2-phenyl-1H-benzimidazole-5-sulphonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative for gene toxicity in two Ames tests and negative in an in vitro chromosome aberration test and in an in vitro gene mutation test with V79 Chinese Hamster cells. Thus, the substance is considered non mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from July 18, 2002 to Oct. 07, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

Chromosomal aberations in human peripheral blood lymphocytes

Species / strain / cell type:

other: human peripheral blood lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

a liver homogenate fraction (S9) from Aroclor 1254 treated male rats

Test concentrations with justification for top dose:

0.33 mM, 3.3 mM, and 10 mM in the tests with 24 hours of treatment without a metabolic activation system,
1.0 mM, 3.3 mM, and 10 mM in the tests with 3.5 hours of treatment with and 4 hours of treatment without a metabolic activation system.

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/ml

Positive control substance:

mitomycin C

Remarks:

without s9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

25 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

with s9

Details on test system and experimental conditions:

Test system:
Lymphocyte cultures
Samples of human peripheral blood were obtained by venipuncture from a male healthy donor, who was not undergoing any drug treatment and collected in heparinised vessels. Lymphocyte cultures were established by addition of small inocula of whole blood (0.4 ml) to each culture tube containing 5 ml of Ham's F10 complete medium, to which 0.1 ml of phytohaemagglutinin was added. The tubes were sealed and incubated at 37 °C.
Metabolic Activation System
The rat liver homogenate fractions used were obtained from King & Hamasch GmbH (Lot KH1501)S Kirchzarten. The preparation of the 9000g supernatant of liver homogenates (S9) from Sprague Dawley male rats (8-10 weeks old, Harlan Winkelman, D-33176 Borchen) induced with Aroclor 1254 (500 mg/kg body weight) is in accordance with the method recommended by Ames et al. (1975).
The S9 preparations (in 0.15 M KC1) were stored in liquid nitrogen.
S9-mix containing 25% S9 was freshly prepared for each mutagenicity assay. The concentrations of the cofactors in the S9-mix are: NADP, 4 mM; glucose-6-phosphate, 25 mM, MgCl2, 8 mM; KC1, 33 mM; phosphate buffer pH 7*4, 100 mM. This solution is filter sterilized by passage through a 0.2 µm filter. S9-mix was kept on ice.

Evaluation criteria:

A significant increase of mutant cells by the test compound will be stated, when the frequency of cells with aberrations in the concurrent negative controls and the frequency of cells with aberrations at each dosage level differ significantly.

Statistics:

The statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).

Key result

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The highest concentration tested induced 69% (1st exp., 24h) and 27 % cytotoxicity (2nd exp., 4h) in the absence and 58 % (1st exp.) and 52 % (2nd exp.) cytotoxicity in the presence of S9-mix.
Test substance did not induce any increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system.

Remarks on result:

other:

Conclusions:

Based on the results of this study, test article did not cause clastogenic effects in human peripheral blood lymphocytes with and without metabolic activation system.

Executive summary:

The chromosome aberration study was undertaken to examine the mutagenic effect of test article for its capability to induce chromosome damage in human peripheral blood lymphocytes in vitro. All tests were carried out both in the presence and absence of metabolic activation system. Two independent experiments were performed. A sampling time at 1.5 times the normal cell-cycle length from the beginning of the treatment (24 h) was used in the first and second experiment. The two experiments without S9-mix differ in the duration of the exposure to the test substance. In the first experiment the cells were treated in the absence of S9 for 24 hours with the test compound, whereas in the second experiment without S9 the test compound was washed away 4 hours after the start of the treatment. In the test with a metabolic activation system, the cells were always exposed to the test substance for 3.5 hours. The following concentrations of test article were tested: 0.33 mM, 3.3 mM, and 10 mM in the tests with 24 hours of treatment without a metabolic activation system 1.0 mM, 3.3 mM, and 10 mM in the tests with 3.5 hours of treatment with and 4 hours of treatment without a metabolic activation system.

