Benzenesulfonic acid, 4-chloro-2-[2-[4,5-dihydro-3-methyl-5-oxo-1-(3-sulfophenyl)-1H-pyrazol-4-yl]diazenyl]-5-methyl-, ammonium salt (1:2)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test, mutagenicity assays in mammalian cells and a chromosome aberration test were performed under GLP and according or similar to OECD guidelines 471, 476, and 473, respectively, to evaluate the genotoxic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

26 May 1983

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: soluble in DMSO; stability not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in dimethyl sulphoxide

Target gene:

his / trp

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rodent liver microsomes fractions

Type and composition of metabolic activation system:
- source of S9 : livers of rats (induced with Aroclor 1254) and hamsters
- method of preparation of S9 mix: Mixed function oxidase systems in the livers of a group of rats were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed, and their livers aseptically removed.The rat livers were placed in 0.15 M KCl (3 ml KCl : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at —80°C until required. Rat S-9 mix contained: S-9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM), NADH (4 mM). All the cofactors were filter-sterilised before use.

Following an overnight starvation, the hamsters were killed, and their livers aseptically removed. The following steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 0.1 M sodium phosphate buffer (pH 7.4) (3 ml buffer : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for
10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at —80°C until required. Hamster S-9 mix contained: S-9 fraction (30% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer (pH 7.4) (100 mM), glucose-6-phosphate (20 mM), NADP (4 mM), NADH (2 mM), flavin mononucleotide (2 mM), glucose-6-phosphate dehydrogenase (2.8 units/ml). All the cofactors were filter-sterilised before use.

- concentration or volume of S9 mix and S9 in the final culture medium: An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml of rat or hamster S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added and the mixture incubated at 30°C for 30 minutes. 2 ml of histidine/tryptophan deficient agar was then added and the mixture thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The efficacy of each batch of rat S-9 fraction was tested with the carcinogens 7,12-dimethylbenzanthracene and
2-aminoanthracene before use. The efficacy of each batch of hamster S-9 fraction was tested with the carcinogens Congo Red and 2-aminoanthracene before use.

Test concentrations with justification for top dose:

5000, 2500, 1250, 625, 312.5 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)

- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test substance was assessed at 50 mg/ml in water and dimethyl sulphoxide. The test substance formed a poor suspension in water but dissolved in dimethyl sulphoxide after warming to ca 50°C. Therefore dimethyl sulphoxide was chosen as the solvent for this study.

- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S-9 mix, in DMSO; for TA1535, TA100 and WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S-9 mix, in DMSO; for TA1538 and TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S-9 mix, in DMSO; for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

In the presence of S-9 mix, in DMSO; for TA1538 and TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene; in the presence of S-9 mix, in DMSO; for TA1535, TA100 and WP2 uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments. 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 min
- Exposure duration/duration of treatment: 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: reduction in revertant colony counts or absence of a complete background bacterial lawn

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9
mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers.

Statistics:

No statistical analysis was performed.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Water solubility: 0.6 g/L
- Precipitation and time of the determination: Precipitation was observed from test substance concentrations of 625 µg/plate

RANGE-FINDING/SCREENING STUDIES (if applicable): Four concentrations of test substance were assessed for toxicity using the six tester strains. The highest concentration was 50 mg/ml of test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Three 10-fold serial dilutions of the highest concentration were also tested. The chosen solvent, dimethyl sulphoxide, was used as the negative control. The test substance was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests.

Conclusions:

It is concluded that, when tested in dimethyl sulphoxide, the test item shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test article, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats and un-induced Syrian hamsters. A 30 minutes at 30°C preincubation stage was included in both tests.

In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, 312.5 µg/plate.

No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in dimethyl sulphoxide the test substance was not mutagenic in this bacterial system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: IAL 3154
- Expiration date of the lot/batch: 01.07.2020
- Purity test date: 20.08.2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under storage conditions: The stability of the test substance under storage conditions was guaranteed until 01 Jul 2020 as indicated by the sponsor.
- Stability under test conditions: This study was performed in an aqueous test system. Due to the use of culture medium (Ham’s F12) as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was suspended in culture medium (Ham’s F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration.

