sodium hydrogen [5-(diethylamino)-2-[(2-hydroxy-4-nitrophenyl)azo]phenolato(2-)][5-hydroxy-6-[(2-hydroxy-4-nitrophenyl)azo]-1-naphthalenesulfonato(3-)]-, chromate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across - OECD TG 471 NR deficient strains - key study - with and without S9 - KS1.

Read-across - OECD TG 471 - supporting study - with and without S9 - KS2. 

Read-across - OECD TG 476 - key study - KS2.

Read-across - OECD TG 473 - key study - KS2. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction

Test concentrations with justification for top dose:

dose levels 2.5 to 300 μg/mL in the non-activated 4-hour exposure group, and at doses 5 to 200 μg/mL in the S9-activated 20-hour exposure group

Vehicle / solvent:

Dimethyl formamide (DMF)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Chromosome Aberration Assays
Seven to nineteen dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was either based on cytotoxicity (cell growth inhibition relative to the vehicle control) or visible precipitate at the conclusion of the treatment period. Two or three additional dose levels were included in the evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Chromosome Aberration Assay)
The pH at the highest test substance concentration was measured prior to dosing using a pH meter or test strips. Treatment was carried out by re-feeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 50 μL of test substance dosing solution or vehicle alone. Untreated controls were re-fed with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.
After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium were aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium, and returned to the incubator under standard conditions.
For the chromosomal aberration assay only, two hours prior to cell harvest, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts, and then Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL. Thus the treatment time for the precipitating dose levels was 18 hours instead of 20 hours.

Collection of Metaphase Cells (Preliminary Toxicity Test and Chromosome Aberration Assayd)
For the preliminary toxicity test and chromosome aberration assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data was presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity).

Evaluation criteria:

The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations.

The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Negative for the induction of structural and numerical chromosome aberrations.

Executive summary:

Method

The test substance was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473.

A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dose formulations were adjusted for the purity of the test substance (65%), using a correction factor of 1.54.

Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a workable suspension in water at a maximum concentration of approximately 10 mg/mL. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥ 50 % reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 600 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 2.5 to 300 μg/mL for the non-activated 4-hour exposure group, from 5 to 200 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 300 μg/mL for the non-activated 20-hour exposure group.

 

Results

In the initial chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at 200 μg/mL in the S9-activated 4-hour exposure group and at dose levels ≥ 200 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 75 μg/mL in all three treatment groups. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

The percentage of cells with structural or numerical aberrations in the non-activated 4 and 20-hour exposure groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (3.0%) relative to vehicle control at 75 μg/mL (p ≤ 0.01, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at dose levels ranging from 25 to 100 μg/mL. In the repeat assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 55 μg/mL. The highest dose analyzed exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

In the repeat assay, the percentage of cells with structural aberrations was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (6.0% and 5.7%) relative to vehicle control at dose levels 25 and 50 μg/mL, respectively (p ≤ 0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the percentage of cells with numerical aberrations was within the historical control range of 0.0% to 9.5% and also within the 95% control limit of historical data. Therefore, the statistically significant induction was considered to have no biological relevance.

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

 

Conclusion

Under the conditions of the assay described in this report, the test substance was considered to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From May 02 to August 30, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Mutagenic effects are detected by the appearance of cells resistant to 6-TG

Species / strain / cell type:

other: CHINESE HAMSTER CELLS V79, clone 65/3

Details on mammalian cell type (if applicable):

Origin: Dr. D. Wild, Freiburg, Germany.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Cytotoxicity test
Range with metabolic activation:
0.24 to 500.0 ug/ml
Range without metabolic activation:
0.24 to 500.0 ug/ml
Mutagenicity test
Original experiment:
Range with metabolic activation:
18.52 to 500.0 ug/ml
Range without metabolic activation:
11.11 to 300.0 ug/ml
Confirmatory experiment:
Range with metabolic activation:
62.5 to 500.0 ug/ml
Range without metabolic activation:
37.5 to 300.0 ug/ml

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Nitrosodimethylamine

Remarks:

