a dominant lethal effect of BHT (Sheu et al., 1986).

A group of 10 male JCL-ICR mice fed on a diet containing 1.5% BHT (ca. 2000 mg/kg per day) for 9 months was mated with untreated females. A 20% reduction in the number of implants and live embryos per dam were observed. The dominant lethal index, which is the ration between the number of live fetuses and implantations in control and treated animals, showed no statistically significant differences from the control group (Masubuchi et al., 1976).

Groups of 20 male Sprague-Dawley rats were fed diets containing 0.04%, 0.13% and 0.4% (ca. 50, 166 and 500 mg/kg per day) of BHT for 10 weeks and each male was then mated with 2 virgin females. No significant effects were observed in the 0.04% treatment group. First week data on dead implants per pregnant female showed non-dose-related increases for the intermediate and the high dose group (Sheu et al., 1986). The authors concluded that BHT may be a mutagen at 0.13 and 0.4% in the diet. The results were judged positive on the basis of increased rates of dead implants. According to recent guidelines for the method of evaluating results on calculations of the rates of dominant lethal mutations, the numbers of live implants of treated animals should be compared to controls. The number of live implants was not reduce in BHT treated animals.

Single oral administration of 30, 900 and 1400 mg/ BHT/kg bw or multiple administration (5 daily doses) of 30, 250 or 500 mg/kg bw to male Sprague-Dawley rats did not show BHT to be a mutagen in the dominant lethal test (Bruce and Heddle, 1979).

No heritable translocations were induced in the progeny (n=507) of (101 x C3H)F1 male mice after feeding a diet containing 1% BHT for 8 weeks (Sheu et al., 1986).

A study on the induction of sperm-head abnormalities was reported. Four hybrid (C57BL/6 x C3H/He)F1 mice per group were given intraperitoneal injections of 125, 250, 500 and 1000 mg BHT/kg on 5 consecutive days (Bruce and Heddle, 1979). The animals were killed on the 35th day after the last injection. From each animals 333 sperms were examined. A low non-dose related incidence up to 3.5% (controls up to 1%) of abnormal spermatozoa was observed (maximum effect at 250 mg/kg). Extreme conditions, such as high temperature, cause sperm-head abnormalities. The multiple high doses of BHT may have generated a similar stress in testicular tissue. No clear correlation between a low incidence of sperm abnormalities and hereditary effects has been established (Wyrobek, 1984). In the view of the general lack of clastogenic potential of BHT in the established in vivo systems at the high dose levels described, the sperm-head abnormalities cannot be considered to be a consequence of genotoxicity.

Conclusions:

In the view of the general lack of clastogenic potential of BHT in the established in vivo systems at the high dose levels described, the sperm-head abnormalities cannot be considered to be a consequence of genotoxicity.

Executive summary:

Single intraperitoneal administration of 0, 250, 500, 1000 or 2000 mg/kg of BHT to groups of 7 or 9 male ICR/Ha Swiss mice and subsequent mating with 3 untreated virgin female mice, did not cause any significant effects on the number of implantations or fetal deaths (Epstein et al., 1972; Epstein and Shafner, 1968).

In a preliminary range finding study 11 male (101 x C3H)F1 mice were given 0 or 1% BHT in their diets for periods of 8 weeks (ca. 1500 mg BHT/kg per day). After this period they were allowed to mate with (SEC x C57BL)F1 females. The same dose regimen was used in the main study using 36 male mice and mating them after the 8 -week treatment period with 66 -76 (SEC x C57BL)F1 hybrid female mice. In both studies there was no evidence of a dominant lethal effect of BHT (Sheu et al., 1986).

A group of 10 male JCL-ICR mice fed on a diet containing 1.5% BHT (ca. 2000 mg/kg per day) for 9 months was mated with untreated females. A 20% reduction in the number of implants and live embryos per dam were observed. The dominant lethal index, which is the ration between the number of live fetuses and implantations in control and treated animals, showed no statistically significant differences from the control group (Masubuchi et al., 1976).