At the concentrations tested, the test article did not induce any increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system. In conclusion, the results indicate that, under the experimental conditions described, the test article was not mutagenic in the in vitro mammalian chromosome aberration test with human peripheral blood lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - May 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

guideline test with GLP, but only plate incorporation method was used

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only plate incorporation method was done

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium histidine system

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

deep rough and reduced UV repairedbility

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

s9 mix induced from rat liver microsome

Test concentrations with justification for top dose:

300, 1000, 3000, 10000, 75000 µg per plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg per plate for TA 1535

Positive control substance:

sodium azide

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg per plate for TA 100

Positive control substance:

other: nitrofurantoin

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg per plate for TA 98 and TA 1535, 10 µg per plate for TA 1537 and TA 1538

Positive control substance:

other: 4-nitro-1,2-phenylene diamine (4-NPDA)

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

3 µg per plate for all stains

Positive control substance:

2-acetylaminofluorene

Remarks:

with S9 mix

Details on test system and experimental conditions:

1, s9 mix preparation:
S9 mix was made from the livers of at least six adult male sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of AROCLOR 1254, dissolved in corn oil, at a dose of 500 mg/kg bw, five days prior to sacrifice. The animals were prepared unfasted. The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4 ºC until all animals had been prepared. All the remaining stedps were carried out under sterile conditions at 4 ºC.
Seventy 7 ml of cofactor solution are composed as follows:
MgCl2 x 6 HaO 162.6 mg
KC1 246.0 mg
glucose-6-phosphate, disodium salt 179.1 mg
NADP, disodium salt 315.0 mg
phosphate buffer 100.0 mM
2, For the mutant count, four plates were used, both with and without s9 mix, for each strain and dose. An equal number of plates, filled with the solvment minus the test substance, comprised the negative control. Each positive control also contained four plates per strain. Generally, the amount of solvent for the test substance and for the controls is 0.1 ml per plate. However, in the present study the amount of solvent per plate used for the highest dose of test article was 0.2 ml per plate since it could not be pipetted in 0.1 ml.
The doses for the first trial were determined by the sponsor of this study. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
3, The toxicity of the substance was assessed in three ways. The first method was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed without any other values, it represents four plates with reduced background growth. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37 °C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure by determining the titers. The results of these determinations may be seen in the negative controls.
The tests were performed both with and without S9 mix.
The count was made after the plates had been incubated for 48 hours at 37 °C. If no immediate count was possible, plates were temporarily stored in a refrigerator.

Evaluation criteria:

A reproducible and dose-related increase in mutant founts of at least one strain is considered to be a positive result. For TA 1535, TA 1538, TA 100 and TA 98 this increase should be about twice that of negative control whereas for TA 1537, at least a three fold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect of test article at dose of up to and including 75000 µg per plate. The total bacteria counts did not vary to a biologically relevant degree. No inhibition of growth was noted as well.
None of the five strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without s9 mix and was confirmed by the results of the repeat tests.

Conclusions:

In conclusion, test article was considered not to be mutagenic based on the result given in this study.

Executive summary:

This study was conducted following OECD guideline 471 and GLP principle to investigate the mutagenic potential of the test substance with histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 at dose levels of 300, 1000, 3000, 10000, 75000 µg per plate. Doses of up to and including 75000 µg per plate did not cause any bacteria toxic effects. Total bacteria counts were not changed to a biologically relevant degree and no inhibition of growth was observed. Evidence of mutagenic activity of test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed, Therefore, test article was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Based on the results of the pre-experiment, the main experiments were started with 6 concentrations. A series of concentrations spaced by a factor of 2 was placed into the lower range. Concentrations tested were 87.5, 175.0, 350.0, 700.0, 1400.0 and 2800.0 µg/mL in all main experiments
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation was noted at 700 µg/mL and above in the presence and absence of metabolic activation following 4 hours treatment.
In experiment I and II the cultures at the lowest concentration with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