With metabolic activation

Untreated negative controls:

yes

Remarks:

vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethansulfonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Preliminary cytotoxicity test
A cytotoxicity test was performed on V79 cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. For each concentration and the untreated controls, 2.5x10s V79 cells were seeded in 5 ml growth medium into a 25 cm2 tissue culture flask and incubated overnight. The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (DMSO) controls were tested. The highest concentration was determined in a preliminary solubility test. Lower concentrations were prepared by serial dilution by a factor of 0.5. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment. The cultures were counted and diluted so that 100 cells were seeded per 9.6 cm2 in 3 ml of growth medium. After seven to eight days of growth the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter (Artek Counter®, Fisher Scientific) or by the naked eye. The sensitivity of the colony counter was adjusted to detect clones of about twenty or more cells. The concentration to be selected as the highest for the mutagenicity assay was the one causing about 50-90% reduction of viable cells in comparison with the mean of the two negative controls or corresponds to the substance's solubility limit (precipitates in the culture).
Mutagenicity test
Depending on the toxicity of the test compound 2.5-5.0x10ʌ6 cells of passage 25 (original experiment) and passage 25 (confirmatory experiment) were plated in 30 ml growth medium into 175 cm2 flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone. In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 ul/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 ul/ml. The treatment was terminated by washing the cell layer extensively with PBS. After washing, the cells were suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM), diluted with fresh growth medium and replated into flasks at 2x10ʌ6 cells. The cultures were incubated at 37°C for seven to eight days during which the cells could recover and divide to express the mutant phenotype. The cultures were subcultered after the second or third day transferring 2x10ʌ6 cells to a fresh flask to maintain exponential growth during the expression phase.
In parallel cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The counted cell suspension of each concentration level was further diluted so that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and incubated at 37 °C. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values).
At the end of the expression period the cultures were trypsinised, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM). The cell suspension of each culture was diluted with fresh growth medium and an aliquot replated into four flasks (75 cm2 growth area) each containing 2x10ʌ6 cells for the mutant selection. The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 ug/ml 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control). After seven to eight days incubation at 37 °C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.
In parallel the viability at the end of the expression period was estimated from the cloning efficiency. The remaining cell suspensions from the various expression cultures were further diluted such that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and were incubated at 37 °C. The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).

Evaluation criteria:

Assay acceptance criteria
• The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.
• From each experiment, at least three concentrations of the test substance, one positive and one solvent control should be evaluated.
• The mutant frequency of the solvent controls (spontaneous mutant frequency) should not exceed 35xl0-6.
• The positive control should fulfil the criteria for a mutagenic substance.
• The highest concentration of the test substance applied in the mutagenicity test should either reduce the viable cells by about 50-90% or correspond to the test substance's solubility limit (precipitates in the culture). In case of non-toxic freely soluble compounds the highest tested concentration
will be 5 mg/ml. In special cases the highest concentration can be determined by the sponsor.
Assay evaluation criteria
All mutant frequencies are normalized to a virtual cloning efficiency of 100 % at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15 %, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the
result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.

Statistics:

Assessment of statistical significance of mutation frequency
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.  

Species / strain:

other: CHINESE HAMSTER CELLS V79, clone 65/3

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Cytotoxicity test

A preliminary range finding test was run assessing cytotoxicity. The test substance was tested at concentrations up to 500.0 µg/ml. Higher concentrations could not be applied due to solubility limitations in the culture vehicle. In the part with metabolic activation, at the highest concentration of 500.0 µg/ml an acute growth inhibiting effect of 40.2 % could be seen. Without metabolic activation treatment with the test substance proved growth inhibiting by 99.98 % at the concentration of 500.0 µg/ml. The next lower concentration of 250.0 µg/ml revealed an acute inhibition of growth of 70.1 %. Accordingly, 500.0 µg/ml with and 300.0 µg/ml without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500.0 µg/ml. No toxicity was found at any concentration. In the confirmatory experiment the concentrations applied were 62.5, 125.0, 250.0 and 500.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 33.7%. N-Nitrosodimethylamine (DMN, 1.0 µl/ml) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 11.11, 33.33, 100.0 and 300.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 82.0% and 19.5% respectively. In the confirmatory experiment the concentrations applied were 37.5, 75.0, 150.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3%. The mean growth inhibition after the expression period was 24.0%. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant increase of the mutant frequencies as determined by the screening with 6-TG.

Conclusions:

Non mutagenic

Executive summary:

Method

The test substance was tested for its mutagenic effect by the gene mutation test with chines hamster cells V79, according to OECD Guideline 476.

The test system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-TG and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, an experiment is performed, in which the metabolic turnover of the test material is simulated in vitro by the addition of an activation mixture to the cell cultures containing rat-liver post mitochondrial supernatant (S9 fraction) and cofactors.