Groups of 20 male Sprague-Dawley rats were fed diets containing 0.04%, 0.13% and 0.4% (ca. 50, 166 and 500 mg/kg per day) of BHT for 10 weeks and each male was then mated with 2 virgin females. No significant effects were observed in the 0.04% treatment group. First week data on dead implants per pregnant female showed non-dose-related increases for the intermediate and the high dose group (Sheu et al., 1986). The authors concluded that BHT may be a mutagen at 0.13 and 0.4% in the diet. The results were judged positive on the basis of increased rates of dead implants. According to recent guidelines for the method of evaluating results on calculations of the rates of dominant lethal mutations, the numbers of live implants of treated animals should be compared to controls. The number of live implants was not reduce in BHT treated animals.

Single oral administration of 30, 900 and 1400 mg/ BHT/kg bw or multiple administration (5 daily doses) of 30, 250 or 500 mg/kg bw to male Sprague-Dawley rats did not show BHT to be a mutagen in the dominant lethal test (Bruce and Heddle, 1979).

No heritable translocations were induced in the progeny (n=507) of (101 x C3H)F1 male mice after feeding a diet containing 1% BHT for 8 weeks (Sheu et al., 1986).

A study on the induction of sperm-head abnormalities was reported. Four hybrid (C57BL/6 x C3H/He)F1 mice per group were given intraperitoneal injections of 125, 250, 500 and 1000 mg BHT/kg on 5 consecutive days (Bruce and Heddle, 1979). The animals were killed on the 35th day after the last injection. From each animals 333 sperms were examined. A low non-dose related incidence up to 3.5% (controls up to 1%) of abnormal spermatozoa was observed (maximum effect at 250 mg/kg). Extreme conditions, such as high temperature, cause sperm-head abnormalities. The multiple high doses of BHT may have generated a similar stress in testicular tissue. No clear correlation between a low incidence of sperm abnormalities and hereditary effects has been established (Wyrobek, 1984). In the view of the general lack of clastogenic potential of BHT in the established in vivo systems at the high dose levels described, the sperm-head abnormalities cannot be considered to be a consequence of genotoxicity.

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

Review of chromosome aberrations and micronucleous formation in somatic cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of the mutagenicity / genotoxicity

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

other: chromosome aberration assays and micronucleus assays

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

After intraperitoneal injection of 75 mg BHT/kg into (CBA x C57BL/6J)F1 hybrid male and female mice bone marrow was sampled at 24, 48, 72 and 96 h from 5 animals at each time point. The number of micronucleated cells among the 1000 polychromatic erythrocytes examined for each animal was not increased at any time point (Paschin et al., 1986).

Intraperitoneal injections on 5 consecutive days into (C57BL/6 x C3H/He)F1 female mice at dose levels of 125, 250, 500 or 1000 mg/kg did not increase the rate of micronuclei in 1000 reticulocytes scored from each animal (Bruce and Heddle, 1979).

Also no adverse effects on rat bone marrow chromosomes were evident after single oral administration fo 30, 900 and 1400 mg/kg or multiple oral doses of 30, 250 and 500 mg/kg (Newell and Maxwell, 1972). Metaphases were investigated 6, 24 and 48 hours after single administration and once after multiple treatment. According to Kawachi et al. (1980) BHT did not induce chromosomal aberrations in rat bone marrow.

Similarly high dose chronic administration of BHT to mice (ICR strain) and rats (Wistar and Sprague-Dawley) did not induce clastogenic effects in bone marrow cells. In these studies groups of 5 animals received BHT in concentrations of 0 or 1.5% in the diet for 9 months, a dose approximately equivalent to 750 mg/kg per day for rats and 2000 mg/kg for mice (Masubuchi et al., 1976).

