Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Red 066 was weakly mutagenic in the reverse mutation assay using bacteria (Salmonella typhimurium) and was also clastogenic in the in vitro mammalian chromosome aberration test.

However, in the in vitro mammalian cell gene mutation test (HPRT locus) in Chinese hamster V79 cells, Reactive Red 066 and its metabolites did not show any mutagenic activity in this forward mutation system.

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

Study period:

January 26, 1993 to February 05, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 287380.26
- Expiration date of the lot/batch: November 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: stable

Target gene:

Preliminary Toxicity/Range-Finding test:
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. 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, as well as each negative control was used.

Mutagenicity test:
The mutagenicity test was performed with strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without and with microsomal 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 as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 µg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in the dark. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Species / strain / cell type:

other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver microsomal fraction S9

Test concentrations with justification for top dose:

Concentrations for cytotoxicity test:
20.5761 µg/plate
61.7284 µg/plate
185.1852 µg/plate
555.5556 µg/plate
1666.6667 µg/plate
5000.0000 µg/plate

Concentration for mutagenicity test:
61.7284 ug/plate
185.1852 µg/plate
555.5556 µg/plate
1666.6667 µg/plate
5000.0000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 and TA 1538 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 98, TA 100, TA 1537 and TA 1538 with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 1535 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
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 substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 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. It was supplemented with 10 % of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water.

The mutagenicity test was performed with strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without and with microsomal 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 as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 /zg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. 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 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 is considered to be mutagenic in this test system if the following conditions are 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 following strains:
S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538.
Generally a concentration-related effect should be demonstrable.

Statistics:

No appropriate statistical method is available

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

not valid

Additional information on results:

Toxicity test/Range finding test:
In the experiments without and with activation, Lanasol Rot 5B Roh Trocken (FAT 92354/A) led to a slight increase in the number of revertants at the highest concentrations. The test material exerted no toxic effect on the growth of the bacteria.

Toxicity test/range finding test

Six concentrations of FAT 92354/A ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. Normal background growth was observed. Slightly increased numbers of revertant colonies were observed in the experiments without and with activation at the highest concentration. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

 

Mutagenicity test, original experiment

In the experiments without microsomal activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. However, treatment of strain TA 100 with the test material lead to a slight increase in the number of revertant colonies at the concentration of 5000 µg/plate. In the experiments with activation performed on strains TA 98, TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine prototrophic mutants was observed in comparison with the negative control. Treatment of strains TA 100 and TA 1535 with the test material lead to a slight increase in the number of revertant colonies at the concentration of 5000 µg/plate.

Mutagenicity test, confirmatory experiment

In the experiments without microsomal activation performed on strains TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. However, treatment of strains TA 98 and TA 1535 with the test material lead to a slight increase in the number of revertant colonies at the concentration of 5000 µg/plate. With strain TA 100 this effect occurred at the concentrations of 1666.7 and 5000 µg/plate.

In the experiments with microsomal activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. Treatment of strain TA 100 with the test material lead to a slight increase in the number of revertant colonies at the concentrations of 1250 and 5000 µg/plate.

In the mutagenicity tests, normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

The various mutagens, pro-mutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

Conclusions:

In Ames assay, FAT 92354/A exerted a very weak mutagenic effect.

Executive summary:

The test was carried out to evaluate mutagenic activity of FAT 92354/A in bacterial test systems in the absence and presence of a rat liver S9 activity system. The test was performed following OECD, EEC and EPA guidelines in accordance to GLP principles. This test system permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium.

The concentration range of Lanasol Rot 5B Roh Trocken (FAT 92354/A) to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 ug/plate. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations.

In the toxicity test/range finding test experiments without and with activation Lanasol Rot 5B Roh Trocken (FAT 92354/A) led to a slight increase in the number of revertants at the highest concentrations. The test material exerted no toxic effect on the growth of the bacteria.

Mutagenicity test, original experiment:

In the original experiments without metabolic activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. However, in the experiment without activation carried out on strain TA 100, treatment with FAT 92354/A led to a slight increase of revertant growth at the highest concentration.

In the original experiments with metabolic activation performed on strains TA 98, TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiments with activation on strains TA 100 and TA 1535, treatment with FAT 92354/A led to a slight increase of revertant growth at the highest concentration.