 

Observation

The original experiment was performed at the following concentrations: 11.11, 33.33, 100.0 and 300.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 82.0 % and 19.5 % respectively. In the confirmatory experiment the concentrations applied were 37.5, 75.0, 150.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3 %. The mean growth inhibition after the expression period was 24.0 %. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control.

 

Results

Non mutagenic

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 12 to May 20, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(No E. Coli strain used; no statistical analysis)

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver metabolic fraction S9

Test concentrations with justification for top dose:

Concentration range in the range finding test: 20.6 to 5000.0 µg/plate.
Concentration ranges in the mutagenicity tests:
Original experiment 61.7 to 5000.0 μg/plate - Confirmatory experiment: 61.7 to 5000.0 μg/plate.

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Details on test system and experimental conditions:

Preparation of the bacterial cultures:
Inoculates from frozen master copies were set up monthly. They were grown in liquid nutrient broth medium (NB-medium) overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

Setting up of the test plates:
0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl and was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water.

Preliminary range finding test:
A range finding test was carried out with strain TA 100 with and without metabolic activation at six concentrations of the test substance and one negative control according to a Standard Operating Procedure of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of three. The plates Were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Mutagenicity test:
The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Evaluation criteria:

Assay acceptance criteria: a test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response: the test substance will be considered to be positive in the test system if the following condition is met: at least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the strains tested. Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis of the test data was not performed.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

other: marginal increased revertant counts

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

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

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Mutagenic

Executive summary:

Method:

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium, according to the OECD Guideline 471. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 1535 and TA 1537.

The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in the range of 61.7 to 5000.0 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 61.7 to 5000.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

 

Observation:

In both experiments, performed with and without metabolic activation, the test substance and its metabolites led to a very strong increase in the number of back-mutants with strains TA 98, TA 100 and TA 1537. The metabolites of the test substance led to weak increased revenant counts with strain TA 1535.

 

Conclusion:

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the substance and its metabolites induced gene mutations in the strains of Salmonella typhimurium.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 06 January 2021 to 19 January 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The reliability of the original study report is 1 (acceptable without restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD, 1997, as corrected in 2020

Deviations:

yes

Remarks:

Due to the nature of the test article, a modified (reductive Prival) metabolic activation system was employed, and only a single experiment was performed. Use of NR-deficient strains.

Principles of method if other than guideline:

The traditional strains used for OECD 471 will be checked in parallel with the same strains deficient in the nitro-reductase enzyme (present only in bacteria) as to avoid the NO2 group reduction present in the test substance. Reduction of the nitro groups in fact produce in the substance aromatic amines that typically give false positive for the traditional tested strains.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

TA 98NR, TA100NR

Metabolic activation:

with and without

Metabolic activation system:

- Type and composition of metabolic activation system: reductive (Prival) metabolic activation system.
- Source of S9: the metabolic activation system was prepared from uninduced male Golden Syrian hamsters.
- Method of preparation of S9 mix: the S-9 was stored frozen at <-50°C, and thawed prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 30% reductive (Prival) S-9 mix respectively. The composition of the mix and buffer solutions are reported in the section "Any other information on material and methods incl. tables".
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of 30% reductive S-9 mix or buffer solution.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Mutation Experiment treatments of all the tester strains were performed using a
pre-incubation methodology in the absence and presence of a modified (reductive)
S-9 mix using final concentrations of test substance at 5, 16, 50, 160, 500, 1600 and
5000 μg/plate.

Highest dose tested: 5000 μg/plate unless limited by cytotoxicity or solubility. This is the maximum recommended concentration according to current regulatory guidelines.

Vehicle / solvent:

Purified water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

mitomycin C

other: metronidazole (MTZ), 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: pre-incubation methodology.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: quantities of test article or control suspension, bacteria and S-9 mix or buffer solution detailed above, were mixed together and placed in an orbital incubator set to either 37°C (for the treatments in the absence of S-9) or 30°C (for treatments in the presence of S-9) for 30 minutes.
- Exposure duration/duration of treatment: 3 days.
- Harvest time after the end of treatment (sampling/recovery times): not reported.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition.
- Any supplementary information relevant to cytotoxicity: the background lawns of the plates were examined for signs of toxicity. Revertant plate count data were also assessed, as a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response was also considered as evidence of toxicity.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance.

Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as intensely coloured agar, precipitation, contaminated plate or absence of background lawn affected the accuracy of the automated counter.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if: a concentration related increase in revertant numbers was ≥ 1.5-fold (in strain TA 102), ≥2-fold (in strains TA 98, TA 98NR, TA 100 or TA 100NR) or ≥3-fold (in strains TA 1535 or TA 1537) respect to the concurrent vehicle control values.
The test article was considered positive in this assay if the above criterion was met. The test article was considered negative in this assay if the above criterion was not met.
Results which only partially satisfied the above criteria were dealt with on a case-by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations. Data from strain TA98 were compared (non-statistically) with that from TA98NR, and data from strain TA100 were compared with that from TA100NR. Where a mutagenic response was seen in one or both parent strains but was absent or much reduced in the corresponding NR variant strain(s), this was considered to be indicative that bacterial nitroreduction enzymes play a significant role in the mutagenicity of the test compound as observed in this study.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 100NR

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity, Solubility and Concentration Selection: mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity ranging from a thinning of the background bacterial lawn with or without a concurrent marked reduction in revertant numbers to a complete killing of the test bacteria was observed in all strains in the absence of S-9 and in most strains in the presence of S-9, with the toxic effects (where observed) extending down to either 500, 1600 or 5000 μg/plate in each case. Further evidence of toxicity in the form of a tailing off of the mutagenic response was also seen at the higher concentration(s) in all strains where a clear mutagenic response was observed. Precipitation of test article was observed on all the test plates treated at 1600 μg/plate and above in the absence of S-9 and at 500 μg/plate and above in the presence of S-9.

Data Acceptability and Validity: from the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception of a few isolated vehicle control counts that fell slightly outside the laboratory control ranges. In each case, these counts were comparable to the other vehicle control replicate counts and the laboratory historical control ranges, and therefore these data were accepted as characteristic and valid. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls, and ≥2-fold with NQO, B[a]P and CR in strain TA98NR and NaN3, AAN and CR in strain TA100NR. The positive control treatments with 2NF in TA98NR and with MTZ in TA100NR each demonstrated a reduced response compared to the equivalent treatments in the parent strains TA98 and TA100, confirming the nitroreductase deficient status of strain TA98NR and TA100NR. The control treatments therefore confirmed discrimination between different strains, and an active S-9 preparation, and the correct strain and assay functioning was demonstrated. The study data were therefore accepted as valid.

Mutation: following test substance treatments of all the test strains in the absence and presence of S-9, notable and concentration-related (up to the lower limit of toxicity and/or precipitation) increases in revertant numbers were observed in all strains except TA98NR in the absence of S-9 and TA102 in the presence of S-9. Where observed, these increases were all ≥1.5-fold (in strain TA102 in the absence of S-9), ≥2-fold (in strains TA98, TA98NR (in the presence of S-9 only), TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values, although it should be noted that in strain TA100NR in the absence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 5000 μg/plate. These increases were therefore all sufficient to be considered as clear evidence of test substance mutagenic activity in these strains in this assay system.
The mutagenic responses in the nitroreductase deficient strain TA100NR in the absence and presence of S-9 were much reduced in magnitude compared to those with the corresponding treatments in the parent (nitroreductase proficient) strain TA100. An even clearer difference in mutagenic activity was evident for the strain TA98 and TA98NR treatments in the absence of S-9, where a clear mutagenic response was observed in the parent (nitroreductase proficient) strain TA98, but no mutagenic response was observed in the nitroreductase deficient strain TA98NR. In relation to the strain TA100 and TA100NR data in particular, it should also be noted that strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the relative mutagenic responses in each of these cases were considered as a clear indication that nitroreduction plays a significant role in the mutagenic activity of the test substance observed in this assay system. When the mutagenic responses in strain TA98 and TA98NR in the presence of S-9 were compared, there appeared to be a greater magnitude of response (in terms of fold increase over the concurrent vehicle control level) in the nitroreductase deficient strain TA98NR than in the parent (nitroreductase proficient) strain TA98. However, the vehicle control counts in strain TA98NR in the presence of S-9 were much lower than those in strain TA98 (although the counts in both strains were all within the laboratory historical range), which serves to exaggerate the magnitude of response in the strain with the lower vehicle control level. When the increases in revertants are assessed in terms of number of induced revertants (above the vehicle control level), the maximum increase in the parent (nitroreductase proficient) strain TA98 is approximately 1.75 times that seen in the nitroreductase deficient strain TA98NR (177.3 revertants/plate in strain TA98 compared to 101.3 revertants/plate in strain TA98NR), which therefore provides further evidence that nitroreduction plays a significant role in the mutagenic activity of the test substance.