Conclusions:

Despite some shortcomings in some of the above studies, e.g., low number of animals per group, relatively low doses, limited description of experimental details, the results show that in vivo BHT does not possess clastogenic potential in somatic cells of mammalian species.

Executive summary:

After intraperitoneal injection of 75 mg BHT/kg into (CBA x C57BL/6J)F1 hybrid male and female mice bone marrow was sampled at 24, 48, 72 and 96 h from 5 animals at each time point. The number of micronucleated cells among the 1000 polychromatic erythrocytes examined for each animal was not increased at any time point (Paschin et al., 1986).

Intraperitoneal injections on 5 consecutive days into (C57BL/6 x C3H/He)F1 female mice at dose levels of 125, 250, 500 or 1000 mg/kg did not increase the rate of micronuclei in 1000 reticulocytes scored from each animal (Bruce and Heddle, 1979).

Also no adverse effects on rat bone marrow chromosomes were evident after single oral administration fo 30, 900 and 1400 mg/kg or multiple oral doses of 30, 250 and 500 mg/kg (Newell and Maxwell, 1972). Metaphases were investigated 6, 24 and 48 hours after single administration and once after multiple treatment. According to Kawachi et al. (1980) BHT did not induce chromosomal aberrations in rat bone marrow.

Similarly high dose chronic administration of BHT to mice (ICR strain) and rats (Wistar and Sprague-Dawley) did not induce clastogenic effects in bone marrow cells. In these studies groups of 5 animals received BHT in concentrations of 0 or 1.5% in the diet for 9 months, a dose approximately equivalent to 750 mg/kg per day for rats and 2000 mg/kg for mice (Masubuchi et al., 1976).

Reference 3

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of the mutagenicity / genotoxicity

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

Type of assay:

other: specific locus test

Key result

Sex:

male/female

Remarks on result:

other: Only one mutation was found among the 20402 offspring alive at weaning.

Remarks:

This incidence is not different from the background incidence of mutations at the 7 loci among over half a million offspring produced in the laboratory. It is concluded that BHT is not likely to induce point mutations in germ cells and based on several worst-case assumptions, it is estimated that mutation induced by BHT of 8% or more over background mutation frequency is excluded with 95% confidence.

Mean litter size was 5.2 which was reported to be within normal range for this hybrid strain of mice in this laboratory. Only one mutation was found among the 20402 offspring alive at weaning. This incidence is not different from the background incidence of mutations at the 7 loci among over half a million offspring produced in the laboratory.

Conclusions:

It is concluded that BHT is not likely to induce point mutations in germ cells and based on several worst-case assumptions, it is estimated that mutation induced by BHT of 8% or more over background mutation frequency is excluded with 95% confidence.

Executive summary:

To study effects of BHT in the specific locus test, a group of 206 male (C3H x 101) F1 mice were fed a diet containing a nominal concentration of 0.75% BHT (approximately 1000 mg/kg bw). During their reproductive life, in most cases more than 2 years, the males were caged with T-stock females. Offspring was scored at weaning for specific locus mutations by means of 7 genetic markers (Cumming et al., 1976). Mean litter size was 5.2 which was reported to be within normal range for this hybrid strain of mice in this laboratory. Only one mutation was found among the 20402 offspring alive at weaning. This incidence is not different from the background incidence of mutations at the 7 loci among over half a million offspring produced in the laboratory. It is concluded that BHT is not likely to induce point mutations in germ cells and based on several worst-case assumptions, it is estimated that mutation induced by BHT of 8% or more over background mutation frequency is excluded with 95% confidence.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to the recommended guidelines (number of animals, positive controls, number of cells examined, etc.). However, the results are taken into account for the weigh of evidence analysis.

Principles of method if other than guideline:

Groups of female (C57BL/6 x C3H/He) F1 mice were given injections of the agent or vehicle on five consecutive days intraperitoneally. Animals were killed on the 5th day, about 4 hours after the last injection. Touch preparations of bone marrow cells were prepared from the femur to a clean microscope slide. The preparations were fixed and stained. Three hundred and thirty-three reticulocytes were scored from each of the three marrow preparations for a total of 1000 per treated group.