In the confirmatory experiments without metabolic activation performed on strains TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. While in the experiment without activation carried out on strains TA 98, TA 100 and TA 1535, treatment with FAT 92354/A led to a slight increase of revertant growth at the upper concentrations. In the experiments with activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation on strain TA 100 treatment with FAT 92354/A led to a slight increase of revertant growth at the upper concentrations.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 92354/A exerted a very weak mutagenic effect in this test system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 20, 1995 to January 15,1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, Annex (November 18, 1987) Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test.

Version / remarks:

Official Journal of the European Communities, No L 133. Vol. 31, 61-63, May 30, 1988

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 287380.26
- Expiration date of the lot/batch: November 30, 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Stability of the test substance in the solvent/vehicle: Not determined

Target gene:

V79 Chinese hamster cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: cells resistant to 6-TG

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Range with metabolic activation:
0.244 to 500.0 µg/ml
Range without metabolic activation:
0.244 to 500.0 µg/ml
Mutagenicity test
Original experiment:
Range with metabolic activation:
18.52 to 500.0 µg/ml
Range without metabolic activation:
18.52 to 500.0 µg/ml
Confirmatory experiment:
Range with metabolic activation:
62.5 to 500.0 µg/ml
Range without metabolic activation:
62.5 to 500.0 µg/ml

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Nitrosodimethylamine with Metabolic activation and Ethylmethansulfonate without Metabolic activation

Details on test system and experimental conditions:

V79 Chinese hamster cells were originally derived from embryonic lung tissue. The cells were cultured in Ham's F10 medium supplemented with 10 % pre-tested foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin in tissue culture (plastic) flasks. The humidity in the incubator was adjusted to >85 % rH, the air was enriched to 5 ±2.0 Vol% C02 and the temperature was 37±1 °C. Twice per week the growth medium was replaced by fresh one. The laboratory cultures were passaged weekly in low number (about 5x10E4 cells per 175 cm²) to keep the level of spontaneous mutants low and to prevent the cells of reaching a stationary phase of cell growth. Large stocks of the V79 cell line have been stored in liquid nitrogen allowing the repeated use of the same cell culture batch in experiments. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. The frozen cell suspension contains 10% dimethylsulfoxide (DMSO). All stock cells were cultured in cleansing medium for three days to purge the cultures of existing hprt" mutants. Cleansing medium was growth medium supplemented with 3 uM aminopterin. The cells have a stable karyotype with a modal chromosome number of 22±1. All stock cells were checked for mycoplasma contamination, using the Hoechst- Dye staining method or the 6-MPDR method, before being frozen. Thawed stock culture cells are kept not longer than for twelve passages (three months) in culture.

Solubilisation of the test substance:
FAT 92354/A was dissolved in DMSO by warming to about 50 °C. The highest concentration of FAT 92354/A was determined in a preliminary solubilisation test to be 50.0 mg/ml soluble in DMSO. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO. The respective solutions were added 1:100 to the cell culture medium. The final concentration of DMSO in the culture medium was 1%. The test substance solutions were prepared immediately before the start of the test.

Preparation and composition of the metabolic activation mixture:
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male RAI rats (Tif: RAIf[SPF]) [7], reared at the Animal Farm of CIBA-GEIGY, Sisseln, Switzerland. The animals were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged at 9000x g for 15 minutes and the resulting supernatant (S9 fraction) was stored at approximately minus 80 °C for no longer than one year. The protein content of the S9 fraction was 36.64 mg/ml. S9 fraction was thawed immediately before use. The S9 mixture was prepared just prior use in an activation experiment and kept on ice [8]. The S9 mixture consisted of:
• Rat liver S9 fraction 250.0 µl/ml
• Glucose-6-phosphate 10.0 µmol/ml
• NADP 8.0 µmol/ml
• CaCl2 20.0 µmol/ml
• MgCl2 20.0 µmol/ml
• Na2HPC»4 1.0 µmol/ml
• FCS 30.0 µl/ml
Unused portions of S9 fraction and S9 mixture were discarded and not saved for another experiment.
The S9 mixture was immediately sterilised by filtration through a 0.45 µm filter. The activation mixture was added to the medium at a concentration of 10% in both the cytotoxicity test and the mutagenicity test and the final concentration of S9 fraction was 2.5 % during the treatment.