Conclusions:

It was concluded that the test substance induced mutation in histidine-requiring strains TA98, TA100, TA100NR, TA1535 and TA1537 of Salmonella typhimurium when tested in the absence and in the presence of a reductive (Prival) hamster liver metabolic activation system (S-9) under the conditions of this study, and also in Salmonella typhimurium strain TA98NR when tested in the presence of S-9 and in Salmonella typhimurium strain TA102 when tested in the absence of S-9. The conditions employed in this study included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines and a precipitating, and in most cases toxic, concentration). It may be noted that an increase in revertant numbers achieving the threshold level for an increase to be considered as clear evidence of mutagenic activity in strain TA100NR in the absence of S-9 was only achieved at the maximum treatment concentration of 5000 μg/plate. The relative mutagenic responses between the nitroreductase proficient and nitroreductase deficient strains used in this study indicated that nitroreduction plays a significant role in the observed mutagenic activity of the test substance.

Executive summary:

The test substance was assayed for mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of a reductive hamster liver metabolising system (S-9), in a single experiment. All the test substance treatments in this study were performed using formulations prepared in purified water. As the test substance is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic. Mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these treatments, evidence of toxicity was observed in all strains in the absence of S-9 and in most strains in the presence of S-9, with the toxic effects (where observed) extending down to either 500, 1600 or 5000 μg/plate in each case. Precipitation of test article was observed on all the test plates treated at 1600 μg/plate and above in the absence of S-9 and at 500 μg/plate and above in the presence of S-9.

 

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control
treatments, and were elevated by positive control treatments.

Following test substance treatments of all the test strains in the absence and presence of S-9, notable and concentration-related (up to the lower limit of toxicity and/or precipitation) increases in revertant numbers were observed in all strains except TA98NR in the absence of S-9 and TA102 in the presence of S-9. Where observed, these increases were all ≥1.5-fold (in strain TA102 in the absence of S-9), ≥2-fold (in strains TA98, TA98NR (in the presence of S-9 only), TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values, although it should be noted that in strain TA100NR in the absence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 5000 μg/plate. These increases were therefore all sufficient to be considered as clear evidence of the test substance mutagenic activity in these strains in this assay system.

The mutagenic responses in the nitroreductase deficient strain TA100NR in the absence and presence of S-9 were much reduced in magnitude compared to those with the corresponding treatments in the parent (nitroreductase proficient) strain TA100. An even clearer difference in mutagenic activity was evident for the strain TA98 and TA98NR treatments in the absence of S-9, where a clear mutagenic response was observed in the parent (nitroreductase proficient) strain TA98, but no mutagenic response was observed in the nitroreductase deficient strain TA98NR. In relation to the strain TA100 and TA100NR data in particular, it should also be noted that strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the relative mutagenic responses in each of these cases were considered as a clear indication that nitroreduction plays a significant role in the mutagenic activity of the test substance observed in this assay system. When the mutagenic responses in strain TA98 and TA98NR in the presence of S-9 were compared, there appeared to be a greater magnitude of response (in terms of fold increase over the concurrent vehicle control level) in the nitroreductase deficient strain TA98NR than in the parent (nitroreductase proficient) strain TA98. However, the vehicle control counts in strain TA98NR in the presence of S-9 were much lower than those in strain TA98 (although the counts in both strains were all within the laboratory historical range), which serves to exaggerate the magnitude of response in the strain with the lower vehicle control level. When the increases in revertants are assessed in terms of number of induced revertants (above the vehicle control level), the maximum increase in the parent (nitroreductase proficient) strain TA98 is approximately 1.75 times that seen in the nitroreductase deficient strain TA98NR, which therefore provides further evidence that nitroreduction plays a significant role in the mutagenic activity of the test substance.