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: (C57BL/6 x C3H/He) F1

Sex:

female

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

Five consecutive days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Touch preparations of bone marrow cells were prepared from the femur to a clean microscope slide. The preparations were fixed and stained. Three hundred and thirty-three reticulocytes were scored from each of the three marrow preparations for a total of 1000 per treated group.

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Conclusions:

The bone marrow micronucleus assay with female mice was negative.

Executive summary:

Groups of female (C57BL/6 x C3H/He) F1 mice were given injections of the agent or vehicle (0, 125, 250, 500 or 1000 mg/kg bw) on five consecutive days intraperitoneally. Animals were killed on the 5th day, about 4 hours after the last injection. Touch preparations of bone marrow cells were prepared from the femur to a clean microscope slide. The preparations were fixed and stained. Three hundred and thirty-three reticulocytes were scored from each of the three marrow preparations for a total of 1000 per treated group. The bone marrow micronucleus assay with female mice was negative.

Reference 5

Endpoint:

genetic toxicity in vivo, other

Remarks:

Sperm abnormalities assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to the recommended guidelines (number of animals, positive controls, number of cells examined, etc.). However, the results are taken into account for the weigh of evidence analysis.

Principles of method if other than guideline:

Groups of male (C57BL/6 x C3H/He) F1 mice were given injections of the agent or vehicle on five consecutive days intraperitoneally. Animals were killed on the 35th day after the last injection. Sperm suspensions were prepared by mincing the cauda epididymis in 2 ml phosphate buffered physiological saline, pipetting the resulting suspension and filtering it through a stainless steel mesh to remove tissue fragments. A fraction of each suspension was mixed 10:1 with 1% eosin-Y in water. Thirty minutes later smears were made. Three hundred and thirty-three sperm were examined from each animal for a total of 1000 per treated group.

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

other: (C57BL/6 x C3H/He) F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

Five consecutive days

Frequency of treatment:

Daily

Post exposure period:

Animals were killed on the 35th day after the last injection.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Sperm suspensions were prepared by mincing the cauda epididymis in 2 ml phosphate buffered physiological saline, pipetting the resulting suspension and filtering it through a stainless steel mesh to remove tissue fragments. A fraction of each suspension was mixed 10:1 with 1% eosin-Y in water. Thirty minutes later smears were made. Three hundred and thirty-three sperm were examined from each animal for a total of 1000 per treated group.

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

The substance is considered positive in the reference. However, the only effects observed were a low non-dose related incidence up to 3.5% (controls up to 1%) of abnormal spermatozoa (maximum effect at 250 mg/kg bw day).

Conclusions:

The substance is considered positive in the reference. However, the only effects observed were a low non-dose related incidence up to 3.5% (controls up to 1%) of abnormal spermatozoa (maximum effect at 250 mg/kg bw day).

Executive summary:

Groups of male (C57BL/6 x C3H/He) F1 mice were given injections of the agent or vehicle on five consecutive days intraperitoneally. Animals were killed on the 35th day after the last injection. Sperm suspensions were prepared by mincing the cauda epididymis in 2 ml phosphate buffered physiological saline, pipetting the resulting suspension and filtering it through a stainless steel mesh to remove tissue fragments. A fraction of each suspension was mixed 10:1 with 1% eosin-Y in water. Thirty minutes later smears were made. Three hundred and thirty-three sperm were examined from each animal for a total of 1000 per treated group. The substance is considered positive in the reference. However, the only effects observed were a low non-dose related incidence up to 3.5% (controls up to 1%) of abnormal spermatozoa (maximum effect at 250 mg/kg bw day).

