Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CA: negative

CA: negative

UDS: negative

Transformation assay: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Qualifier:
according to guideline
Guideline:
other: Methods described in Tsutsui et al. (1991), AATEX 1 and Tsutsui et al. (1997), Mutat. Res.
Principles of method if other than guideline:
Chemicals were tested on Syrian Hamster Embryo (SHE) cells. Cytotoxicity was determined by measuring colony-forming efficiency. For determination of chromosomal aberrations, cells were treated with substance for 24 hours at varying concentrations. 100 Metaphases per experimental group were scored with regard to chromatid gaps, isochromatid gaps, chromatid breaks, isochromatid breaks, exchanges, ring chromosomes, dicentric chromosomes, and fragmentations. Chemicals not showing clastogenic activity were tested additionally in the presence of metabolic activation.
GLP compliance:
not specified
Type of assay:
other: chromosomal aberration test
Species / strain / cell type:
primary culture, other: syrian hamster embryo cells
Details on mammalian cell type (if applicable):
Cells were grown as described in Tsutsui et al. (1991), AATEX and Tsutsui et al. (1997), Mutat. Res.
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
rat liver post-mitochondrial supernatant
Test concentrations with justification for top dose:
Test concentrations were 30, 100, 300, and 1000 uM. High dose was selected based on decreased colony forming efficiency suggesting overt cytotoxicity at doses greater than 1000 uM.
Vehicle / solvent:
Substance was diluted with Ca(2+)- and Mg(2+)-free phosphate-buffered slaine (pH 7.4) at 100 mM.
Untreated negative controls:
yes
Positive controls:
not specified
Remarks:
no specific postive control substance was mentioned, but panel of 14 substances was tested with substantial positive reactions of several test substances, showing the sensitivity of the test.
Details on test system and experimental conditions:
SHE cells (500000) in tertiary culture were plated overnight and then treated for 24 hours with substance. In case of low cytotoxicity, test substance concentrations were increased up to maximum solubility or 10 mM. After treatment, the cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid was administered at 0.2 ug/mL and metaphase chromosomes were prepared. For determination of structural chromosome aberrations, 100 metaphases were scored per experimental group. The aberrations scored were chromatid gaps, isochromatid gaps, chromatid breaks, isochromatid breaks, exchanges, ring chromosomes, dicentric chromosomes, and fragmentations. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks. 100 metaphases were also scored for examining the induction of polyploidy and endoreduplication.
Substances with a negative response in the chromosome aberrations assay were also examined for their clastogenic activity in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant. Cells were plated as before and after overnight incubation they were treated for 3 hours with chemical agents at varying concentrations in a 5% PMS mixture. Following treatment, the cells were washed twice with PBS and incubated with fresh medium for 21 hours followed by chromosome preparations.
Evaluation criteria:
see above.
Statistics:
Statistical analysis was performed by x2-test to assess the significance of the difference in the incidences of chromosome aberrations between control cultures and cultures treated with chemical agents. The level of significance in the statistical analysis was determined at p<0.05.
Key result
Species / strain:
primary culture, other: Syrian Hamster Embryo Cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
colony-forming efficiency decreased to 59% at 1000 uM in the test without metabolic activation.
Untreated negative controls validity:
valid
Additional information on results:
Increased frequency of polyploidy and endoreduplication in testing with metabolic activation, but no increases in frequency of aberrant metaphases.

Results for p-phenolsulfonic acid (4 -hydroxybenzenesulfonic acid):

metabolic activation

Concentration

Relative colony-forming efficiency (%)

metaphases scored (#)

Mitotic index (%)

Aberrant metaphases (%)

Polyploidy and endoreduplication (%)

without

0

100

100

10.5

0

1.0

 

30

93

100

11.6

2.0

4.0

 

100

91

100

9.8

0

7.0

 

300

63

100

8.7

1.3

7.0

 

1000

59

100

4.8

2.0

5.0

with

0

100

100

11.1

2.0

2.0

 

30

ND

100

ND

0

2.0

 

100

96

100

12.4

1.0

7.0

 

300

94

100

14.7

2.0

ND

 

1000

112

100

11.0

0

11.0*

ND: not done

*: Significantly different from control (P<0.05; x2 -test)

Executive summary:

4 -hydroxybenzenesulfonic acid did not induce increased frequencies of chromosome aberrations, neither in the absence nor in the presence of metabolic activation. In the presence of metabolic activation, increased frequencies of polyploidy and endoreduplication were observed, however the dose/response relationship as well as the biological significance of this observation cannot be assessed based on the data at hand. Therefore, 4 -hydroxybenzenesulfonic acid was not clastogenic under the test conditions employed.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006
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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No information on controls provided.
Principles of method if other than guideline:
No precise information on positive, negative, vehicle controls given.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Syrian hamster embryo cells
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital induced rat liver post-mitochondrial supernatants.
Test concentrations with justification for top dose:
0; 30; 100; 300; 1000 µM
Key result
Species / strain:
other: Syrian hamster embryo cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Incubation without metabolic activation system:

Type of structural aberrations (%)
name concentration [µM] relative colony-forming efficiency (%) mitotic index (%) number of metaphases scored Chromatid gaps Isochromatic gaps Chromatic breaks Isochromatic breaks Exchanges Ring chromosomes Dicentric chromosomes Fragmentations Aberrant metaphases (%) Polyploidy and endoreduplication (%)
p-Phenolsulfonic acid 0 100 10.5 100 0 0 0 0 0 0 0 0 0 1
30 93 11.6 100 2 0 0 0 0 0 0 0 2 4
100 91 9.8 100 0 0 0 0 0 0 0 0 0 7
300 63 8.7 78 1.3 0 0 0 0 0 0 0 1.3 7
1000 59 4.8 100 2 0 0 0 0 0 0 0 2 5

