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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is negative in assays for mutation in Salmonella and mammalian cells (references 7.6.1-1 and 7.6.1-2), for DNA adducts and DNA strand breaks. In vitro chromosome aberrations are observed at cytotoxic concentrations. At high doses, a weak induction of micronuclei in mouse bone marrow increase with chronic treatment. A mechanistic study on chromosome aberrations 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The detailed protocol and these data are presented in Mortelmans, K.; Haworth, S.; Lawlor, T.; Speck, W.; Tainer, B.; Zeiger, E., Salmonella mutagenicity tests. II. Results from the testing of 270 chemicals. Environ. Mutagen. 1986, 8 (Suppl 7) 1-119.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al., 1983]. The S-9 mixes were prepared immediately prior to use and contained either 10% or 30% S-9; occasionally, 5% S-9 was also used. All chemicals were tested in the absence of metabolic activation, and with rat and hamster S-9 fractions.
Test concentrations with justification for top dose:
0, 3.3, 10, 33, 100, 333, and 1000 µg/plate (Laboratory Case Western Reserve University)
0, 3.3, 10, 33, 100, and 220 µg/plate (Laboratory EG&G Mason Research Institute)
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
Salmonella typhimurium strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and stored as recommended [Maron and Ames,1981]. Cultures were grown at 37°C overnight, with shaking, in Oxoid #2 broth, or in defined minimal medium supplemented with biotin (0.8 µg/mL) and histidine (40 µgiml). The phenotypes of the strains were analyzed at the time of their use in mutagenicity assays.
The preincubation procedure was performed [Haworth et al., 1983], with some differences, as described below.
The test chemical (0.05 mL), overnight culture of Salmonella (0.10 mL), and S-9 mix or buffer (0.50 mL), were incubated at 37°C, without shaking, for 20 min. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium [Vogel and Bonner, 1956]. Histidine-independent (his+) colonies arising on these plates were counted following two days incubation at 37°C. Plates were machine counted (New Brunswick; Artek) unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar. At the discretion of the investigators, plates with low numbers of colonies, containing precipitated test chemical, or having excessivelyreduced contrast because of chemical color, were counted by hand.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: no data
Evaluation criteria:
1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

The initial determination of mutagenic, nonmutagenic, or equivocal was made by the testing laboratory; the final determination was made by the project officer (E.Z.). The chemicals were decoded by the chemical repository (Radian Corporation) only after the mutagenicity or nonmutagenicity of the chemicals had been determined.
Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>220 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>220 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>220 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True 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:
cytotoxicity
Remarks:
>220 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid

Laboratory Case Western Reserve University

Dose
[µg/plate]
TA 100
NA 10 % HLI 10 % RLI
0 87±0.9 113±12.8 128±3.5
3.3 82±1.2 96±5.6
10 88±4.3 102±10.1 107±2.7
33 82±7.8 89±6.7 128±2.9
100 70±7.3 108±10.7 118±6.2
333 t 117±9.3 126±4.0
1000 103±15.4
 
Control 1641±38.6 2007±217.0 1586±45.0




Dose
[µg/plate]
TA 1535
NA 10 % HLI 10 % RLI
0 5±1.8 13±0.3 11±1.2
3.3 6±1.7
10 7±1.5 12±3.5 11±1.9
33 5±0.7 10±1.7 7±1.0
100 4±1.5 9±2.4 11±2.1
333 t 11±2.0 10±2.1
1000 12±3.3 16±2.3
 
Control 1081±144.9 133±6.8 287±19.9



Dose
[µg/plate]
TA 1537
NA 10 % HLI 10 % RLI
0 9±2.6 11±1.5 15±1.0
3.3

10 9±1.8 9±1.5 11±0.6
33 9±0.9 12±0.9 11±1.2
100 10±1.2 9±0.3 13±1.8
333 7±0.9 9±2.1 13±1.8
1000 t t t
 
Control 113±39.5 194±5.9 158±41.7



Dose
[µg/plate]
TA 98
NA 10 % HLI 10 % RLI
0 25±2.2 30±4.3 31±5.8
3.3

10 21±2.9 30±4.2 30±1.5
33 21±3.5 27±2.0 31±2.3
100 20±4.1 31±0.9 22±3.5
333 17±0.9 23±4.0 19±2.8
1000 t t t
 
Control 92±4.0 966±33.4 681±66.8



Laboratory EG&G Mason Research Institute

Dose
[µg/plate]
TA 100
NA 10 % HLI 10 % RLI
0 176±6.2 197±12.8 169±6.8
3.3 170±4.5 196±5.6 173±11.8
10 180±4.7 211±10.1 166±14.7
33 163±3.4 198±6.7 177±1.5
100 184±79.0 204±10.7 191±7.2
220 132±2.7 s 226±9.3 s 168±6.9 s
 
Control 1072±48.0 691±21.4 617±8.8




Dose
[µg/plate]
TA 1535
NA 10 % HLI 10 % RLI
0 28±7.0 11±1.2 14±0.6
3.3 26±1.9 12±0.0
12±2.3
10 19±3.3 12±1.9 11±0.7
33 21±3.7 13±4.2 17±1.8
100 25±2.6 14±0.6 12±1.2
220 t 14±0.6 s 15±4.7 s
 
