Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Discussion

The genotoxicty of Phenolphthalein was studied in numerous assays in vitro and in vivo and the results are available in published literature. Summaries of those data have been generated by NTP, IARC and other expert committees. Below a short overview of the data is given that provides key information.

Phenolphthalein was not mutagenic in several assays in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence or absence of exogenous metabolic activation[1, 2]. It did not induce DNA damage in DNA repair-deficient strains of Bacillus subtilis [3].

Phenolphthalein induced a positive increase in hprt but not Na+/K+ ATPase gene mutations or aneuploidy in Syrian hamster embryo (SHE) cells exposed for 48 hours [4].

Phenolphthalein did not induce sister chromatid exchange in Chinese hamster ovary cells in the presence or absence of exogenous metabolic activation but a highly significant reproducible increase in chromosomal aberrations in phenolphthalein-exposed Chinese hamster ovary cells in the presence but not the absence of S9 activation was reported [5, 6]. Phenolphthalein also induced a positive increase in chromosomal aberrations but not aneuploidy in SHE cells exposed for 6 (aberrations) or 48 (aneuploidy) hours [4]. Similarly, a dose-dependent increase in morphological transformation was induced by phenolphthalein in SHE cells exposed for 48 hours [4].

In vivo, phenolphthalein induced an equivocal increase in DNA single-strand breaks in the peripheral blood leukocytes of female p53-deficient transgenic mice (C57B1/6 background) following dosed-feed exposure for 26 weeks and sampling at days 39, 92, 137, and 183 [7]. No increase was observed in the livers of treated mice sampled upon termination of exposure.

Micronuclei were induced by phenolphthalein in male and female B6C3F1 mouse peripheral blood polychromatic (PCE) and normochromatic (NCE) erythrocytes via a variety of exposures including gavage for 2 to 3 days, or dosed feed for 4 to 14 days.
Similarly, phenolphthalein induced micronuclei in the blood PCE of CD-1 mice after 14 weeks of continuous feed treatment. Micronuclei were also induced in the bone marrow of male B6C3F1 mice treated by gavage for 3 days or feed for 4 to 14 days. However, it should be noted that the doses applied in those studies were in general significantly higher than those to which humans would be exposed. No significant increases in micronuclei were observed in mouse bone marrow after only 2 days of gavage treatment or 3 days of feed treatment [6].

A significant increase in micronucleated erythrocytes was observed in a study on the effects of phenolphthalein at various concentrations in the diet of transgenic female mice heterozygous for the p53 gene, over a six-month period. Most of the micronuclei arise from whole chromosomes rather than chromosomal damage as shown by kinetochore analysis. The lowest effective dose (LED) for the induction of micronucleated erythrocytes was 200 ppm. Inconclusive evidence was found for DNA damage in blood leukocytes, and there was no evidence for DNA damage, apoptosis or necrosis in liver parenchymal cells [7].

Overall, Phenolphthalein is negative in assays for mutation in Salmonella and mammalian cells, for DNA adducts and DNA strand breaks. In vitro chromosome abberations are observed at cytotoxic concentrations. At high doses, a weak induction of micronuclei in mouse bone marrow increases with chronic treatment.
A mechanistic study on chromosome abberations in vitro strongly suggests that chromosome damage occurs by indirect mechanisms above a threshold concentration [8]. Such indirect mechanisms may well contribute to the reported cytogenetic effects in vivo at high dose above a threshold concentration.

The weight of evidence demonstrates that Phenolphthalein possess no direct mutagenic potential both in vitro and in vivo. However, evidence exists that Phenophthalein's generates free radicals responsible for the indirect mutagenic potential observed.


References

1.    Bonin, A.M., J.B. Farquharson, and R.S.U. Baker, Mutagenicity of arylmethane dyes in salmonella. Mutation Research/Genetic Toxicology, 1981. 89(1): p. 21-34.
2.    Mortelmans, K., et al., Salmonella mutagenicity tests. II. Results from the testing of 270 chemicals. Environ. Mutagen., 1986. 8 (Suppl 7) p. 1-119.
3.    Kada, T., K. Tutikawa, and Y. Sadaie, In vitro and host-mediated "rec-assay" procedures for screening chemical mutagens; and phloxine, a mutagenic red dye detected. Mutation Research, 1972. 16(2): p. 165-74.
4.    Tsutsui, T., et al., Cell-transforming activity and genotoxicity of phenolphthalein in cultured Syrian hamster embryo cells. International Journal of Cancer, 1997. 73(5): p. 697-701.
5.    National Toxicology Program (NTP), Toxicology and Carcinogenesis Studies of Phenolphthalein (CAS no. 77-09-8) in F344/N Rats and B6C3F1 Mice (Feed Studies) - Technical Report 465, in Technical Report 465. 1996.
6.    Witt, K.L., et al., Phenolphthalein: induction of micronucleated erythrocytes in mice. Mutation Research/Genetic Toxicology, 1995. 341(3): p. 151-160.
7.    Tice, R.R., et al., Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene. Environmental & Molecular Mutagenesis, 1998. 31(2): p. 113-24.
8.    Armstrong, M.J., et al., Induction of chromosome aberrations in vitro by phenolphthalein: mechanistic studies. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2000. 457(1-2): p. 15-30.




Short description of key information:
Phenolphthalein is negative in assays for mutation in Salmonella and mammalian cells, for DNA adducts and DNA strand breaks. In vitro chromosome abberations are observed at cytotoxic concentrations. At high doses, a weak induction of micronuclei in mouse bone marrow increaes with chronic treatment. A mechanistic study on chromosome abberations in vitro suggests that chromosome damage occurs by indirect mechanisms above a threshold concentration. Such indirect mechanisms may well contribute to the reported in vivo cytogenetic effects in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Provided key and supporting studies with Phenolphthalein did show genotoxic potential in vitro and in vivo at high concentration via indirect mechanisms. Therefore it can be concluded that the substance is mutagenic in somatic cells in vivo thus justifying the current legal classification for mutagenicity Cat 2 according to REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006.