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO (Chinese hamster ovary) cell line; source not specified
- Suitability of cells: given
- Normal cell cycle time (negative control): doubling time of about 12 - 16 hours

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: not specified
- Methods for maintenance in cell culture: Routine passage (preparation of a single cell suspension from a 75 cm² flask): Cell medium was removed and washed with 5 mL PBS or HBSS (both Ca-Mg-free). Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks. This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS. Cells were pipetted to separate the cells and to prepare a homogeneous single cell suspension. Cells were counted in a counting chamber or using a cell counter.
Cell suspension was diluted with complete culture medium to the desired cell count.
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes: not specified
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by
pretreatment with "HAT" medium.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Treatment medium (4-hour exposure period): Ham's F12 medium containing stable glutamine and hypoxanthine

Culture medium and Treatment medium (24-hour exposure): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10%
(v/v) fetal calf serum (FCS)

Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10-3 mg/mL)
- aminopterin (0.18 x 10-3 mg/mL)
- thymidine (3.88 x 10-3 mg/mL)
- 10% (v/v) fetal calf serum (FCS)

Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with:
- 6-thioguanine (10 μg/mL)
- 1% (v/v) stable glutamine (200 mM)
- 10% (v/v) fetal calf serum (FCS)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: liver of rats induced with phenobarbital (i.p.) and β-naphthoflavone (orally)
- method of preparation of S9 mix: At least 5 male Wistar rats received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. 24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (so-called S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in both main experiments. The mixture of both components, so-called S9 mix, was kept on ice until use. The concentrations of the cofactors in the S9 mix were:
− MgCl2 8 mM
− KCl 33 mM
− glucose-6-phosphate 5 mM
− NADP 4 mM
− phosphate buffer (pH 7.4) 15 mM

- concentration or volume of S9 mix in the final culture medium: 20%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not specified, but as the positive control yielded the expected results, appropriate metabolic capability of the S9 mix is expected

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 2.5; 5.0; 10.0; 20.0; 40.0; 1 300.0; 2 600.0; 5 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 2.5; 5.0; 10.0; 20.0; 40.0 μg/mL


2nd Experiment
without S9 mix (4-hour exposure period)
0; 250.0.; 500.0; 1 000.0; 2 000.0; 3 000.0; 4 000.0; 5 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 3.1; 6.3; 12.5; 25.0; 50.0 μg/mL

3rd Experiment
without S9 mix (24-hour exposure period)
0; 3.1; 6.3; 12.5; 25.0; 125.0; 250.0; 500.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

- Justification for choice of solvent/vehicle: Using deionized water as solvent a homogeneous suspension of the test substance was obtained. Therefore, culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: methylcholanthrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): about 1x10^6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours both with and without metabolic activation and 24 hours without metabolic activation

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): about 6 - 8 days
- Selection time (if incubation with a selective agent): about 1 week
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration: 6-thioguanine (10 μg/mL)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency (CE) (pre-experiment), Cloning efficiency 1 (CE1; survival), Cloning efficiency 2 (CE2; viability)

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and the historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within the historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative/vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all experiments in the absence of S9 mix at least in the highest applied concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Osmolarity and pH values were not influenced by test substance treatment
- Effects of osmolality: Osmolarity and pH values were not influenced by test substance treatment
- Precipitation: The test substance was poorly soluble in the most suitable vehicle culture medium. The stock preparation was a homogeneous suspension. In this study, in the absence and the presence of S9 mix, test substance precipitation in culture medium determined macroscopically at the end of treatment occurred from about 10 μg/mL onward in all experiments under all test conditions.

RANGE-FINDING/SCREENING STUDIES: pre-test for dose selection

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The substance test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese

hamster ovary (CHO) cells in vitro. Three experiments were carried out independently of each other.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment

without S9 mix (4-hour exposure period)

0; 2.5; 5.0; 10.0; 20.0; 40.0; 1 300.0; 2 600.0; 5 200.0 μg/mL

with S9 mix (4-hour exposure period)

0; 2.5; 5.0; 10.0; 20.0; 40.0 μg/mL

2nd Experiment

without S9 mix (4-hour exposure period)

0; 250.0.; 500.0; 1 000.0; 2 000.0; 3 000.0; 4 000.0; 5 200.0 μg/mL

with S9 mix (4-hour exposure period)

0; 3.1; 6.3; 12.5; 25.0; 50.0 μg/mL

3rd Experiment

without S9 mix (24-hour exposure period)

0; 3.1; 6.3; 12.5; 25.0; 125.0; 250.0; 500.0 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the

frequencies of forward mutations. The test substance was poorly soluble either in all tested vehicles or in culture medium. Therefore, in the pretest using culture medium as most suitable vehicle precipitation occurred from the lowest applied concentration onward. In all experimental parts in the absence of metabolic activation cytotoxicity was observed at strongly precipitating concentrations only. Due to lacking cytotoxicity in the pretest in the presence of metabolic activation both experiments with S9 mix were performed using concentrations at the border of saturation of culture medium.

