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GENETIC TOXICITY IN VITRO

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). All studies are also presented in IUCLID Section 7.6.1.

 

Bacterial test systems detecting gene mutations

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, see robust study summary in IUCLID Section 7.6.1). 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 (see robust study summary in IUCLID Section 7.6.1). 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

An overview on chromosomal mutations in mammalian cells 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; see robust study summary in IUCLID Section 7.6.1) 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 mutagenic in the chromosome aberration assay in CHO cells with metabolic activation (Ivett et al., 1989; see robust study summary in IUCLID Section 7.6.1).

In further in vitro micronucleus tests (see also Table 4.20 in EU-RAR 2006) only weak positive results were obtained without S-9 mix (Glatt et al., 1989; Yager et al., 1990).

Tsutsui et al. (1997) reported negative results in the chromosome aberration assay using SHE (Syrian hamster embryo) cells without metabolic activation; however, cells were not treated up to cytotoxicity threshold (max. concentration 9.4 µg/ml; see also Table 4.19 in EU-RAR 2006).

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

 

Gene mutations in mammalian cells

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; see robust study summary in IUCLID Section 7.6.1) 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 further HPRT gene mutation studies in mammalian cells weak positive (Paschin & Bahitova, 1982) or negative results (Glatt et al., 1989) were reported, in both studies dose levels up to the cytotoxicity threshold were used (see Table 4.17 in EU-RAR 2006).

In the mouse lymphoma assay (Wangenheim & Bolcsfoldi, 1988; see robust study summary in IUCLID Section 7.6.1) 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. (see robust study summary in IUCLID Section 7.6.1).

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

 

Aneuploidy in mammalian cells

No induction of aneuploidy was detected in SHE cells exposed to dose levels up to 9.3 µg/ml without MA. However, the threshold for cytotoxicity was not reached (Tsutsui et al., 1997).

 

Assays indicating DNA damage

These studies are summarized in Table 4.22-4.26 (EU-RAR 2006, page 117 ff). In EU-RAR (2006) all test systems were considered to give positive results.

In the SCE assay (sister chromatid exchange) reported by Khalil & Odeh (1994; see detailed documentation in IUCLID Section 7.6.1) primary cultures of rat bone marrow cells were incubated with 0, 0.094, 0.94, 9.4, or 94 µg/ml without MA. A statistically significant increase in the SCE frequency was detected at 9.4 µg/ml in experiment 1 & 2. A doubling of the mean SCE rate was found only at 94 µg/ml in experiment 1 & 2, a concentration resulting also in a decrease of the mitotic index to 35 or 27% of control value in experiment 1 & 2, respectively. In summary, phenol resulted in an increase in the SCE rate at cytotoxic dose levels.

Further SCE assays were published (see Table 4.22 and 4.23 in EU-RAR 2006). In human lymphocytes (Morimoto et al., 1983) and CHO cells (Ivett at al., 1989) weakly positive effects were reported with MA. Without MA negative results (Glatt et al., 1989; Jansson et al., 1986), marginal/weakly positive results (Morimoto & Wolff, 1980) as well as positive results (Erexson et al., 1985; Tsutsui et al., 1997; Ivett et al., 1989) were obtained in different mammalian cell lines.

Tsutsui et al. (1997; Table 4.24 in EU-RAR) measured the UDS induction in SHE cells (only without MA); genotoxic effects were observed from 0.09μg/ml upwards but no data on toxicity were given.

A positive result in a test for induction of DNA strand breaks in mouse lymphoma cells (L5178Y) with MA at 140μg/ml and 470 μg/ml was reported by Garberg et al. (1988); without MA doses up to 470μg/ml were negative (Garberg et al., 1988; Pellack-Walker & Blumer 1986; see also Table 4.25 in EU-RAR). No toxic effects were observed with and without MA.

A test on formation of DNA adducts in HL60 cells was described by Kolachana et al. (1993; Table 4.26 in EU-RAR). At the only tested dose of 9.4 μg/ml DNA adducts were detected without MA; no data on toxicity were given.

Conclusion: Indicator tests on phenol suggested genotoxic activity.

 

GENETIC TOXICITY IN VIVO

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). However, all these studies are also presented in IUCLID Section 7.6.2.

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 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. The tested concentrations of 265 and 300 mg/kg bw are close to the oral LD50 in mice.
Recent studies which were not available for the documentation in EU-RAR were included in the IUCLID Phenol (robust study summaries were presented) and evaluated concerning the mode of action.

 

In the micronucleus assay presented by Ciranni et al. (1988; see robust study summary in IUCLID Section 7.6.2) 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; see robust study summary in IUCLID Section 7.6.2) 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; robust study summary in IUCLID Section 7.6.2) 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; limited documentation; see detailed documentation in Section 7.6.2) 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. Gad-El-Karim et al. did not detect in two independent studies (1985 & 1986) clastogenic effects in male Swiss CD-1 mice 30 h after p.o. 250 mg/kg bw. Weak positive results were demonstrated in pregnant Swiss CD-1 mice 24 h after administration of 265 mg/kg bw via gavage (Ciranni et al., 1988). The slight increase in micronuclei reported by Marrazzini et al. (1994) in Swiss CD-1 mice 18 h after i.p. injection of 120 mg/kg bw has no toxicological relevance comparing the data with historical controls (MN vehicle control range in CD-1 mice: 0.9-3.1 o%; Krishna et al., Mutat Res. 453:45-50). In NMRI mice negative results were obtained after 2 i.p. injections of up to 188 mg/kg bw (24 h interval; Wild et al., 1980). In B6C3F1 mice three i.p. injection of up to 180 mg/kg bw (24 h interval) induced only a slight (but significant) increase in micronuclei (Shelby et al., 1993). Weak positive results were also reported by Chen and Eastmond (1995) in male Swiss CD-1 mice after three i.p. injections at 24 h intervals of 160 mg/kg bw.

No mutagenic activity was found in the rat bone marrow chromosome aberration test (Thompson &Gibson, 1984; see Table 4.30 in EU-RAR 2006, page 125) 20 h after p.o. 450-510 mg/kg bw (male Sprague-Dawleys) or 300 - 410 mg/kg bw (females) and 20 h after i.p. injection of up to 180 mg/kg bw in males and 110 mg/kg bw in females. However, the validity of the study has limitations (3 rats per group and only 30 mitoses per rat scored).

No increase in DNA strand breaks in cells of the testis were detected in Sprague-Dawley rats 2-24 h after a single i.p. injection of 8 -79 mg/kg bw or after 5 i.p. applications of 4-40 mg/kg bw (Skare & Schrotel, 1984; Table 4.31 in EU-RAR 2006, page 125).

Woodruff et al. (1985; Table 4.32 in EU-RAR 2006, page 126) reported negative results in the sex-linked recessive lethal assay (SLRL) in Drosophila at toxic dose levels in feeding studies and after injection of the test substance.

Using the P32-postlabeling method no DNA adducts were detected in bone marrow, zymbal gland, and liver of female Sprague-Dawley rats after 4 oral applications via gavage (24 h interval) of 75 mg/kg bw/day ( Reddy et al., 1990). No oxidative DNA damage in bone marrow cells of male B6C3F1 mice were observed 1 h after a single i.p. injection of 75 mg/kg bw (Kolachana et al., 1993; both studies in Table 4.33 in EU-RAR 2006, page 126).


Short description of key information:
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. Also in chromosome mutation assays positive results were obtained mainly at cytotoxic concentrations. A test for induction of aneuploidy was negative. Evidence for sister chromatid exchange was given in mammalian cells at cytotoxic dose levels. Further indicator tests suggested genotoxic effects of phenol.
Tests with negative outcome:
bacterial gene mutation, induction of aneuploidy in mammalian cells
Tests with positive outcome:
chromosome aberration and gene mutation in mammalian cells, DNA damage in mammalian cells

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. Furthermore in indicator test for systemic genotoxicity no DNA strand breaks were detected in rats and no DNA adduct formation was found in rats and mice. The SLRL assay in Drosophila revealed also negative results.
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. These results suggested a possible threshold mechanism above 100 mg/kg bw/d for the induction of micronuclei by phenol treatment in mice via prolonged hypothermia.

Endpoint Conclusion:

Justification for classification or non-classification

According to the available data phenol is currently classified as Muta 2, but the results from in vivo test systems suggested a possible threshold mechanism above 100 mg/kg bw/d for the induction of micronuclei via prolonged hypothermia.