Control 840±53.4 68±1.5 59±0.9



Dose
[µg/plate]
TA 1537
NA 10 % HLI 10 % RLI
0 7±1.2 9±2.0 9±2.1
3.3 6±2.3
9±2.2
8±0.3
10 7±0.7 7±1.2 10±1.0
33 5±2.0 11±1.2 7±0.6
100 11±1.5 9±0.3 11±1.8
200 4±1.0 s 10±1.8 s 5±0.3 s
 
Control 188±17.2 54±2.5 42±2.5



Dose
[µg/plate]
TA 98
NA 10 % HLI 10 % RLI
0 19±2.7 23±4.0 22±2.3
3.3 18±2.3
23±1.0
28±3.1
10 12±2.0 32±2.1 25±2.6
33 14±3.8 25±2.0 25±0.6
100 11±0.9 25±1.2 20±0.3
220 10±2.9 s 23±5.0 s 25±2.6 s
 
Control 1335±27.2 966±33.4 402±49.7

Abbreviations
NA, not activated
HLI, Aroclor 1254-induced hamster liver S-9
RLI, Aroclor 1254-induced rat liver S-9
s, slight clearing of background lawn
t, complete clearing of background lawn (colonies not counted)
p, precipitate present in plates
x, precipitate present with toxicity
+, mutagenic
+W, weakly mutagenic
?, questionable response

- , nonmutagenic.

Conclusions:
With and without addition of S9 mix as the external metabolizing system, Phenolphthalein was not mutagenic under the experimental conditions described.
Executive summary:

The purpose of this assay was to provide information on the genotoxic potential of the test item. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains Salmonella strains TA 98, TA 100, TA1535, and TA1537. Studies were performed in two different laboratories. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254 -pretreated rats and hamster was used. Two independent experimental series were performed without and with 10 % S9 mix, respectively. The test item was dissolved in DMSO and tested at concentrations up to 1000 µg/plate. Toxicity to the bacteria was observed at concentrations larger than 220 µg/plate with and without S9 mix.

Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test item showed no increase in the number of revertants of any bacterial strain. These published data clearly indicate that the test item was not mutagenic under the described experimental conditions. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
A supernatant fraction of homogenised liver centrifuged at 9000 g/10 min (S9) was prepared from Sprague-Dawley rats pretreated with Aroclor 1254 (Ames et al., 1975). A single batch of homogenized liver S9 fraction was employed in this study. Each day fresh S9 mix was prepared consisting of the following ingredients: MgCl2 (0.4 M), 20 µL, KCl (1.65 M) 20 µL, Glucose-6-phosphate (0.5 M) 5 µL, NADP (0.05 M) 40 µL, phosphate buffer (0.2 M, pH 7.4) 815 µL, S9 fraction 100 µL.This mix was stored on ice until required, then incorporated together with the test material and each of the bacteria strains as follows: 100 µL bacterial broth culture, 100 µL test compound solution in DMSO at various concentrations, and 500 µL S9 mix as required were added to each 2 mL of top agar at 42 °C
Test concentrations with justification for top dose:
0, 32, 100, 320, and 1000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Aflatoxin B1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
plate incorporation test; no further data available

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Plates were incubated at 37 °C for 72 h before counting

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: no data available
Evaluation criteria:
(a) dose dependent response
(b) reproducibility of the result
Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid


Dose
[µg/plate]
TA98 TA100 TA1535 TA1537 TA1538
NA 20 %
RLI
NA 20 %
RLI
NA 20 %
RLI
NA 20 %
RLI
NA 20 %
RLI
  - - - - - - - - - -
1000 t 44 t 105 t 14 t 7 t 13
320 32 47 154 134 28 21 8 16 23 31
100 30 46 139 124 39 24 9 12 20 28
32 31 47 145 126 35 22 10 17 17 26
0 29 40 146 116 36 22 10 17 21 33

Abbreviations
NA, not activated
RLI, Aroclor 1254-induced rat liver S-9
t, indicates toxicity
- , nonmutagenic.

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.
Executive summary:

The purpose of this assay was to provide information on the genotoxic potential of the test item. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1538, TA 1535 and TA 1537. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254 -pretreated rats was used. Two independent experimental series were performed with and without S9 mix, respectively. The test item was dissolved in DMSO and tested at concentrations ranging from 32 to 1000 µg/plate. Toxicity to the bacteria was observed at 100 µg/plate without S9 mix. Daunomycin, 9 -aminoacridine, aflatoxin B1, 2 acetylaminofluorene, 4 -N-nitroquinoline-N-oxide, and methylmethansulfonate served as strain specific positive control test materials. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test item showed no increase in the number of revertants of any bacterial strain. These published data clearly indicate that the test item was not mutagenic under the described experimental conditions.

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Discussion

The genotoxicty of the test substance 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. 

The test substance 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]. The test item 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]. The test substance 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 test item-exposed Chinese hamster ovary cells in the presence but not the absence of S9 activation was reported [5, 6]. The test item 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 the test item in SHE cells exposed for 48 hours [4]. In vivo, the test substance 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, the test item 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 the test item 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, the test substance is negative in assays for mutation in Salmonella and mammalian cells, for DNA adducts and DNA strand breaks. In vitro chromosome aberrations 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 aberrations 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 the test substance possess no direct mutagenic potential both in vitro and in vivo. However, evidence exists that the test item 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.   

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The substance is harmonized classified for mutagenicity Cat.2 (H341) according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation).