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The reaction mass of phenol and 4,4'-isopropylidenediphenol (BPA) contains phenol and BPA.

For considerations on the analogue approach used please refer to the reporting form attached to section 13 of the IUCLID.

The mammalian toxicity of this product is expected to be affected most by the presence of phenol although BPA is discussed at the end of this section.

 

GENETIC TOXICITY IN VITRO FOR PHENOL

A summary of data on genotoxicity in vitro is presented in the EU-RAR Phenol (2006) in Section 4.1.2.7.1 (page 106) and an overview on in vitro findings in Table 4.13 (page 108). 

Bacterial test systems detecting gene mutations on phenol

The available data on the Salmonella microsome assay (Ames test) have been reported in a summary table in the EU-RAR Phenol (page 111, Table 4-16); the authors concluded that phenol has no mutagenic activity in this test system. The most informative and valid study was documented by Glatt et al. (1989). S. typhimurium strains TA97, TA98, TA100, TA102, TA104, and TA1535 were exposed with and without metabolic activation (MA) to varying concentrations of phenol (100 -5000 µg/plate without S-9 mix and 20 -5000 µg/plate with S-9 mix). Cytotoxic effects (LD50 measured in his+ mutants as internal standards) were detected at 3000 µg/plate without MA and at 1800 µg/plate with MA. No genotoxic effects were recorded in any strain.

No gene mutagenic effects were detected in further Salmonella microsome studies (see Table 4.16 in EU-RAR; Gilbert et al., 1980; Haworth et al., 1983).

Ambiguous test results were presented by Wild et al. (1980) and Gocke et al. (1981) Both publications referred to the same experiments. Using a standard medium all experiments gave negative results in this Ames test but the ZLM medium (used in E. coli culture) resulted in weak mutagenic effects (2 -2.5 -fold increase in revertants) in TA98 with metabolic activation at high dose levels of 3.3 -7.5 mg/plate (a max. concentration of 5 mg/plate is recommended in OECD 471).

In conclusion, phenol resulted in no induction of gene mutation in the bacterial reverse mutation assay at concentrations up to 5 mg/plate. 

Chromosome mutations in mammalian cells by phenol

An overview on chromosomal mutations in mammalian cells by phenol is given in EU-RAR (2006) in Table 4.19 (page 115; chromosome aberration assay, 2 studies) and Table 4.20 (page 116; micronucleus assay, 3 studies). Phenol was considered to have mutagenic activity concerning this endpoint (EU-RAR 2006, Table 4.14 on page 108).

In the micronucleus assay (Miller et al., 1995) CHO cells were exposed at dose levels of 350 -2.000 µg/ml (with metabolic activation [MA]) or 10 -250 µg/ml (without MA). Phenol caused slight increase in MN accompanied by cytotoxicity; without MA 250 µg/ml increased the MN frequency by factor 3-3.5 and with MA 2000 µg/ml was highly toxic and increased the MN rate by a factor of 4.8-7.0. In summary, phenol induced increased incidence in micronuclei in CHO cells at high dose levels resulting also in cytotoxic effects.

Chinese hamster ovary cells were exposed without metabolic activation (MA) to 600, 700, 800 µg/ml (exposure duration 8 hours) and with MA to 2000, 2500, 3000 µg/ml (exposure duration 2 hours) (solvent DMSO). No increase in chromosome aberrations were recorded without MA. However, increased incidence of aberration were found with MA. A slight reduction in cell confluency at the two top doses were observed with and without MA. Conclusion: Phenol was clastogenic in the chromosome aberration assay in CHO cells with metabolic activation (Ivett et al., 1989).

Conclusion; in chromosome mutation assays positive results were obtained mainly at cytotoxic concentrations. 

Gene mutations in mammalian cells on phenol

Data on this endpoint were summarized in Table 4.17 and 4.18 (EU-RAR 2006, page 112). In the EU-RAR (2006) phenol was considered to induce gene mutation in mammalian cells.

In the HPRT assay (Tsutsui et al., 1997) SHE cells were exposed without metabolic activation to phenol at dose levels of 0, 0.28, 0.94, 2.8 µg/ml. A clearly increased mutation frequency was detected at the high dose level. Data on cytotoxicity in the main experiment were not available. In a separate cell growth experiment no cytotoxicity was found 48 h after incubation with dose levels up to 9.4 µg/ml. Conclusion: Phenol at a dose level of 30 µM (2.8 µg/ml, not cytotoxic) resulted in an increased gene mutation frequency in SHE cells at the HPRT locus. Similar results were obtained in the same study using the Na+/K+ locus of SHE cells.

In the mouse lymphoma assay (Wangenheim & Bolcsfoldi, 1988) cells were exposed to 5.2 - 41.8 µg/ml with MA and to 178 - 887 µg/ml without MA. Phenol resulted with and without MA in increases of the mutation frequency between 2 - and 3 -fold of control value at concentrations that reduced total growth to 10 -55%. Conclusion: Phenol resulted in weak mutagenic effects at dose levels decreasing the total growth.

Similar results were presented by McGregor et al. (1988). Ambiguous results in the 1st-trial and positive results in the 2nd-trial were found in the mouse lymphoma assay with MA but ambiguous results without MA in both trials; the authors concluded that the assay was incapable of providing a clear indication of whether phenol is a mutagen.

Conclusion: Phenol has gene mutagenic activity in mammalian cells but most studies revealed only weak positive results at cytotoxic dose levels. 

GENETIC TOXICITY IN VIVO (Phenol)

Several studies are available investigating the induction of micronuclei in the mouse bone marrow. In the EU-RAR (2006) a more detailed summary was given in Tables 4.27-4.29 (page 122-124) and a general overview on micronucleus tests in Table 4.14 (page 108 of EU-RAR 2006). In the EU-RAR (2006) it is concluded that the results from micronucleus tests were weakly positive or negative and the frequency of micronuclei is low even at near-lethal doses. The authors suggested that the induction of micronuclei by phenol at high doses may be based on an indirect mode-of-action; possible mechanisms for the induction of micronuclei at high doses are given by hypothermia and metabolic overload.
Recent studies which were not available for the documentation in EU-RAR were included in the IUCLID Phenol and evaluated concerning the mode of action: In the micronucleus assay presented by Ciranni et al. (1988) male CD-1 mice received 265 mg/kg bw phenol via gavage or i.p. injection. 18, 24, 42, 48 h after application mice were killed and bone marrow prepared for light microscopical evaluation. The negative control received the vehicle (distilled water) via gavage. After gavage slight increases of micronuclei at 24 h were found which are statistically significant but of questionable toxicological relevance because the value is within published historical vehicle control data of the same strain and application route (no historical data presented of this lab). A bone marrow depression (reduced PCE/NCE ratio) was evident after 18 h and persists even 48 h after treatment. After i.p. administration phenol produced weak genotoxic effects at 18 h post exposure time and which decreased thereafter; bone marrow depression is obvious and constant with time. In conclusion, at a dose level inducing myelotoxic effects weak positive effects were found after i.p. injection but not after gavage.

In a further mouse bone marrow micronucleus test (McFee et al., 1991) male B6C3F1 mice received a single i.p. injection of 0 or 300 mg/kg bw phenol. Bone marrow samples were prepared 26 h after application. Concerning the cytotoxic effects in bone marrow the % PCE (polychromatic erythrocytes) decreased from 50.0+-4.8 (mean +- S.E.) in vehicle control to 19.8 +- 2 4 in treated mice. A 3.3-fold increase was found in the micronucleated PCE per 1000 PCE: 3.3 +-0.2 in control and 11.4 +- 1.9 in treated mice. Conclusion: The i.p. injection of 300 mg/kg bw induced a weak positive effects in the mouse bone marrow micronucleus assay combined with clear myelotoxic effects (decreased PCE/NCE ratio).

In a recent study by Spencer et al. (2007) the dose dependent effects of phenol on body temperature and clinical signs & survival after a single i.p. injection were studied in male and female CD-1 mice for 48 h (0, 50, 100, 150, 200, 300, 400, or 500 mg/kg bw). At >= 400 mg/kg bw mice died within 24 h after application. Clinical signs occurred at >=100 mg/kg bw but survivors appeared normal approximately 1 h after application. However, at 300 mg/kg bw (or above) significant and prolonged hypothermia in male and female mice (up to 7°C decrease) was detected. In the following micronucleus (MN) assay males and females were killed 24 and 48 h after a single i.p. application ( i.p. 0, 30, 100, 300 mg/kg bw) and the incidence of MN in bone marrow was measured. Prolonged hyperthermia was found only in the high dose group as well as a significant increase in micronuclei. No clastogenic effects were reported at lower dose levels. These results suggested a threshold mechanism for the induction of MN by phenol treatment in mice via prolonged physiologic hypothermia. In additional experiments a significant increase in kinetochore-positive MN was observed at 300 mg/kg bw, but the response was considerably less than that the known spindle poison vinblastin indicating that the interruption of the cell spindle apparatus appeared to play only a minor role in MN formation. The relationship between hypothermia and clastogenicity has been demonstrated by Asanami et al. (1998) using chlorpromazine, a drug which was negative in an in vitro chromosome aberration test. Conclusion: Micronucleus formation exhibited a dose threshold which might be correlated with phenol-induced hypothermia.

Spencer et al. (2003) evaluated the effects of thermoregulatory support on the induction of micronuclei in the bone marrow of phenol-treated mice. Doses of phenol that did not induce substantial hypothermia in mice were not associated with an increase frequency of micronuclei ( see above, Spencer et al., 2007). It should be shown in this study that the prevention of hypothermia will inhibit the induction of micronuclei in the bone marrow of phenol-treated mice. Mice housed in standard environment or under thermoregulatory support conditions received a single injection of i.p. 0 or 300 mg/kg bw and bone marrow was prepared 24 or 48 h after application. In unsupported males and females the body temperature (BT) decreased to ca. 31.5°C 24 h after application; BT of 29°C in males and ca. 30.5 in females were measured 48 h after application. Only a slight decrease in BT of ca. 1°C was detected at the same time in male and female mice using thermoregulative support. The mortality rate was increased in supported mice. Thermoregulatory support did not prevent MN induction in phenol-treated mice at 24 h post-dosing but at 48 h post-dosing. BT measurement at 0, 24 and 48 h did not allow full characterization of BT profiles. Therefore, additional experiments were conducted on the effectiveness of thermoregulatory support (BT measured every 5 minutes). Also in mice receiving thermoregulatory support a dose of 300 mg/kg bw resulted in an initial decrease in BT (min. 32.5°C) reaching average BT again after approx. 3 h. Conclusion: Thermoregulatory support prevented the induction of micronuclei measured 48 h after application of phenol. Initial sustained hypothermia in phenol-treated animals was not prevented by thermoregulatory support and likely accounts for the increase in micronuclei at 24 h. However, differences in clastogenic effects after 24 versus 48 h in relation to body temperature need further investigations.

In further mouse bone marrow micronucleus studies (Tables 4.27-4.29 and Table 4.14 in EU-RAR 2006) negative or weak positive results were reported.

The phenol EU RAR 2006 concludes as follows: "Results from in vivo micronucleus tests were weakly positive or negative. The frequency of micronuclei is extremely low even in doses which correspond to the LD50. The induction of micronuclei at high doses may be based on an indirect mode-of-action.

The EU Classification and Labelling Working Group decided in 2001 to classify phenol as a category 3 mutagen. Based on the available evidence, it is considered that this classification still stands and that phenol should still be regarded as a somatic cell mutagen. It is noted that although the high dose positive micronuclei results being secondary to phenol-induced hypothermia is a plausible hypothesis, no definite conclusions about this mechanism can be drawn due to the limited nature of the available data (abstract form and lack of a confirmatory test showing that prevention of hypothermia by maintaining the animals body heat also prevents the induction of micronuclei).

Furthermore, it is deemed that the available in vivo genotoxicity data are unable to address remaining concerns about mutagenicity at the initial site of contact following inhalation or dermal exposure."

BPA

The 2003 EU RAR concluded that when all of the availalable genotoxicity data are considered, and in the absence of significant tumour findings in animal carcinogenicity studies, it does not appear that BPA has significant mutagenic potential in vivo. Any aneugenic potential of BPA seems to be limited to in vitro test systems and is not of concern. The relevance of the finding that BPA can produce rat hepatic DNA adduct spots in a post-labeling assay is not entirely clear. Given the absence of positive results for gene mutation and clastogenicity in cultured mammalian cell tests, it seems unlikely that these are of concern for human health. No human data regarding mutagenicity are available.

The 2008 updated EU RAR concluded that new data from a study indicating effects of BPA on meiosis in female mice cannot be taken as conclusive evidence of an effect of BPA on germ cell meiosis because of the several methodological weaknesses and flaws identified in the study, the reporting inadequacies, and the known mutagenicity and toxicity profile of BPA. In addition, these findings have not been confirmed in more recent publications. Thus, the original conclusion that BPA has no significant mutagenic potential in vivo is still valid.

Additional recent information concerning the observations reported by the Hunt group on congression failure: Two in vivo studies(Hunt et al. (2003) and Susiarjo et al., (2007)) evaluated during the 2008 EU RAR reported that short-term oral exposure to low doses of Bisphenol A (≥ 0.020 mg/kg bw/day) in peripubertal or pregnant mice can interfere with meiotic divisions in development of female germ cells (“egg” or “oocyte”). An increase in hyperploid (aneuploid) metaphase II oocytes was observed following treatment with 0.020 mg/kg bw/day. There was not a significant increase in aneuploid embryos. 

Two subsequent in vivo studies(Pacchierotti et al.(2008), Eichenlaub-Ritter et al. (2008)) attempted to replicate these findings. Consistent with the previous findings, they detected no significant effects of Bisphenol A exposure on the frequency of aneuploidy in “zygotes” (fertilised oocytes) produced from female mice treated before puberty or as adults with a similar range of doses. In addition, Eichenlaub-Ritter et al. (2008) found no effects of Bisphenol A exposure on aneuploid oocytes and Pacchierottiet al. (2008) found no increase in aneuploid or diploid sperm following exposure of male mice to Bisphenol A. The authors concluded that the aneuploidy predicted by the Hunt group could not be confirmed. 

In addition in a recent study published by the Hunt group, Mulhauser et al. (2009), the authors could not replicate their initial findings on “congression failure” but report effects on chromosome alignment and/or spindle formation. The authors state “After publishing our findings [Hunt et al., 2003], we initiated studies to assess the effect of long term BPA exposure on the growing follicle. To our surprise, levels of BPA that were sufficient to elicit an effect on meiotic chromosome dynamics during the previous two years of study suddenly produced little or no effect. In an analysis of possible changes in experimental protocol, the only change identified was the lot of animal feed.” The authors report frequencies of abnormal oocytes in the absence and presence of BPA in two different diets (casein based and soy based). The reported frequencies of abnormal oocytes of the BPA/casein group are lower than the background value reported in the soy-based diet. 

Overall, the initial observations reported by the Hunt laboratory were not reproduced in the same laboratory or in other independent laboratories. Therefore, the conclusion from the 2003 EU RAR and the 2008 EU RAR Update is still valid; Bisphenol A has no significant mutagenic potential in vivo.


Short description of key information:
The reaction mass of phenol and 4,4'-isopropylidenediphenol (BPA) contains phenol and BPA. The mamalian toxicity of this product is expected to be affected most by the presence of phenol.

In vitro test systems
Phenol has no mutagenic properties in bacterial gene mutation tests. There is evidence for gene and chromosome mutagenic effects in mammalian cells, mainly in the presence of MA. However, concerning gene mutation assays most studies resulted only in weak positive results at cytotoxic dose levels.

In vivo test systems
In studies investigating the systemic chromosome mutagenic activity of phenol after oral or parenteral administration weak positive or negative results were reported.
The weak positive results in micronucleus tests were found at dose levels inducing severe signs of intoxication. In the EU-RAR (2006) it was suggested that these weak clastogenic effects " may be based on an indirect mode-of-action"; possible mechanisms for the induction of micronuclei at high doses are given by hypothermia and metabolic overload. In a recently published study (Spencer et al., 2007) a significant increase in micronuclei was found only in the high dose groups with prolonged hypothermia. No clastogenic effects were reported at lower dose levels. Note that carcinogenicity studies with phenol in mice and rats were negative.

BPA is not of concern regarding genotoxicity. Due to the content in phenol, the reaction mass will be classified as Mutagen Cat 3.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

The major constituent phenol carries an official classification of Muta Cat 3 according to DSD and Muta Cat 2 according to CLP. The same classification will be assigned to the reaction mass.