4-tert-butylphenol

Endpoint summary

Additional information

In Vitro Bacterial Gene Mutation Data

Three bacterial gene mutation studies (tests) are presented in the dossier.

AMES.001 - 1992 key: Klimisch 1, GLP-compliant guideline study without deviation. Sponsored by Dow and conducted by Safepharm (Jenkinson PC). In both the presence and absence of metabolic activation (S9), S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2uvrA were exposed to ptBP at 0, 1.6, 8, 40, 200 and 1000 µg/plate in a first experiment, and then at 0, 31.25, 62.5, 125, 250, 500 and 1000 µg/plate in an independent second experiment. DMSO was used as the solvent, and solvent only treatment groups were used as negative controls, all of which gave revertant colony counts within the normal range. Appropriate positive controls all markedly increased revertant colony counts, validating both the sensitivity of the bacteria and the efficacy of the S9. Cytotoxicity was seen in all experiments with all strains from 1000 µg/plate, so ptBP was tested up to its toxic limit. No genotoxicity was observed in any bacterial tester strain in the presence or absence of metabolic activation.

AMES:002, 1985: An Ames assay with S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 as well as E. coli WP2 and WP2uvrA was conducted by Dean et al.(1985). The amount of ptBP used was 125, 250, 500, 1000, 2000 and 4000 µg/plate in the presence and absence of metabolic activation. No genotoxicity was observed in any bacterial tester strain in the presence or absence of metabolic activation. Several positive controls were used in this study but there is no further information about the doses used and for which bacteria strain they were used. Positive controls were ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS), cyclophosphamide (CP), benzo(a)pyrene (B(a)P), neutral red (NR), sodium azide (SA), 7,12-dimethylbenzanthracene (DMBA), and 4-nitroquinoline-N-oxide (NQO). B(a)P, NQO and DMBA were all dissolved in DMSO while the other positive controls were dissolved in aqueous solutions. The purity of the test substance was reported to be >95% and DMSO was used as a solvent.

AMES:003, 1996: Another Ames assay (summarized in the EU Risk Assessment for ptBP) reported negative results for ptBP in the presence and absence of metabolic activation. This study (OECD, SIDS program, 1996; Ministry of Health and Welfare Japan, 1996) used S. typhimurium strains TA 100, TA 1535, TA 98, and TA 1537 as well as E. coli WP2 uvrA. No gene mutations were induced in any strain in the presence or absence of metabolic activation. The cytotoxic concentration for bacteria in the presence of metabolic activation was 500 µg/plate for all five strains; while without metabolic activation it was 500 µg/plate for TA 100, TA 1535, TA 1537 and 1000 µg/plate for WP2uvrA and TA 98. The test was performed according to the guidelines for Screening Mutagenicity Testing of Chemicals and OECD guidelines 471 (471/472) and was according to GLP. The amounts of ptBP per plate were; 0, 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA strains, and 0, 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the WP2uvrA strain. The purity of the test substance was 99.9%. DMSO was used as a solvent in the plate incorporation method. Tests were performed in the presence as well as in the absence of an external metabolic activation system (rat liver S9 mix). Three plates per concentration were used and all tests were performed in duplicate. There was no information available regarding the use of positive controls in this study.

In Vitro Chromosomal Aberration Data

Two mammalian cytogenicity studies (Chromosome Aberration tests) are presented in the dossier.

Chrom. Ab.004 - 1992, key: Klimisch 1, GLP-compliant guideline study without deviation. Sponsored by Dow and conducted by Safepharm (Jenkinson PC). Lymphocytes from male CD rats were exposed to ptBP in the presence and absence of metabolic activation (S9). A first experiment investigated effects following a 20-hour continuous exposure to ptBP at concentrations up to 62.5 µg/mL in the absence of metabolic activation, and the effects of a 4-hour exposure to ptBP at concentrations up to 60.0 µg/mL in the presence of S9 (2% v/v), followed by a 16-hour or 26-hour expression period. The ptBP exhibited a very steep toxicity dose-response curve, and these concentrations represent the toxicity limits for the treatments described. In a second experiment, ptBP was tested at concentrations up to 31.25 µg/mL for a 20-hour continuous exposure time in the absence of S9, and at concentrations up to 30.0 µg/mL and 60.0 µg/mL for a 4-hour exposure time in the presence of S9 (2% v/v), followed by a 16-hour or 26-hour expression period, respectively. These values again represent the toxicity limits for the treatments described, with the apparently increased toxicity shown by two of the groups being attributed to the steepness of the toxicity dose-response curve and the use of smaller blood volumes in the second experiment. DMSO was used as the solvent, and solvent only treatment groups were used as negative controls, all of which gave aberration counts within the normal range. All positive controls markedly increased the proportion of aberrant cells, validating both the sensitivity of the lymphocytes and the efficacy of the S9. No significant increases in the proportion of metaphase figures containing chromosomal aberrations were seen, at any concentration, either in the absence or presence of S9 in either test. Nor were any significant increases in the proportion of polyploid cells seen during metaphase analysis in either test.