Reference 6

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although the study was not conducted according to the recommended guidelines and that metaphases were examined only once (6 h after last administration), it provides some scientific valid information to take into account for the weight of evidence analysis.

Principles of method if other than guideline:

Cytogenetic evaluations were made on bone-marrow cells in metaphase from random-bred male albino rats. The positive control compound triethylenemelamine (TEM) was injected intraperitoneally at a dose of 0.5 mg/kg dissolved in normal saline. Six, 24 and 48 hours subsequent to single administration, five rats each from the maximum tolerated dose, intermediate and low groups and three rats from the negative control group were sacrificed and bone marrow was obtained. In conjunction with this single-administration acute study a subacute study was undertaken simultaneously by administering compound every 24 hours for five days to five animals per group. All rats in the subacute study were sacrificed six hours after the last intubation and bone marrow was obtained. Duplicate slides were prepared for each animal or treatment.

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

Single administration and five-day administration

Dose / conc.:

30 mg/kg bw (total dose)

Remarks:

Single oral dose

Dose / conc.:

900 mg/kg bw (total dose)

Remarks:

Single oral dose

Dose / conc.:

1 400 mg/kg bw (total dose)

Remarks:

Single oral dose

Dose / conc.:

30 mg/kg bw/day

Remarks:

Five oral administrations

Dose / conc.:

250 mg/kg bw/day

Remarks:

Five oral administrations

Dose / conc.:

500 mg/kg bw/day

Remarks:

Five oral administrations

No. of animals per sex per dose:

Five males per dose

Control animals:

yes

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Duplicate slides were prepared for each animal or treatment.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Conclusions:

Under the conditions of this in vivo chromosome aberration assay BHT showed no clastogenic effects on bone marrow chromosomes of rats.

Executive summary:

Cytogenetic evaluations were made on bone-marrow cells in metaphase from random-bred male albino rats. The positive control compound triethylenemelamine (TEM) was injected intraperitoneally at a dose of 0.5 mg/kg dissolved in normal saline. Six, 24 and 48 hours subsequent to single administration, five rats each from the maximum tolerated dose, intermediate and low groups and three rats from the negative control group were sacrificed and bone marrow was obtained. In conjunction with this single-administration acute study a subacute study was undertaken simultaneously by administering compound every 24 hours for five days to five animals per group. All rats in the subacute study were sacrificed six hours after the last intubation and bone marrow was obtained. Duplicate slides were prepared for each animal or treatment. Under the conditions of this in vivo chromosome aberration assay BHT showed no clastogenic effects on bone marrow chromosomes of rats.

Reference 7

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according the recommended guidelines, it provides some scientific information to be taken into account for the weight of evidence analysis.

Principles of method if other than guideline:

For the acute study, ten experienced male rats per treatment group were administered a single dose of test material orally. Within two to three hour, each male was mated with two virgin females for a period of seven days. Breeding and implant data were obtained for eight weeks. Control groups were included in the experiment. The positive reference control was triethylenemelamine (TEM) given at a dose of 0.2 mg/kg as a single peritoneal injection. For the subacute assay, the same experimental parameters as those used in the acute test were employed, with two exceptions: five dosings were administered at 24-h intervals and weekly matings were for seven weeks. One fourth of the pregnant females in each group were sacrificed on each of four days starting on the 15 th day after the first day of breeding. A complete autopsy was performed and the following parameters were scored: early fetal deaths, late fetal deaths, living fetuses, corpora lutea and preimplantation loss.

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

rat

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

Single administration and five-day administration

No. of animals per sex per dose:

Ten males per dose

Control animals:

yes

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Conclusions:

Under the conditions of this dominant lethal assay BHT was considered negative.