Incubation with metabolic activation system:

Type of structural aberrations (%)
name concentration [µM] relative colony-forming efficiency (%) mitotic index (%) number of metaphases scored Chromatid gaps Isochromatic gaps Chromatic breaks Isochromatic breaks Exchanges Ring chromosomes Dicentric chromosomes Fragmentations Aberrant metaphases (%) Polyploidy and endoreduplication (%)
p-Phenolsulfonic acid 0 100 11.5 100 2 0 0 0 0 0 0 0 2 2
30 ND ND 100 0 0 0 0 0 0 0 0 0 2
100 96 12.4 100 0 0 1 0 1 0 0 0 1 7
300 94 14.7 100 1 0 0 0 1 0 0 0 2 ND
1000 112 11 100 0 0 0 0 0 0 0 0 0 11
Conclusions:
Interpretation of results (migrated information):
negative

The results lead to the conclusion that the test compound is not mutagenic in the chromosome aberration test system in vitro with cells of the Syrian hamster embryo cell line.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)
Deviations:
yes
Remarks:
historical controls are lacking, detailed reporting is not available
Qualifier:
according to guideline
Guideline:
other: Method described in Tsutsui et al. (1984)
GLP compliance:
not specified
Type of assay:
other: unscheduled DNA synthesis assay
Specific details on test material used for the study:
Purity: unknown
Species / strain / cell type:
primary culture, other: syrian hamster embryonic cells
Details on mammalian cell type (if applicable):
isolated from 13-day gestation fetuses and grown as described in Tsutsui et al. (1983)
Metabolic activation:
with and without
Metabolic activation system:
5% rat-postmitochondrial supernatant and other cofactors as described in Tsutsui et al. (1984)
Test concentrations with justification for top dose:
1, 3, 10 uM with no justification for top dose given
Vehicle / solvent:
cell culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
100000 cells were plated in quadruplicate on 15-mm-diameter glass coverslips in 16-mm tissue culture cluster dishes in complete medium. After overnight incubation, complete medium was replaced with medium containing 1% fetal bovine serum (FBS), and then the cultures were incubated for 48h. The cells were treated with the indicated concentrations of the agents for 1h. When exogenous metabolic activation was used, the cultures were treated for 1h in a reaction mixture consisting of 5% rat-postmitochondrial supernatent and other cofactors prepared by the method as described in Tsutsui et al (1984). To measure UDS, the cells were washed after treatment with 1 mL of PBS and 1 mL of FBS medium containing 10mM hydroxyurea. Following addition of [3H]thymidine in 1% urea medium to the cultures, the cells were incubated for 6h. After washing 3 times with cold trichloroacetic acid (5%), the coverslips were put into scintillation vials to determine the readioactivity in the samples. All experiments were repeated at least twice with similar results. The number of cells on a coverslip was counted using the culture cluster dishes treated with the same conditions used in the experiments for detection of UDS with [3H]thymidine. No significant difference in the number of cells was found between the control and experimental groups.
Statistics:
t-test
Key result
Species / strain:
primary culture, other: syrian hamster embry cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
not examined

concentration

Relative cell survival (%)

Induced UDS (cpm) in the absence or presence of exogenous metabolic activation

 

 

Absence

Presence

1

95.4

0

0

3

101.3

0

0

10

100.5

0

0

Executive summary:

The ability of p-phenolsulfonic acid (4 -hydroxybenzenesulfonic acid) to induce unscheduled DNA synthesis was tested in primary syrian hamster embryonic cells. Under the conditions tested, the substance was not cytotoxic and did not induce unscheduled DNA synthesis.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data available for 4-hydroxybenzenesulfonic acid (p-phenolsulfonic acid) are publications addressing chromosomal aberration (CA), sister chromatid exchange (SCE), cell transformation, and unscheduled DNA synthesis (UDS) in Syrian hamster embryo (SHE) cells. These publications are of acceptable quality, however the interpretation of the data for SCE is difficult since no historical control data are available. The methodology used for the cell transformation assay, especially the scoring criteria were not provided. Therefore, these two studies were assigned a Klimisch 4 score. 4-hydroxybenzenesulfonic acid was not genotoxic in the tow available CA tests, UDS and cell transformation assays, but increased frequencies of SCEs were observed. However, as mentioned above, the interpretation of the biological relevance is not possible in the absence of historical control data. The molecular basis of SCEs is not known, although it is generally assumed that it involves DNA breakage and reunion. The occurrence of DNA strand breaks through 4-hydroxybenzenesulfonic acid is unlikely, since neither the CA nor the UDS test showed a reaction. Therefore, the results in the SCE are difficult to interpret and cannot be explained with the other data obtained. Consequently, a weight of evidence approach is applied, suggesting no genotoxic concern for 4-hydroxybenzenesulfonic acid.

This is underlined by data from similar substances. The closely related p-toluoenesulfonic acid did not cause mutation in an Ames test conducted according to guideline and GLP. Further, 4-methylbenzenesulfonic acid was tested in an in vitro mammalian chromosome aberration test and did not show genotoxicity.

Overall, it is concluded that 4-hydroxybenzenesulfonic acid does not raise a concern for genotoxicity and is neither mutagenic nor clastogenic.

Justification for classification or non-classification

Based on the data available, the classification criteria according to Regulation (EC) 1272/2008 (CLP) are not fulfilled, therefore non-classification is warranted.