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, 2.5x10E5 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 2.5-5.0x10E6 cells of passage 28 (original experiment) and passage 26 (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 µl/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 2x10E6 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 2x10E6 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 2x10E6 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 artefact.
• 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 35x10E-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.

Statistics:

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.

Assessment of statistical significance of mutation frequency:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines [9] Criteria for a positive response
The test substance will be considered to be mutagenic if:
• The assay is valid (see assay acceptance criteria)
• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.
• There is a significant dose-relationship as indicated by the linear trend analysis.
• The effects described above are reproducible.

Exceptions:
By extreme cases or if the results only partially satisfy the above criteria the Study Director (if necessary after consulting the person who will approve the report) will interpret the results from his own experience. Positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance.

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 h and in the experiments without metabolic activation for 21 h. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

Cytotoxicity test:
A preliminary range finding test was run assessing cytotoxicity. FAT 92354/A was tested at concentrations up to 500.0 µg/ml. Higher concentrations could not be applied due to solubility limitations in the vehicle. In the part with metabolic activation, at the highest concentration of 500.0 µg/ml an acute growth inhibiting effect of 37.2 % could be seen. Without metabolic activation treatment with FAT 92354/A at the highest concentration of 500.0 µg/ml revealed an acute inhibition of growth of 85.9 %. Accordingly, 500.0 µg/ml with and without metabolic activation was 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. 500.0 ug/ml represents the solubility limit of the test chemical. The highest concentration revealed no substantial toxicity after treatment. After expression, a mean inhibition of growth of 38.0 %* was determined. 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 15.6 %. The mean growth inhibitory effect after the expression period was 27.7 %*. 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 relevant 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: 18.52, 55.56, 166.67 and 500.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 87.7%* and 23.9 %* respectively. 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 inhibitory effect of 62.1 %*. The mean growth inhibition after the expression period was 30.9 %*. Ethylmethansulfonate (EMS, 0.3 ul/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 relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Conclusions:

FAT 92354/A and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

FAT 92354/A was tested to determine mutagenic effects on V79 Chinese hamster cells in vitro according to OECD 476, EU and EPA guidelines. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 h and in the experiments without metabolic activation for 21 h. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment). N-Nitrosodimethylamine (DMN, 1.0 µl/ml) was used as positive control. A preliminary range finding test was run assessing cytotoxicity. FAT 92354/A was tested at oncentrations up to 500.0 µg/ml and at the highest concentration of 500.0 µg/ml an acute growth inhibiting effect of 37.2 % could be seen while in without metabolic activation treatment at the highest concentration of 500.0 µg/ml revealed an acute inhibition of growth of 85.9 %. Accordingly, 500.0 µg/ml with and without metabolic activation was 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. 500.0 µg/ml represents the solubility limit of the test chemical. The highest concentration revealed no substantial toxicity after treatment. After expression a mean inhibition of growth of 38.0 % was determined. 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 15.6 %. The mean growth inhibitory effect after the expression period was 27.7 %. 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 relevant 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: 18.52, 55.56, 166.67 and 500.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 87.7 % and 23.9 % respectively. 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 inhibitory effect of 62.1 %. The mean growth inhibition after the expression period was 30.9 %. 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 relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 92354/A and its metabolites did not show any mutagenic activity in this forward mutation system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Reactive Red 066 did not lead to increased DNA damage at and up to 2000 mg/kg bw/day when assessed in the in vivo mammalian alkaline comet assay, neither it was clastogenic in the in vivo micronucleus erythrocyte test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 27 August 2022; Experimental starting date: 01 September 2022; Study completion date - 23 January 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

Name of Test Item: FAT 92354
CAS No: 70210-39-8
Physical Appearance (with colour): Dark brown powder
Batch No: C/TE (MC-PD-R5B-03-21)
Purity (as per certificate of analysis): 67.6 %
Date of Manufacture: July 2021
Date of Expiry: 02 August 2026
Storage Conditions: Cool and dry (+2 to +8 ºC)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.
Source of supply: In house bred animals

Sex:

male

Details on test animals or test system and environmental conditions:

Animal species: Rat (Rattus norvegicus)
Strain: Wistar
Justification for selection of species: The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.
Source of supply: In house bred animals
No. of groups: Main Study: 6 groups (1 vehicle control, 2 positive control and 3 treatment groups)
No. of animals / group and sex: 6 males/group (36 males)
Age at treatment: 9 weeks
Body weight range at receipt (g): 181.05 to 229.99 g
Animal identification: Acclimatization period: All the animals were identified by tail marking using a black marker pen. Additionally, a cage card was displayed which included study no., cage no., sex, animal no. (temporary), start date and end date of acclimatization period.
Treatment period: All the animals were identified by body marking using turmeric solution, potassium permanganate and additionally, a cage card was displayed which included at least study no., cage no., sex, animal no. (permanent), treatment date and date of necropsy.