On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in three experiments performed independently of each other.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Version / remarks:

26 May 1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: soluble in DMSO; stability not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in dimethyl sulphoxide

Species / strain / cell type:

lymphocytes: cultured human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human blood from healthy male donors
- Suitability of cells: given
- Normal cell cycle time (negative control): not specified

For lymphocytes:
- Sex, age and number of blood donors: male, age and number of donors not specified
- Whether whole blood or separated lymphocytes were used: separated lymphocytes
- Whether blood from different donors were pooled or not: pooled
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Medium: RPMI 1640 + 20% foetal calf serum + phytohaemagglutinin;
CO2 concentration: 5%;
humidity level: humid atmosphere;
temperature: 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: rat liver (induced with Aroclor 1254)
- method of preparation of S9 mix: Mixed function oxidase systems in the liver of a group of rats were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed. The following steps were carried out at 0 - 4°C under aseptic conditions. The livers were placed in 0.15 M KCl (3 ml KCl : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at -80°C until required.
S-9 mix contains: S-9 fraction (10% v/v), MgCl2 (8 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): All batches of S-9 fraction were tested using the carcinogens 2-aminoanthracene and 7,12-dimethylbenzanthracene before use.

Test concentrations with justification for top dose:

First test
Without S-9 mix, 18 hour harvest: 18.8, 75 and 150 µg/ml.
With S-9 mix, 18 hour harvest: 37.5, 150 and 300 µg/ml

Second test
Without S-9 mix, 18 hour harvest: 37.5, 75 and 150 µg/ml.
With S-9 mix, 18 hour harvest: 37.5, 150 and 225 µg/ml.
Without S-9 mix, 32 hour harvest: 37.5, 150 and 225 µg/ml.
With S-9 mix, 32 hour harvest: 37.5, 150 and 225 µg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)

- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in solvents compatible with the test system was assessed. The test substance dissolved in dimethyl sulphoxide at a concentration of 40 mg/ml. At a final concentration of 400 µg/ml in tissue culture medium a precipitate formed after incubation. When 30 mg/ml was dosed at 1% v/v into medium to give a final concentration of 300 µg/ml, and incubated, only a slight precipitate formed.

- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 18 h and 32 h
- Harvest time after the end of treatment (sampling/recovery times): Three hours after dosing the cultures containing the S-9 mix were centrifuged and the cell pellets resuspended in fresh medium and incubated for a further 15 hours. The cultures treated in the absence of S-9 mix were incubated for 18 hours. After a 2-hours incubation with colchicine the cells were harvested by centrifugation.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Two hours before the cells were harvested mitotic activity was arrested by addition of colchicine to each culture at a final concentration of 0.25 µg/ml.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After the centrifugation step (10 minutes at 200 g), the cell pellets were treated with a hypotonic solution (20% Hanks balanced salt solution). After a 10 minute period of hypotonic incubation the suspensions were centrifuged at 110 g for 10 minutes and the cell pellets fixed by addition of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid). The pellets were allowed to fix for at least two hours. The pellets were aspirated through a 20 gauge needle, then centrifuged at 200 g for 10 minutes and finally resuspended in a small volume of fresh fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air-dry. The slides were then placed in buffered distilled water (pH 6.8) prior to staining in Giemsa (dilution factor: 1 part Giemsa to 9 parts buffered distilled water). After rinsing in buffered distilled water the slides
were left to air-dry and then mounted in DPX.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value or, if there was no decrease, the maximum achievable concentration was used as the highest dose level for the metaphase analysis. The intermediate and low doses were also selected. The concentration of positive control compounds selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells. 100 metaphase figures were examined from each culture, with a maximum of 25 from each slide.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only cells with 44 - 46 chromosomes were analysed.
- Determination of polyploidy: no data
- Determination of endoreplication: no data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Statistics:

The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Water solubility: not specified
- Precipitation and time of the determination: precipitation was observed at the highest dose level (300 µg/ml)
- Definition of acceptable cells for analysis:
First experiment: In the absence of S-9 mix, the test substance reduced the mitotic index to 26% of the solvent control value at the highest concenttation dosed, 300 µg/ml. This proved to be unanalysable as the metaphase figures were pycnotic. 150 µg/ml was therefore selected as the highest concentration to be used for metaphase analysis.
In the presence of S-9 mix the test substance reduced the mitotic index to 61 % of the solvent control value at the highest concentration dosed, 300 µg/ml. This was selected for analysis. The lower concentrations selected were 150 and 37.5 µg/ml.

