C.I. Reactive Yellow 2

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2020 - 18 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of trisodium 4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate and trisodium 4-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate

EC Number:

943-063-8

Molecular formula:

C25H15Cl3N9Na3O10S3 (both components have the same molecular formula)

IUPAC Name:

Reaction mass of trisodium 4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate and trisodium 4-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate

Test material form:

solid: particulate/powder

Details on test material:

Physical Appearance: Orange powder.
Storage conditions: The test material was stored in a dark storage place at room temperature.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was put into a tight container and stored at room temperature of the test item storage room (permissible range: from 10 to 30 °C)
- Stability under storage conditions: stable.
- Solubility in dimethylsulfoxide: ≤ 439 g/L
- Solubility in acetone: ≤ 0.03 g/L
- Water solubility: ≥ 250 mg/mL (confirmed in a fourteen day repeated dose oral toxicity study in rats)

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Mice are widely used in micronucleus tests because the observation of the micronuclei is easy and the historical data is abundant.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks.
- Weight at study initiation: Male: 31.6-37.1 g, Female: 25.2-26.8 g
- Assigned to test groups randomly: yes, under following basis: after acclimation, healthy mice were allocated by a body weight-stratified random sampling method on the day before administration.
- Housing: Animals were housed 5 or less by cage (before allocation) and 3 animals or less by cage (after allocation) in polycarbonate cages with flat bottom (210 W x 320 D x 130 H mm).
- Diet: ad libitum
- Water: ad libitum (Hita City water supply, chlorinated at about 3 to 5 ppm by adding sodium hypochlorite (Purelox))
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 23.1 – 24.3 °C
- Humidity: 51.1 – 63.4 %
- Air changes: 10 - 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: distilled water.
- Justification for choice of vehicle: The test material was soluble in water at 250 mg/mL and the test material solution of 250 mg/mL prepared with water was considered to be stable as there was no change in colour until 19 hours after preparation.
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle: 10 mL/kg based on the body weight on the administration day

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test material (2.765 g) was weighed and dissolved in distilled water using a laboratory mixer and ultrasonicator to make a 10 mL solution of 200 mg/mL test material solution. This solution was serially diluted with distilled water to prepare 100 and 50.0 mg/mL test material solutions, using a magnetic stirrer.
- The test material solutions were prepared on each administration day, stored in a cold place and used within 19 hours of preparation.

Duration of treatment / exposure:

2 days.

Frequency of treatment:

Test material solution: 2 doses within a 24 hour interval.
Negative control: 2 doses within a 24 hour interval.
Positive control (MMC): 1 dose within a 24 hour interval during the second administration of the test material.

Post exposure period:

Animals were observed frequently until 1 hour after each administration. In addition, the animals were observed at 1 day after each administration. Body weights were measured once a day for 3 days (from the first administration day to the day after the second administration) using an electronic balance (Sartorius). The specimens of the negative and the positive control groups, and all 3 doses in the test material group were observed microscopically (x 1000) in a blinded manner.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

500 mg/kg/day: 5 male animals.
1000 mg/kg/day: 5 male animals.
2000 mg/kg/day: 5 male animals and 3 female animals.

Control animals:

yes

Positive control(s):

- Positive control: Mitomycin C (MMS)
- Justification for choice of positive control: MMC is listed as a positive control material in the test method described in “5. TEST METHOD”, and historical data has been accumulated in the testing facility.
- Route of administration: intraperitoneal injection.
- Doses / concentrations: MMC (2 mg) was weighed, added into distilled water, and mixed with a laboratory mixer to make a 10 mL solution of 0.2 mg/mL MMC solution. The positive control material solution was treated in the safety cabinet. One dose a day was administered (2 mg/kg/day).

Examinations

Tissues and cell types examined:

4000 polychromatic erythrocytes (PCE) per animal and 500 erythrocytes (TE) per animal.

Details of tissue and slide preparation:

TIMING OF PREPARATION
- Specimens were prepared 24 hours after the second administration in the test material groups.

DETAILS OF SLIDE PREPARATION
- The animals were euthanised by a cervical dislocation.
- The femur was removed and the bone marrow cells were collected with approximately 0.8 mL of a heat-inactivated foetal bovine serum into a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes.
- A small amount of the cell suspension was smeared on a glass slide.
- The smears were air-dried and fixed with methanol.
- The specimens were stained with 3 vol % Giemsa solution prepared with 1/15 mol/L phosphate buffer solution (pH 6.8) and treated with 0.004 w/v % citrate aqueous solution.
- The specimens of the negative and positive control groups, and all three doses in the test material group were observed. Slide numbers were allocated randomly.

METHOD OF ANALYSIS
- The frequency of micronucleated polychromatic erythrocytes (MNPCE/PCE) was calculated.
- The ratio of PCE to TE (PCE/TE) was calculated.

Evaluation criteria:

EVALUATION CRITERIA
- The test was considered as negative if either a) or b) were satisfied for the MNPCE/PCE in the test material dose:
a) all results are inside the distribution of the historical data of the negative control group.
b) outside the distribution of the historical data of the negative control group, but none of the doses of the test material exhibit a statistically significant increase compared with the concurrent negative control.
- The test was considered to be positive is all c), d), e) and f) were fulfilled:
c) outside the distribution of the historical data of the negative control group.
d) the doses of the test material exhibit a statistically significant increase compared with the concurrent negative control.
e) the increase of the MNPCE/PCE is dose-related.
f) both the micronucleus test and confirmation test are fulfilled in case of c) and d).