Husbandry:

Environmental Conditions: Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.3 to 22.5 ºC and relative humidity 49 % to 64 % in main study, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.

Housing: Maximum of three animals of same sex and group were housed in a standard polysulphonate rat cage (L 430 × B 280 × H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized corn cob was used as a bedding material.
Feed: Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.

Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula.

All the doses were administered in an equal volume of 10 mL/kg bw/day the dose of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg bw/day as low, mid and high dose, respectively. Vehicle control group (G1) animals were administered with vehicle.

Cyclophosphamide monohydrate were dissolved in distilled water and administered at a dose of 250 and 100 mg/kg bw/day, respectively.

Duration of treatment / exposure:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula.

Frequency of treatment:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula.

Post exposure period:

Post 3 hours of the last day dosing, terminal sacrifice was done for all animals and all the animals were subjected to gross pathological examination.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

High dose group

No. of animals per sex per dose:

6 males/group (36 males)

Control animals:

yes

Positive control(s):

Cyclophosphamide Monohydrate

Details of tissue and slide preparation:

Minimum of three slides were prepared. The smears before staining were fixed by immersing the slides in methanol for 5 minutes. The air dried slides were stained with May-Gruenwald and Giemsa stain for evaluation.

All the slides including those of positive and vehicle controls were coded before microscopic evaluation to avoid group bias during evaluation.
For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCEs: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).

Evaluation criteria:

For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCEs: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).
The average numbers of micronucleated polychromatic erythrocytes (MNPCEs) were observed for a minimum of 4000 polychromatic erythrocytes (PCEs). The average percentage of MNPCEs in animals dosed with vehicle was 0.07. In the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.07, 0.09 and 0.10. There was no statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at any of the doses, in comparison with vehicle control.

Statistics:

Body weight of day 1, 2 and 3 was analysed by SPSS, at a 95 % level (p≤0.05) of significance. Intergroup comparison of body weight of Day 1, 2 and 3 were done. The slides from main study were decoded after analysis, the number of PCEs (Polychromatic erythrocytes), RBC (Red blood corpuscles), MNPCEs (Micronucleated Polychromatic erythrocytes) and PCEs/ total erythrocytes ratio (Polychromatic erythrocytes/ total erythrocytes) and frequency of MNPCEs was calculated. The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95 % level (p ≤0.05) of significance. All analysis and comparisons were evaluated at the 95 % level of confidence (p <0.05). The clinical chemistry examination was analysed by SPSS at a 95 % level (p ≤0.05) of significance. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gamma-glutamyl transpeptidase (GGT) were subjected to statistical analysis.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The average percentage of MNPCEs in animals dosed with vehicle was 0.07. In the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.07, 0.09 and 0.10. There was no statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at any of the doses, in comparison with vehicle control.

The dose formulation samples were analyzed for the dose concentration by HPLC and the results are found within the range of ± 15% (i.e. 85% to 115%) recovery to the nominal concentration.

Clinical Signs of Toxicity and Mortality
The administration with the test item resulted in no clinical signs or mortality in any of the treated animals.

Body Weight
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

Gross Pathology
Grossly, pinkish discoloration of the stomach contents was observed in animals of G2, G3 and G4 group and this was attributable to the physical nature of the test item.
However, the mucosa of stomach was found to be within normal limits. There were no other gross pathological observations in the study.

Histopathology Findings
There was no test item-related histopathological findings in the study. The minimal, multifocal increased apoptosis in duodenal crypt epithelium in G5 group of animals (2/6), is considered to be related to administration of ethyl methanesulfonate which is used as positive control in the study.
Few microscopic findings observed in the study such as infiltration of mononuclear cells in liver and other findings were considered incidental as they occurred randomly across the groups including vehicle control group and/or were expected for laboratory rats.

Clinical Chemistry
No adverse treatment related variation was noted in clinical chemistry parameters. There were no statistically significant variations noted.

Conclusions:

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 92354, is neither clastogenic nor aneugenic at and up to 2000 mg/kg bw/day.

Executive summary:

A study was conducted according to OECD test guideline 474 to determine the genotoxic potential of FAT 92354 (test item) in the micronucleus test using bone marrow cells of Wistar rat.

The test item, vehicle control and positive control were dosed for three consecutive days by oral route using oral gavage cannula. After the last dosing, animals were sacrificed and bone marrow cells were collected. The slides of bone marrow cells were stained with May-Gruenwald and Giemsa stain and observed for incidences of micronucleated polychromatic erythrocytes (MNPCEs). 

This study used 6 groups of rats and each group consisted of 6 males. The animals received designated treatment for 3 consecutive days by oral route using gavage canula. The animals designated as group G1 animals were administered with corn oil as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 92354, respectively.

The animals in group G6 were administered 100 mg/kg bw/day of the positive control cyclophosphamide monohydrate (for micronucleus test).

Bone marrow was collected for G6 group.

In the micronucleus test, the average percentage of MNPCEs was 0.07 in males dosed with vehicle. For the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.07, 0.09 and 0.10, respectively. There was no statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at the doses of 500, 1000 and 2000 mg/kg bw/day of test item, in comparison with the vehicle control.

The positive control group (G6), cyclophosphamide monohydrate at 100 mg/kg bw/day exhibited statistically significant increase in the numbers of MNPCEs when compared to vehicle control and the average percentage of MNPCEs (per 4000 PCEs scored) in positive control was 0.79. This demonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate.

There was no statistically significant variation in body weight for treated animals.

No test item related gross or histopathological changes were seen in any of the treated animals.

No adverse treatment-related changes were observed in clinical chemistry parameters.

The dose formulation samples were analyzed for dose concentration by HPLC and the results are found within the range of ± 15 % (i.e. 85 % to 115 %) recovery to the nominal concentration.

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 92354, is neither clastogenic nor aneugenic at and up to 2000 mg/kg bw/day.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 27 August 2022; Experimental starting date: 01 September 2022; Study completion date - 23 January 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Name of Test Item: FAT 92354
CAS No: 70210-39-8
Physical Appearance (with colour): Dark brown powder
Batch No: C/TE (MC-PD-R5B-03-21)
Purity (as per certificate of analysis): 67.6 %
Date of Manufacture: July 2021
Date of Expiry: 02 August 2026
Storage Conditions: Cool and dry (+2 to +8 ºC)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.
Source of supply: In house bred animals

Sex:

male

Details on test animals or test system and environmental conditions:

Animal species: Rat (Rattus norvegicus)
Strain: Wistar
Justification for selection of species: The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.
Source of supply: In house bred animals
No. of groups: Main Study: 6 groups (1 vehicle control, 2 positive control and 3 treatment groups)
No. of animals / group and sex: 6 males/group (36 males)
Age at treatment: 9 weeks
Body weight range at receipt (g): 181.05 to 229.99 g
Animal identification: Acclimatization period: All the animals were identified by tail marking using a black marker pen. Additionally, a cage card was displayed which included study no., cage no., sex, animal no. (temporary), start date and end date of acclimatization period.
Treatment period: All the animals were identified by body marking using turmeric solution, potassium permanganate and additionally, a cage card was displayed which included at least study no., cage no., sex, animal no. (permanent), treatment date and date of necropsy.

Husbandry:
Environmental Conditions: Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.3 to 22.5 ºC and relative humidity 49 % to 64 % in main study, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.

Housing: Maximum of three animals of same sex and group were housed in a standard polysulphonate rat cage (L 430 × B 280 × H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized corn cob was used as a bedding material.
Feed: Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.

Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula. All the doses were administered in an equal volume of 10 mL/kg bw/day the dose of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg bw/day as low, mid and high dose, respectively. Vehicle control group (G1) animals were administered with vehicle. Positive control group G5 animals were administered with ethylmethane sulfonate.

The dose volume administered to each animal was calculated for individual animals on the day of treatment based on the individual animal body weight.

Duration of treatment / exposure:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula.

Frequency of treatment:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula.

Post exposure period:

Post 3 hours of the last day dosing, terminal sacrifice was done for all animals and all the animals were subjected to gross pathological examination.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

High dose group

No. of animals per sex per dose:

6 males/group (36 males)

Control animals:

yes

Positive control(s):

yes

Tissues and cell types examined:

Liver, glandular stomach, duodenum and testicles were collected for the comet assay.

The liver, glandular stomach and duodenum for the comet assay was placed into ice-cold mincing and/or homogenization buffer (DPBS) and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and stored in ice-cold mincing buffer until processed. A portion of the liver, glandular stomach and duodenum were collected and preserved in 10 % Neutral Buffered Formalin fixative and testes were preserved in modified Davidson’s fixative for 24 to 48 hours and then transferred to 10 % NBF for histopathology.

Details of tissue and slide preparation:

Three slides were prepared for each tissue from each animal and labelled with the study number, animal no., tissue, slide number (e.g., 1/3 to 3/3) and sex of each animal using pencil. To reduce the possibility of detachment of the agarose during the procedure, slides were pre-coated with 100 µL of liquid agarose and the agarose was allowed to dry to a thin film. Approximately 75 µL of cell suspension with 75 µL of 1.0 % low-melting agarose gel was mixed and rapidly pipetted onto the surface of the pre-coated slides and a coverslip was placed on it. The slides were placed on ice packs until the Agarose layer hardened (5 minutes). The slides were immersed in chilled lysing solution in the dark for overnight in a refrigerator under the light proof conditions. After completion of lysing, the slides were rinsed in distilled water to remove residual detergent and salts prior to alkali unwinding step.

Evaluation criteria:

All the slides were coded before evaluation to avoid group bias during evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes and stain with ethidium bromide, covered with a fresh coverslip and cells were scored under 400 X magnification. At least 150 cells were analyzed per sample. The open comet software was used for analysis. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs was determined for at least 150 cells per sample. After slide observation finished, slides were drained by keeping in cold 100 % ethanol for 20 minutes for dehydration. Slides were air dried and then placed in an oven at 50 °C for 30 minutes.

Statistics:

The percentage of tail DNA was analysed by SPSS at a 95 % level of confidence (p <0.05) of significance*. The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95 % level (p ≤0.05) of significance. All analysis and comparisons were evaluated at the 95 % level of confidence (p <0.05). Statistically significant changes obtained were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (p <0.05).

The clinical chemistry examination was analysed by SPSS at a 95 % level (p ≤0.05) of significance. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gamma-glutamyl transpeptidase (GGT) were subjected to statistical analysis.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results for the assessment of the test item, FAT 92354, to cause DNA strand breaks are provided for the doses of 500 [G2], 1000 [G3] and 2000 [G4] mg/kg bw/day, respectively. The average % tailing for DNA from male liver cells was 3.77, 3.83, 3.84 and 3.83 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. The average % tailing for DNA from glandular stomach cells were 3.27, 3.38, 3.22 and 3.33 respectively and in duodenum, the % tailing observed were 3.58, 3.82, 3.73, and 3.87 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose-dependent or statistically significant increase in the % tailing of DNA from cells of any organ for any of FAT 92354 groups when compared to the vehicle control group.
The positive control [G5], ethyl methanesulfonate at a dose of 250 mg/kg bw/day produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (liver, duodenum and glandular stomach) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the comet assay for this test of FAT 92354 were fully adequate.
The dose formulation samples were analyzed for the dose concentration by HPLC and the results are found within the range of ± 15% (i.e. 85% to 115%) recovery to the nominal concentration.

Clinical Signs of Toxicity and Mortality
The administration with the test item resulted in no clinical signs or mortality in any of the treated animals.

Body Weight
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

Gross Pathology
Grossly, pinkish discoloration of the stomach contents was observed in animals of G2, G3 and G4 group and this was attributable to the physical nature of the test item. However, the mucosa of stomach was found to be within normal limits. There were no other gross pathological observations in the study.

Histopathology Findings
There was no test item-related histopathological findings in the study. The minimal, multifocal increased apoptosis in duodenal crypt epithelium in G5 group of animals (2/6), is considered to be related to administration of ethyl methanesulfonate which is used as positive control in the study.
Few microscopic findings observed in the study such as infiltration of mononuclear cells in liver and other findings were considered incidental as they occurred randomly across the groups including vehicle control group and/or were expected for laboratory rats.