Chrom. Ab.005 - 1996: As summarized in the EU Risk Assessment for ptBP, Honma et al.(1999) demonstrated no significant mutagenic potential for ptBP in the mouse lymphoma TK+/- locus assay in L5178Y cells. PtBP was tested at a concentration of 0, 20, 40, 60 and 80 µg/ml following 3 to 6 hours of exposure, either with or without metabolic activation. However, ptBP 0, 20, 40, 60 and 80 µg/ml was shown to be mutagenic following a 24-hour exposure period. The mutagenic potential was investigated up to a sufficient cytotoxic condition (<20% relative survival (RS) as a rule) and at 40 µg/ml ptBP the RS was less than 20% for the 24-hour exposure. Each experiment was performed with a single culture per treatment without S9 mix. The test was not performed according to OECD test guideline 476. The actual mutant frequencies obtained following 24-hour exposure was for 30 µg/ml about 100 MF(x 10^-6), 40 µg/ml about 150 MF(x 10^-6) and 50 µg/ml about 230 MF(x 10^-6). The actual concentrations appear to be different than from those reported above. These concentrations are extracted visually from Figure 1 in the paper of Honma et al.(1999) and are not consistent with the exposure doses.

As summarized in the EU Risk Assessment for ptBP, Dean et al.(1985) examined ptBP for mitotic gene conversion in Saccharomyces cerevisiae JD1 and for structural chromosomal damage in a cultured rat liver cell line. PtBP did not induce chromosomal aberrations in rat liver epithelial-type cells. PtBP also did not induce mitotic recombination in Saccaromyces cerevisiae JD1 with or without an exogenous metabolic activation system when exposed for 18 hours at 30°C. One stationary and one log-phase conversion assay were performed with 5% solution of the test substance. The test was performed according to EEC Annex V B16 (Commission Directive 84/449/EEC), following GLP.

Two studies summarized in the EU Risk Assessment for ptBP have reported positive findings with ptBP for chromosomal aberrations and polyploidy in Chinese hamster lung cells (CHL/IU cells). In the first study (OECD, SIDS program, 1996), ptBP induced chromosomal aberrations in CHL/IU cells only in the presence of an exogenous metabolic activation system. Polyploidy was reported with and without an exogenous metabolic activation system, but the high incidences of polyploidy were only observed at cytotoxic concentrations. The following experimental conditions were reported:

Solvent: DMSO

Positive control: Mitomycin C without S9 (-S9) and Cyclophospfamide with S9 (+S9).

Doses used: -S9 (continuous treatment, 24 or 48 hours); 0, 0.013, 0.025, and 0.05 mg/ml.

Doses used: -S9 (short term treatment, 6 hours); 0, 0.02, 0.04, 0.08 mg/ml.

Doses used: +S9 (short term treatment, 6 hours); 0, 0.013, 0.025. 0.050 mg/ml.

Cytotoxicity was detected for continuous treatment at 0.025 mg ptBP/ml and for short-term treatment at 0.08 mg ptBP/ml, both without metabolic activation. There was no observation of cytotoxicity with metabolic activation. Lowest concentration producing cytogenetic effects was: (1) without S9 (continuous treatment) using 0.025 mg/ml (polyploidy), (2) without S9 (short-term treatment) 0.02 mg/ml (polyploidy), (3) with S9 (short-term treatment) 0.013 mg/ml (clastogenicity) and 0.025 mg/ml (polyploidy). After 24 hours the percent polyploidy was 7.63% and after 48 hours 93.18%. Further evaluation of the study was not possible since only an English summary was available, the full study report being in Japanese. The study was conducted according to OECD Guideline 473, following GLP. The purity of the test substance was reported to be 99.9%. Cytotoxicity was observed at 0.025 mg ptBP/ml (without metabolic activation, continuous treatment) and 0.08 mg ptBP/ml (without metabolic activation, short-term treatment).

In the second study (Kusakabe et al., 2002), ptBP induced chromosomal aberrations and polyploidy in CHL/IU cells. CHL/IU cells were treated with 100 to 1000 mM (from the paper the range was from 50 mg/ml to 500 mg/ml) ptBP dissolved in aceton or DMSO. However, the experimental concentration and solvent used is not clearly described in the publication. Therefore the concentration might be 100 mM (15 mg/ml) or 50 mg/ml in water. In order to examine a possible role of metabolic activation of ptBP, the proliferating cells were treated with ptBP for 6 hours in serum-free medium with or without S9 mix, then cultured a further 18 hours in fresh medium with serum. The cells were also treated with ptBP for 24 hours and 48 hours continuously in the absence of S9 mix. Duplicate cultures were used for each experiment. The study was conducted according to OECD TG 473. PtBP induced structural chromosomal aberrations (within the range of <20% to =>20%) with the minimum effective dose manifesting severe cytotoxicity (50% or less) in a short-term treatment assay with S9 mix, and 93.2% polyploidy in a 48 hour continuous treatment test.