Deionised water was used as solvent

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

Culture Medium
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) supplemented with Hank’s salts, 10% Fetal Bovine Serum (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.
Seeding
Two to three days after sub-cultivation of the V79 cell stock culture, rinsing with PBS buffer containing 200 mg/L EDTA (ethylene diamine tetraacetic acid) occurred.
Following rinsing, the cells were trypsinized at 37 °C for 5 minutes. The trypsin concentration for all sub-culturing steps was 0.2% in PBS.
Then, the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared.
From this cell suspension, approximately 1.5x10E6 cells were seeded in a plastic culture flask for further mutation rate and cloning efficiency II (viability) analysis. Independently, twice 5x10E2 cells were seeded in two plastic culture flasks for further survival (Cloning Efficiency I) analysis.
The cells were grown for 24 hours prior to treatment with the test item, to enable attach-ment of the cells to the bottles.
The PBS is composed as follows (per litre):
NaCl 8000 mg
KCl 200 mg
KH2PO4 200 mg
Na2HPO4 150 mg
Treatment
After the seeding phase, the complete medium was replaced with serum-free medium containing the test item, either with or without 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel.
In the first experiment the treatment period was 4 hours with and without metabolic activa-tion. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
After the relevant treatment periods, the cultures were washed twice with "saline G" and replaced with complete medium.
The "saline G" solution had the following constituents (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose 1100 mg
Na2HPO4x2H2O 192 mg
KH2PO4 150 mg
The pH was adjusted to 7.2
Cloning efficiency I (Survival) determination:
The colonies contained in the two flasks used to determine the cloning efficiency I (survival) were fixed and stained approx. 7 days after end of treatment. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. When in doubt the colony size was checked with a microscope preparation.
Mutation and Cloning efficiency II (Viability) determination:
Three or four days after end of treatment with the test item, 1.5x10E6 cells from the single flask used for mutation and CE II (viability) analysis were sub-cultivated in a single 175 cm² flask containing 30 mL medium. Following the overall expression time of 7 days after end of treatment, the cells were selected as follow:
- Five 80 cm² cell culture flasks were seeded, with about 3 - 5x10E5 cells each, in medium containing 6-TG to determine mutation.
- Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (CE II).
All seven subcultures per concentration including controls were then incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. Finally, the colonies were stained with 10% methylene blue in 0.01% KOH solution.The stained colonies with more than 50 cells were counted. When in doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
experimental group p-value
experiment I, culture I without S9 mix 0.110
experiment I, culture II without S9 mix 0.032(S)
experiment I, culture I with S9 mix 0.277
experiment I, culture II with S9 mix 0.574
experiment II, culture I without S9 mix 0.549
experiment II, culture II without S9 mix 0.597
experiment II, culture I with S9 mix 0.514
experiment II, culture II with S9 mix 0.480
(S) = Significant trend

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No relevant toxic effects occurred up to the maximum concentration of 2800 µg/mL equal to approximately 10 mM.
No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls.
The induction factor exceeded the threshold of three times the mutation frequency of the solvent control in the first experiment with metabolic activation at 175.0, 700, and 2800 µg/mL in culture I. However, these increases were judged as biologically irrelevant as they were based on a rather low solvent control and remained within the historical range of solvent controls of 3.4 - 36.6 mutant colonies/10E6 cells.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture of the first experiment without metabolic activation. This trend however, was judged as irrelevant as the mutation frequency neither exceeded the threshold described above nor the historical range of solvent controls.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.9 up to 21.0 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.2 up to 22.3 mutants per 10E6 cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 2-phenyl-1H-benzimidazole-5-sulphonic acid is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of 2-phenyl-1H-benzimidazole-5-sulphonic acid to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The study is comprised of a pre-experiment and two independent main experiments. In the pre-experiment the cell cultures were treated with the test item for 4 hours with metabolic activation and for 4 and 24 hours without metabolic activation. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the pre-experiment (2800 µg/mL) was equal to a molar concentration of about 10 mM. No relevant toxic effects occurred up to this concentration. Water was used as solvent.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in either of the main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Therefore, 2-phenyl-1H-benzimidazole-5-sulphonic acid is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance has been tested in two independent in vitro gene mutation tests (Ames tests), one using salmonella typhimurium strains TA 98, 100 1535, 1537 and 1538 and one using salmonella typhimurium strains TA 98, 100 1535, 1537 and 1538 besides E. coli WP2 and WP2 uvr A, always with and without metabolic activation. In both tests all strains tested were found negative for mutagenicity. In addition an in vitro chromosome aberration study was performed which also turned out negative causing no mutagenic effect in the human peripheral blood lymphocytes with and without metabolic activation system. Additionally, an in vitro gene mutation assay in Chinese Hamster V79 cells (with and without metabolic activation) resulted negative.

Based on three different types of in vitro mutagenicity test, the substance is considered non-mutagenic and further in vivo tests are not required.

Justification for classification or non-classification

In three different in vitro assays, with and without metabolic activation, the test substance was found negative for mutagenicity. As a result classification for mutagenicity according to CLP (Regulation EC No 1272/2008) is not required.