Clinical Chemistry
No adverse treatment related variation was noted in clinical chemistry parameters. There were no statistically significant variations noted.

Conclusions:

FAT 92354, did not lead to increased DNA damage at and up to 2000 mg/kg bw/day.

Executive summary:

The test item, FAT 92354, was evaluated in the “In vivo Mammalian Alkaline Comet Assay” as per OECD Guideline No. 489, adopted on 29 July 2016. This study was conducted to determine if the test item, FAT 92354, can cause an increase in DNA damage in cells from specific organs and also to detect damage induced to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes. The comet assay detects single and double stranded breaks when DNA is analyzed under alkaline conditions (>pH 13). These strand breaks, when they occur in vivo, may be repaired, resulting in no persistent effect, or may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. This study used 6 groups of rats and each group consisted of 6 males. The animals received designated treatment for 3 consecutive days by oral route using gavage canula. The animals designated as group G1 animals were administered with corn oil as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 92354, respectively. The animals in group G5 were administered was 250 mg/kg bw/day of the positive control ethyl methanesulfonate (for comet assay). Approximately 3 hours after the last dosing, all rats were sacrificed by cervical dislocation and the designated organs (liver, duodenum, glandular stomach and testis) were collected for G1 to G5 groups. The collected tissues were processed, single cells were isolated, and slides were prepared. Slides were run through submarine-type electrophoresis and drained. Drained slides were stained with ethidium bromide and evaluated for % tailing of DNA, i.e. tail length in microns measured from the estimated edge of the head region closest to the anode. In the comet assay the average % tailing for DNA from male liver cells was 3.77, 3.83, 3.84 and 3.83 at 0, 500, 1000 and 2000 mg/kg bw/day respectively. The average % tailing for DNA from glandular stomach cells were 3.27, 3.38, 3.22 and 3.33 and in duodenum, the % tailing observed were 3.58, 3.82, 3.73 and 3.87 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any of the organs for any of  FAT 92354 treated groups when compared to the vehicle control group. The average % tail DNA observed in the liver, glandular stomach and duodenum of the positive control (at 250 mg/kg bw/day of ethyl methyl sulfonate) dosed animals were 8.80, 8.65 and 9.03, respectively. The positive control [G5], ethyl methanesulfonate at a dose of 250 mg/kg bw/day produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (liver, duodenum and glandular stomach) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the comet assay for this test of FAT 92354 were fully adequate. There was no statistically significant variation in body weight for treated animals. No test item related gross or histopathological changes were seen in any of the treated animals. No adverse treatment-related changes were observed in clinical chemistry parameters. The dose formulation samples were analyzed for dose concentration by HPLC and the results are found within the range of ± 15 % (i.e. 85 % to 115 %) recovery to the nominal concentration. Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 92354, did not lead to increased DNA damage at and up to 2000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The potential of Reactive Red 066 to cause genetic toxicity was assessed in three in vitro studies followed by an in vivo comet assay combined with the micronucleus test.

In a reverse mutation assay using bacteria (Salmonella typhimurium), FAT 92354/A exerted a very weak mutagenic effect in this test system.

In the in vitro mammalian chromosome aberration test, FAT 92354/B induced structural chromosome aberrations in the V79 Chinese hamster cell line in the absence and the presence of metabolic activation.

In the in vitro mammalian cell gene mutation test (HPRT locus) in Chinese hamster V79 cells, FAT 92354/A, and its metabolites did not show any mutagenic activity in this forward mutation system.

In the in vivo comet assay combined with the micronucleus test, FAT 92354 did not lead to increased DNA damage, neither demonstrated any clastogenic nor anuegenic effects at and up to 2000 mg/kg bw/day when administered orally to Wistar rats.

Conclusion:

As Reactive Red 066 did not lead to increased DNA damage nor had clastogenic effect when administered orally to rats. This combined with the negative outcome observed in the in vitro mammalian cell gene mutation assay (HPRT locus), indicate that Reactive Red 066 will be not genotoxic to mammalian cell systems.

Justification for classification or non-classification

Based on the findings of the in vitro and in vivo genetic toxicity studies, Reactive Red 066 can be considered to be non-mutagenic according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.