Second experiment - 18 h harvest: In the absence of S-9 mix, no surviving cells were observed, in cultures treated with 300 µg/ml of the test substance. The relative mitotic index of the next dose level, 225 µg/ml, was 27%. However, the metaphase figures were pycnotic and unanalysable. 150 µg/ml was, therefore, selected as the highest concentration used for analysis. This gave a relative mitotic index value of 77%. The lower concentrations selected were 75 and 37.5 µg/ml.
In the presence of S-9 mix, the mitotic index could not be calculated for the highest dose level, 300 µg/ml, as the test substance obscured the metaphase figures. Hence, the next dose level, 225 µg/ml, was selected for analysis. This was non-toxic to the cells. The lower concentrations selected were 150 and 37.5 µg/ml.

Second experiment - 32 h harvest: In the absence of S-9 mix, the test substance caused a decrease in the mitotic index to 49% of the solvent control value at the highest concentration dosed, 300 µg/ml. However the metaphase figures at this dose level were pycnotic and unanalysable. Hence, 225 µg/ml was selected as the highest concentration for analysis. This gave a relative mitotic index value of 51%. 150 and 37.5 µg/ml were the lower concentrations selected for analysis.
In the presence of S-9 mix, the mitotic index could not be calculated, at a concenttation of 300 µg/ml as the test substance obscured the metaphase figures. At 225 µg/ml the test substance proved to be non-toxic to the cells. This concentration was selected for analysis. The lower concentrations of 150 and 37.5 µg/ml were also selected for analysis.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, Historical control data for human lymphocytes treated with solvent controls from June 1983 until August 1994.

Conclusions:

It is concluded that the test item has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treatment time cell division was arrested using colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage.

In order to assess the toxicity of the test substance to cultured human lymphocytes the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data the following concentrations were selected for metaphase analysis:

First test

Without S-9 mix, 18 hour harvest: 18.8, 75 and 150 µg/ml.

With S-9 mix, 18 hour harvest: 37.5, 150 and 300 µg/ml

Second test

Without S-9 mix, 18 hour harvest: 37.5, 75 and 150 µg/ml.

With S-9 mix, 18 hour harvest: 37.5, 150 and 225 µg/ml.

Without S-9 mix, 32 hour harvest: 37.5, 150 and 225 µg/ml.

With S-9 mix, 32 hour harvest: 37.5, 150 and 225 µg/ml.

The test article did not cause any statistically significant increases in the proportion of metaphase figures with chromosomal aberrations, in either the presence or absence of S-9 mix.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A micronucleus assay in mice was performed under GLP and equivalent or similar to OECD guideline 474 to evaluate the genotoxic potential of the test substance. The test material was negative for induction of micronuclei formation in mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

well documented, meets criteria of corresponding OECD guideline, no information on test substance purity and expiration date

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Within the test report no guideline is mentioned but the assay meets basic criteria of the OECD guideline 474 "Mammalian Erythrocyte Micronucleus Test"

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 9 weeks
- Weight at study initiation: weight range was 28.2-38.8 and 22.9-31.4 grams for the males and females, respectively
- Assigned to test groups randomly: yes
- Fasting period before study: /
- Housing: five per cage during quarantine, and housed up to five per cage at randomization
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 +/- 6
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: due to solubility and homogeneity
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw, 10ml/kg bw for positive control
- Lot/batch no. (if required): Lot # 4H0080; Prep.06/07/1996; Exp. 12/07/1996; Batch #1

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions for the assay were prepared by making a 250.02 mg/ml stock for the high dose (5000 mg/kg). This was prepared by adding MC to 13.7514 g of the test article up to a volume of 55 ml. The mixture was stirred with a flat-end spatula for ~1 minute and then slowly mixed on a stirplate for ~ 10 minutes. A thick viscous yellow-orange suspension was obtained. Dilutions of this stock were prepared for the 2500 and 1250 mg/kg dose levels. All dosing stocks were placed on magnetic stir plates during the dosing procedure.

Duration of treatment / exposure:

Animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow.

Frequency of treatment:

once only

Dose / conc.:

1 250 other: mg/kg bw

Dose / conc.:

2 500 other: mg/kg bw

Dose / conc.:

5 000 other: mg/kg bw

No. of animals per sex per dose:

5 /sex/dose/treatment period

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 80 mg/kg bw

Tissues and cell types examined:

extraction of bone marrow from femur, analysis of erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
no mortalities or signs of toxicity at dose range finder study, maximum dose for micronucleus assay was estimated to be 5000 mg/kg bw

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction ofthe bone marrow.