Executive summary:

A dominant lethal assay was performed. For the acute study, ten experienced male rats per treatment group were administered a single dose of test material orally. Within two to three hour, each male was mated with two virgin females for a period of seven days. Breeding and implant data were obtained for eight weeks. Control groups were included in the experiment. The positive reference control was triethylenemelamine (TEM) given at a dose of 0.2 mg/kg as a single peritoneal injection. For the subacute assay, the same experimental parameters as those used in the acute test were employed, with two exceptions: five dosings were administered at 24-h intervals and weekly matings were for seven weeks. One fourth of the pregnant females in each group were sacrificed on each of four days starting on the 15 th day after the first day of breeding. A complete autopsy was performed and the following parameters were scored: early fetal deaths, late fetal deaths, living fetuses, corpora lutea and preimplantation loss. Under the conditions of this dominant lethal assay BHT was considered negative.

Reference 8

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the in vivo genotoxicity of the substance.

Principles of method if other than guideline:

Mouse bone marrow micronucleus employed 3 treatments of the test chemical administered at 24 h intervals. The route of administration was intraperitoneal injection. Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing. The bone marrow is flushed from the femurs and spread onto slides. 2000 polychromatic erythrocytes are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

3 treatments administered at 24 h.

Frequency of treatment:

3 treatments administered at 24 h.

Post exposure period:

24 h

Remarks:

Doses / Concentrations:
18.75, 37.5, 75.75, 150.15, 300.3, 400.4 and 600.6 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Normally 5 male animals per treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
-Doses / concentrations: 25.25 mg/kg

Tissues and cell types examined:

Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing. At that time, about 50% of the erythrocytes in the bone marrow are immature, newly formed erythrocytes, and these are the cell types that are checked for presence of micronuclei. The animals are euthanized by CO2 inhalation and the femurs are removed.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing.


DETAILS OF SLIDE PREPARATION: The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present.


METHOD OF ANALYSIS: Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity.

Evaluation criteria:

The final conclusion for a micronucleus test is determined by considering the results of statistical analyses, the reproducibility of any observed effects, and the magnitude and biological significance of those effects.

Statistics:

A formal statistical analysis of the data is performed that includes a trend test, to determine if there is an overall increase across all doses in the frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group is statistically different from the control group in frequency of micronucleated cells. Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group. A positive trend test is one in which the P value is equal to or less than 0.025. For the micronucleus frequency in any dose group to be considered significantly elevated over the control group, the P value must be equal to or less than 0.025 divided by the number of chemical-treatment groups. Thus, if the number of treated groups is 3, then the required pairwise P value is 0.008. This adjustment in the pairwise P value is a correction for multiple comparisons of the same data.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The bone marrow micronucleus assay in mice reported in this study was negative.

Executive summary:

Mouse bone marrow micronucleus employed 3 treatments of the test chemical administered at 24 h intervals. The route of administration was intraperitoneal injection. Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing. The bone marrow is flushed from the femurs and spread onto slides. 2000 polychromatic erythrocytes are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity. The bone marrow micronucleus assay in mice reported in this study was negative.

Reference 9

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the in vivo genotoxicity of the substance. Only one single dose level was applied but the negative result is considered as supportive for the other test results taking into account for the weight of evidence analysis.

Principles of method if other than guideline:

The micronucleus test using mouse bone-marrow polychromatic erythrocytes was used. Bone marrow was sampled at 24, 48, 72 and 96 h after ip injection, from five control and five mice. For each animal, 1000 polychromatic erythrocytes were examined at high magnification (x 1000) for the incidence of micronucleated cells.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: (CBA x C57BL/6J)F1 hybrid

Sex:

male/female

Route of administration:

intraperitoneal

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Dose / conc.:

75 mg/kg bw (total dose)

No. of animals per sex per dose:

Five mice per dose

Control animals:

yes

Tissues and cell types examined:

Bone marrow was sampled at 24, 48, 72 and 96 h after ip injection, from five control and five mice. For each animal, 1000 polychromatic erythrocytes were examined at high magnification (x 1000) for the incidence of micronucleated cells.