It was concluded that the test substance induced mutation in histidine-requiring strains TA98, TA100, TA100NR, TA1535 and TA1537 of Salmonella typhimurium when tested in the absence and in the presence of a reductive (Prival) hamster liver metabolic activation system (S-9) under the conditions of this study, and also in Salmonella typhimurium strain TA98NR when tested in the presence of S-9 and in Salmonella typhimurium strain TA102 when tested in the absence of S-9. The conditions employed in this study included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines and a precipitating, and in most cases toxic, concentration). It may be noted that an increase in revertant numbers achieving the threshold level for an increase to be considered as clear evidence of mutagenic activity in strain TA100NR in the absence of S-9 was only achieved at the maximum treatment concentration of 5000 μg/plate. The relative mutagenic responses between the nitroreductase proficient and nitroreductase deficient strains used in this study indicated that nitroreduction plays a significant role in the observed mutagenic activity of the test substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Read-across - OECD TG 489 - Comet assay TPE.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Not yet defined

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: analogue substance 04
This test will be used in read-across on the target substance Acid Blue 284:1 [EC: 947-929-6].

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
This part refers to the available studies on the target substance Acid Blue 284:1 [EC: 947-929-6]:
- Available GLP studies: not available.
- Available non-GLP studies: not available.
- Historical human data: not available.
- (Q)SAR: not available.
- Weight of evidence: not available.
- Grouping and read-across:
In vitro gene mutation study in bacteria (OECD TG 471 – NR deficient strains) – Key study – Read-Across on analogue substance 03.
In vitro gene mutation study in bacteria (OECD TG 471) – Supporting study – Read-Across on analogue substance 02.
In vitro gene mutation in mammalian cells (OECD TG 476) – Key study – Read-Across on analogue substance 02.
In vitro mammalian chromosome aberration test (OECD TG 473) – Key study – Read-Across on analogue substance 02.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Under Annex VIII Section 8.4., column 2 of REACH, further mutagenicity studies must be considered in case of a positive result in an in vitro gene mutation study in bacteria.
Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both the mammalian cell tests are negative but there was a positive result in the bacterial test, it will be necessary to decide whether any further testing is needed on a case-by-case basis. For example, suspicion that a unique positive response observed in the bacterial test was due to a specific bacterial metabolism of the test substance could be explored further by investigation in vitro. Alternatively, an in vivo test may be required.
The submitted dossier contains results for the in vitro gene mutation study in bacteria, following the OECD TG 471 (Traditional Ames Test). The test was conducted on the analogue substance 02. Moreover, an in vitro gene mutation study, OECD TG 471 with NR deficient strains, was performed on the analogue substance 03 and used in read-across to cover the information requirements for the target substance. Both tests gave positive results and this raises the concern for in vivo gene mutation, since the influence of the nitro-reductase is demonstrated but needs further evidence.
In particular, annex VIII, Column 2 requires the registrant to consider appropriate mutagenicity in vivo studies already at the Annex VIII tonnage level, which involves studies mentioned in Annex IX (among OECD TG 474 Mammalian Erythrocyte micronucleus test, OECD TG 488 Transgenic Rodent Mutation Assay, OECD TG 489 In vivo mammalian Alkaline Comet Assay and OECD TG 486 Unscheduled DNA Synthesis).

CONSIDERATIONS ON THE IN VIVO STUDIES INSERTED IN THE DOSSIER AND EXPERT ASSESSMENT ON TESTING PROPOSAL
There are no in vivo studies conducted on the target substance or on similar substances, submitted with the present dossier.
However, in order to further and completely assess gene mutation properties of the target substance in different tissues of the animal, a Comet Assay, OECD TG 489, performed on the analogue substance 04 was presented as testing proposal.
Analogue substance 04 is, in fact, considered as representative of the mutagenic behavior of Acid Blue 284:1 [EC: 947-929-6] as specified in the read-across section.
OECD TG 489 allows to measure DNA strand breaks, that may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognizes primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.
Therefore, a confirmation by the Comet assay (for azo dyes the intestinal tract is the site of major metabolism and dye/metabolites absorption) would be sufficient to assess the genotoxic potential of the substance.
Finally, as reported in literature, from the analysis of 91 chemicals with published data from Comet Assay and Transgenic rodent mutation assay (TGR), the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract (predominantly stomach and colon data) and, hence, can be confidently performed to confirm in vivo gene mutation activity in terms of genotoxicity in general.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Sex:

not specified

Genotoxicity:

other: to be performed

Remarks on result:

other: the test is in read-across from a submitted testing proposal still under evaluation.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For further details refer to the attached document on genotoxicity assessment.

Justification for classification or non-classification

Classification for mutagenicity is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans:

The classification in Category 2 is based on:

- Positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

• Somatic cell mutagenicity tests in vivo, in mammals; or


• Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

 

A new classification will be applied when results of the "In vivo" Comet assay on analogue substance 04 will be available.