DETAILS OF SLIDE PREPARATION:
The marrow was flushed from the bone and transferred to centrifiige tubes containing 3 - 5 ml bovine serum (one tube for each animal). Following centrifiigation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, and stained in May-Grunwald solution followed by Giemsa. The air-dried slides were coverslipped using Depex® mounting medium.

METHOD OF ANALYSIS:
The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this Crl:CD-l(ICR) BR strain is about 0.0 - 0.4%. The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).

The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the
detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.

Statistics:

Data are summarized by sex and dose groups for the different time points. Individual animal data are also presented. The analysis of these data was performed using an analysis of variance on either untransformed (when variances are homogeneous) and rank transformed (when variances are heterogeneous) proportions of cells with micronuclei per animal. If the analysis of variance was significant (p<0.05), a Dunnett's t-test was used to determine which dose groups, if any, were significantly different from the negative control. Analyses were performed separately for each harvest time and sex combination.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: In the dose selection study, the test article was suspended in 1.0% methyl cellulose (MC) and dosed by oral gavage at 1000, 2000, 3000, 4000, and 5000 mg/kg. Six animals (three males and three females) were assigned to each dose group. Animals were observed for three days after dosing for toxic signs and/or mortality.
- Solubility: test item forms a suspension in 1% CMC
- Clinical signs of toxicity in test animals: no

RESULTS OF DEFINITIVE STUDY
The test article induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times, except in the females from the 5000 mg/kg dose group in the 24 hour harvest. This is a statistical anomaly rather than a true test article effect and is due to the very low (0.02%) micronucleated PCE's in the vehicle control group); also the value at this time point (0.14%) is well within the historical vehicle control values of 0.0%-0.22%.

Conclusions:

The test material did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.

Executive summary:

The objective of this in vivo assay was to evaluate the ability of the test article to induce micronuclei in bone marrow polychromatic erythrocytes of Crl:CD-l(ICR) BR mice.

In the dose selection study, the test article was suspended in 1.0% methyl cellulose (MC) and dosed by oral gavage at 1000, 2000, 3000, 4000, and 5000 mg/kg. Six animals (three males and three females) were assigned to each dose group. Animals were observed for three days after dosing for toxic signs and/or mortality. Based on the results of the dose selection study, the maximum tolerated dose was estimated as > 5000 mg/kg. In the micronucleus assay, the test article was suspended in 1.0% methyl cellulose (MC) and dosed by oral gavage at 1250, 2500, and 5000 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive

control groups, euthanized approximately 24 hours after dosing, were included in the assay. The animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow.

The test material did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

There are reliable, GLP-conform in vitro and in vivo studies available to assess the genotoxic potential of the test substance in bacteria, mammalian cells and animals.

The first study was performed to assess the mutagenic potential of the test article. Histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats and un-induced Syrian hamsters. A 30 minutes at 30°C preincubation stage was included in both tests. In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, 312.5 µg/plate. No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in dimethyl sulphoxide the test substance was not mutagenic in this bacterial system.

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three experiments were carried out independently of each other at dose levels of 2.5 - 5200 µg/ml in absence and presence of a metabolic activation system for 4h and 24h, respectively. Thereafter, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. Precipitation occurred from the lowest applied concentration onward. In all experimental parts in the absence of S9 mix cytotoxicity was observed at strongly precipitating concentrations only. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system. However, in the 1st Experiment after 4 hours exposure in the absence of S9 mix an increase in the number of mutant colonies was observed in a single culture which was not corroborated in the culture treated in parallel or in the repeat experiment (2nd Experiment).

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in cultured human lymphocytes. The experiments differed in test concentration, exposure duration and metabolic activation. 100 metaphase figures were examined, from each culture, with a maximum of 25 from each slide.

The test article did not cause any significant increases in the proportion of metaphase figures with chromosomal aberrations, in either the presence or absence of S-9 mix. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. Thus, it is concluded that the test item has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Genetic toxicity in vivo

An in vivo assay was conducted to evaluate the ability of the test article to induce micronuclei in bone marrow polychromatic erythrocytes in mice. Based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg bw. In the micronucleus assay, the test article was suspended in 1.0% methyl cellulose (MC) and dosed by oral gavage at 1250, 2500, and 5000 mg/kg bw. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups, euthanized approximately 24 hours after dosing, were included in the assay. The animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The test item did not induce mutations or chromosome aberrations in vitro. The test material was also negative for induction of micronuclei formation in mice. Therefore, the substance is considered as non-mutagenic under the conditions of these tests. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.