Evaluation criteria:

The hybrid mice used have a stable rate of spontaneous micronuclei in polychromatic erythrocytes of less than 2 per 1000 cells.

Statistics:

The statistical significance of the results was determined by Student's t test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Additional information on results:

Treatment with dibunol had no mutagenic effects on the erythroblasts.

Conclusions:

Treatment with dibunol (BHT) had no mutagenic effects on the erythroblasts in the micronucleus test using mouse bone-marrow.

Executive summary:

The micronucleus test using mouse bone-marrow polychromatic erythrocytes was used. Bone marrow was sampled at 24, 48, 72 and 96 h after ip injection, from five control and five mice. For each animal, 1000 polychromatic erythrocytes were examined at high magnification ( x 1000) for the incidence of micronucleated cells. Treatment with dibunol (BHT) had no mutagenic effects on the erythroblasts.

Reference 10

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the in vivo genotoxicity of the substance.

Principles of method if other than guideline:

Dominant lethal assay with male mouse fed with 1% BHT in the diet (1500 mg/kg bw/day) for 8 weeks. Each male (36/group) was then caged with
one (SEC x C5BL)Fl and one (C3H x C57BL)Fl virgin female, beginning immediately after the end of the 8-wk treatment period. The females were examined for the presence of vaginal plugs each morning, and those with plugs were replaced with new females during this 1 wk of mating. The females were sacrificed for uterine analysis 12-15 days after mating.

Heritable translocations: The protocol for the heritable translocation test was similar to that described by Generoso et al [1980] with a slight modification: steps were introduced to test for preexisting translocations among parental mice.

GLP compliance:

not specified

Type of assay:

other: dominant lethal assay and heritable translocation test

Species:

mouse

Strain:

other: (101 x C3H)F1

Sex:

male

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
1 % w/w (1500 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

Dominant lethal assay: 36 males per group

Heritable translocation test: 50 males per group

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Dominant lethal assay: each male was caged with one (SEC x C5BL)Fl and one (C3H x C57BL)Fl virgin female, beginning immediately after the end of the 8-wk treatment period. The females were examined for the presence of vaginal plugs each morning, and those with plugs were replaced with new females during this 1 wk of mating. The females were sacrificed for uterine analysis 12-15 days after mating.

Heritable translocation test: the male progeny produced from the matings before and after treatment were subjected to the sequential fertility procedure. All progeny classified as having reduced fertility were then subjected to cytological analysis for the confirmation of translocation heterozygosity.

Evaluation criteria:

Heritable translocation test: The male progeny obtained before treatment served as the negative controls as well as for determining the possible existence of translocations in the parental mice. The male or female of a translocation-carrying son was considered normal (free of pre-existing translocation) if a parent produced no other translocation-carrying progeny out of at least 12 male progeny tested. If the number of male progeny from a parent was insufficient for evaluation, additional progeny were generated from this parent by mating to a normal mouse, or the parent was subjected to cytological analysis.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

Dominant lethal assay: BHT did not induce a detectable level of dominant lethal effect in male mice.

Heritable translocation test: A total of 507 control and 475 experimental male progeny were tested in the BHT experiment. In the control group, 503 (99.4%) were found to be normal in the fertility testing. Of the remaining four, one was semisterile, two were sterile, and one initially had low fertility but became sterile and died before cytology was performed. The semisterile male carried a translocation, but its mother was also a translocation carrier. Of the two steriles, one was XYY and the other a translocation carrier with normal parents. In the experimental group, 473 (99.6%) were found to be normal on the basis of fertility testing. The remaining two, which were brothers, were semisterile translocation carriers, but their mother was also a carrier of translocation

Conclusions:

The dominant lethal assay and the heritable translocation test were negative.