InVitro Mammalian Cell Gene Mutation Data

TK Mouse Lymphoma.006 - 1992, key: Klimisch 1, GLP-compliant guideline study without deviation. Sponsored by Dow and conducted by Safepharm (Jenkinson PC). In both the presence and absence of metabolic activation (S9), mouse lymphoma L5178Y cells were exposed for 3 hours at 37°C to ptBP concentrations of 0, 5, 10, 20, 40 and 80 µg/mL in a first experiment, and then to 0, 5, 10, 20, 40 and 60 µg/mL in a second independent experiment. DMSO was used as the solvent, and solvent only treatment groups were used as negative controls, which gave mutant frequencies around the upper end of the normal range. All positive controls markedly increased the mutant frequency, validating both the sensitivity of the lymphoma cells and the efficacy of the S9. Markedly reduced viability was seen in experiment 1 at 80 µg/mL, and reduced viability was also seen in experiment 2 at 60 µg/mL, in both cases with and without S9, so ptBP was tested up to its toxic limit. PtBP was not considered to have produced any dose-related or biologically significant increases in mutant frequency, and was considered to be non-mutagenic under the conditions of the test.

In Vivo Data

A mammalian erythrocyte micronucleus test with ptBP has been conducted (MHLW, 2005) and is summarized in the EU Risk Assessment for ptBP. This study was conducted according to OECD Test Guideline 474. Based on the results of this study ptBP was considered not genotoxic in vivo. In a preliminary range-finding experiment 5 males and 5 females were exposed to 25, 50, 100 and 200 mg/kg body weight ptBP (dissolved in 0.5% methyl cellulose) by intraperitoneal injection. All animals died at 200 mg/kg body weight, and 3 males and 4 females died at 100 mg/kg body weight with severe clinical signs. Based on this preliminary study the maximal tolerable dose (MTD) was considered to be 50 mg/kg body weight. For the main study, a single intraperitonal injection of ptBP at 0, 12.5, 25 or 50 mg/kg body weight was given to male CD-1 mice age 9 weeks (5 animals in the treated group, 5 treated animals per dose), based on severe toxicity at 100 mg/kg body weight and no sex difference on toxicity in the previous range-finding study. Two thousand polychromatic erythrocytes (PCEs) were counted at 24 and 48 hours after ptBP injection and compared to the results from the positive control, Cyclophosphamide (CPA) and the negative control methyl cellulose. No significant differences in signs of toxicity between negative control and ptBP-exposed animals were found. The ptBP-exposed mice showed low locomotor activity at 25 and 50 mg/kg body weight. No increase in the frequency of micronucleated bone marrow cells was observed in any dose groups at 24 and 48 hours after administration compared to control animals. Although the data in this study was not statistically significant, the data indicate that there is a trend to decreased PCE/NCE ratios with dose. In addition, the route of administration in combination with the tested dose (close to MTD) suggests that it is very likely that the material had reached the target organ. Based on these results (see table 1 from the study) ptBP was considered not genotoxic in vivo. In addition, when the authors tested the same specimens for chromosomal aberrations there was no detection of chromosomal aberration or spindle body formation in the mouse marrow cells induced by ptBP.

A mouse micronucleus test also has been conducted with a structural isomer of ptBP, namely o-tert-butylphenol (Cinelli, 2000). The study was conducted according to OECD guideline 474 and according to GLP. Following a range-finding study that tested doses of up to 2000 mg/kg body weight, male and female CD-1 mice (5/sex) were dosed once with o-tert-butylphenol at 250, 500, or 1000 mg/kg body weightviaoral gavage. Negative and positive control animals received either the solvent (corn oil) alone or mitomycin C, respectively, by oral gavage. Preparation of bone marrow cells was done after 24 hours. An additional group of animals (5/sex) received a single dose of 1000 mg/kg body weight and bone marrow cells were prepared after 48 hours. Substance related toxic effects were observed in all groups. No increase in micronuclei was observed in any of the test groups. It was concluded that, under the reported experimental conditions, o-tert-Butylphenol administered orally at the selected dose levels to male and female mice, did not induce micronuclei in the polychromatic erythrocytes. Clinical signs and mortality (observed at the highest dose level) provide evidence of bioavailability of the test item and adequate exposure. O-tert-Butylphenol was not clastogenic under the conditions of the test.

Short description of key information:
PtPB has been assessed for genetic toxicity in in vitro and in vivo systems in proprietary and published studies, some of which were conducted according to OECD test guidelines and GLP. In vitro studies in bacteria (Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538; Escherichia coli WP2 and WP2uvrA) and mouse lymphoma L5178Y cells have demonstrated no genetic toxicity of ptBP when tested in the presence or absence of exogenous metabolic activation. In vitro studies in Chinese hamster lung cells demonstrated chromosomal aberrations only in the presence of exogenous metabolic activation, while an in vitro study in rat lymphocytes in the presence or absence of exogenous metabolic activation, and in vivo mouse micronucleus tests with ptBP and with a structural surrogate (o-tert-butylphenol) all showed no chromosomal aberrations. The overall weight of evidence suggests no genetic toxicity potential for ptBP.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance does not meet the criteria for classification and labelling for this endpoint, as set out in Regulation (EC) NO. 1272/2008.