ACCEPTABLE CRITERIA
- The means of the MNPCE/PCE in the concurrent negative control are “under control” in the quality control method used for the control chart in the historical data.
- The means of the MNPCE/PCE in the concurrent positive control are “under control” in the quality control method used for the control chart in the historical data. Concurrent positive controls produce a statistically significant increase compared with the concurrent negative control.
- In the test material group, the PCE/TE is 20 % or more compared with that of the negative control group at all observation doses.

Statistics:

MNPCE/PCE
- The Conditional Binomial test (Kastenbaum and Bowman) was conducted to compare the MNCPE/OCE in each dose of the test material group and the positive control group with that in the negative control group.
- Upper tailed significance levels of 5 % and 1 %.
- A significant different was not obtained.

PCE/TE
- Statistical tool, “StatLight” was used for all tests.
- All the data except for the positive control group was tested by Bartlett’s test for homogeneity of variance. Since the variances were homogenous the William’s test was conducted.
- Since there were no significant differences within the William’s test the Dunnett’s test was performed to compare the mean in the negative control group with that in each dose of the test material group.
- The comparisons were tested by the F test for homogeneity of variance between the groups.
- Since the results of the F test showed homogeneity, Student’s t test was performed to compare the mean in the positive control group with that in the negative control group.
- Significance levels: 5 % for Bartlett’s test and F test. Both sides of 5 % for Williams test, and both sides of 5 % or 1 % for other tests.

Results and discussion

Test results

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:

CLINICAL SIGNS/ OBSERVATIONS
- No abnormalities were observed at any doses of the test material group, the negative control group or the positive control group.
- No abnormal changes were noted in the body weight of the animals when compared with the negative control group in all doses of the test material group until the day after the second administration.

MNPCE/PCE
- Mean result in negative control group: 0.085 %
- Mean result in the positive control group: 7.195 %
- Mean result at 500 mg/kg/day: 0.035 %
- Mean result at 1000 mg/kg/day: 0.055 %
- Mean result at 2000 mg/kg/day: 0.060 %
- At all doses in the test material group the results were within the range of the historical data of the negative control.
- There was no significant increase in MNPCE/PCE in any doses of the test material group compared with the negative control group at 5 % level.
- At the positive control group, a statistically significant increase was observed at 1 % level.

PCE/TE
- Mean result in the negative control group: 47.0 %
- Mean result in the positive control group: 38.3 %
- At the positive control group a statistically significant decrease was observed at 5 % level, compared with the negative control group.
- Mean result at 500 mg/kg/day: 47.2 %
- Mean result at 1000 mg/kg/day: 47.4 %
- Mean result at 2000 mg/kg/day: 49.2 %

DISCUSSION
- The means of MPCE/PCE in the negative and positive control groups were within the range of the respective historical data in the testing facility.
- In the test material group the PCE/TE was 20 % or more compared with the negative control group at all observation doses therefore it was confirmed that the test was conduced appropriately.
- The means of the MNPCE/PCE of the test material group at all observation doses were within the range of the historical data of the negative control. Therefore, the results were judged to be negative.
- No statistically significant differences were observed in the PCE/TE test at any doses of the test material group compared to the negative control group therefore the exposure of the test material to bone marrow cells was not proven.
- The potential to induce micronuclei of the test material could be evaluated adequately as 2000 mg/kg/day, which was the maximum dose in the test method, was set in the present test.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the test material did not induce micronuclei in mice bone marrow cells.

Executive summary:

The ability of the test material to induce micronuclei in mice was investigated in accordance with the standardised guidelines OECD 474, under GLP conditions.

During the study, the test material was administered to 7 week old CD-1 mice twice in a 24-hour interval by oral gavage. The highest dose for administration was determined to be 2000 mg/kg/day from the results of the acute oral toxicity study in rats, and two lower doses of 1000 and 500 mg/kg/day were set based on a geometric ratio of 2. Five males were used for each dose level. The test material was administered to three females at a dose of 2000 mg/kg/day to evaluate sex differences in the maximum tolerance dose (MTD). Negative and positive control groups were set. As the negative control item, distilled water was administered at 10 mL/kg twice in a 24-hour interval by oral gavage. The positive control, Mitomycin C was administered once, intraperitoneally at 2 mg/kg/day. There was no mortality at the dose of 2000 mg/kg/day in both male and female mice, therefore it was estimated that there were no sex differences and the MTD determined by the death of animals was 2000 mg/kg/day or more. Therefore, the three doses were set for male mice in the micronucleus test. The frequencies of the micronucleated polychromatic erythrocytes to polychromatic erythrocytes (MNPCE/PCE) in bone marrow cells and the ratio of PCE to total erythrocytes (PCE/TE) were examined.

The means of MNPCE/PCE in all observation doses of the test material group were within the range of the historical data of the negative control. Therefore, the results were judged to be negative. As such it was concluded that the test material did not induce micronuclei in mice bone marrow cells under the conditions of this study.