Executive summary:

A dominant lethal assay and a heritable translocation test were conducted with male mouse fed with 1% BHT in the diet (1500 mg/kg bw/day) for 8 weeks. BHT did not induce a detectable level of dominant lethal effect in male mice. In the heritable translocation test, a total of 507 control and 475 experimental male progeny were tested in the BHT experiment. In the experimental group, 473 (99.6%) were found to be normal on the basis of fertility testing. The remaining two, which were brothers, were semisterile translocation carriers, but their mother was also a carrier of translocation. Therefore, the dominant lethal assay and the heritable translocation test were negative in mice.

Reference 11

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

BHT induced statistically significant dominant lethal effects in male rats. However, re-calculation of the dead implants according to recent guidelines for the method of calculating rates of dominant lethal mutations (Dean 1983; EEC 1987; OECD 1984) revealed no differences between BHT-treated animals and controls.

Principles of method if other than guideline:

Dominant lethal assay with male rats fed with 0.04, 0.13 or 0.4% BHT in the diet (50, 166 or 500 mg/kg bw/day) for 10 weeks. Each male was mated with two virgin females per week for 2 successive wk. At 14 days after midweek of mating, the females were sacrificed and the number of live and dead implants were counted.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Duration of treatment / exposure:

10 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.04, 0.13 and 0.4 % w/w (50, 166 or 500 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

Twenty male rats per group

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control group received drinking water containing 0.48 mg of TEM per liter.

Tissues and cell types examined:

Each male was mated with two virgin females per week for 2 successive wk. At 14 days after midweek of mating, the females were sacrificed and the number of live and dead implants were counted.

Evaluation criteria:

1. Dead implants per pregnant female, analyzed by the t-test after transformation of the data by the Freeman-Tukey square-root method;
2. Dead implants per total implants (% dead implants), analyzed by the t-test after transformation of the data by the Freeman-Tukey arc-sine method;
3. Proportion of females with one or more dead implants, analyzed by the chi- square test; and
4. Proportion of females with two or more dead implants, analyzed by the chi-square test.

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

(However, re-calculation of the dead implants according to recent guidelines for the method of calculating rates of dominant lethal mutations (Dean 1983; EEC 1987; OECD 1984) revealed no differences between BHT-treated animals and controls).

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

BHT induced statistically significant dominant lethal effects in male rats. However, re-calculation of the dead implants according to recent guidelines for the method of calculating rates of dominant lethal mutations (Dean 1983; EEC 1987; OECD 1984) revealed no differences between BHT-treated animals and controls.

Conclusions:

BHT induced statistically significant dominant lethal effects in male rats. However, re-calculation of the dead implants according to recent guidelines for the method of calculating rates of dominant lethal mutations (Dean 1983; EEC 1987; OECD 1984) revealed no differences between BHT-treated animals and controls.

Executive summary:

A dominant lethal assay was conducted with male rats fed with 0.04, 0.13 or 0.4% BHT in the diet (50, 166 or 500 mg/kg bw/day) for 10 weeks. Each male was mated with two virgin females per week for 2 successive wk. At 14 days after midweek of mating, the females were sacrificed and the number of live and dead implants were counted. BHT induced statistically significant dominant lethal effects in male rats. However, re-calculation of the dead implants according to recent guidelines for the method of calculating rates of dominant lethal mutations (Dean 1983; EEC 1987; OECD 1984) revealed no differences between BHT-treated animals and controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Weight of evidence: Experimental results from scientific articles and reviews. The in vitro and vivo genotoxicity studies available for BHT were not conducted in accordance with specific testing guidelines. Most of these studies have limitations which have been discussed in the corresponding summaries, but taken together they allow drawing conclusions as to the genotoxicity potential of the substance BHT.

Taking into account all the available genotoxicity information on BHT for the weight of evidence analysis, it is concluded that the substance is not classified as a mutagen in accordance with the CLP Regulation.

Justification for classification or non-classification

Taking into account all the available genotoxicity information on BHT for the weight of evidence analysis, it is concluded that the substance is not classified as a mutagen in accordance with the CLP Regulation.