Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

other: experimental results on same main component, with a different counter ion. The different salificaton which does not influence the characteristics related to the specific end-point

Adequacy of study:

weight of evidence

Study period:

From November 6th to December 12th, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986 "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay."

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Red 253 (NA:TEA salt)

Molecular formula:

C35H42N11NaO12S2

IUPAC Name:

Direct Red 253 (NA:TEA salt)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH- 4414 Füllinsdorf/Basel, Switzerland.
- Age at study initiation: minimum 10 weeks at start of acclimatization.
- Body Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: animals were distributed into the test groups at random and identified by cage number.
- Fasting period before study: approximately 18 h before treatment with the test article the animals received no food but water ad libitum.
- Housing: single, in Makrolon Type I cages, with wire mesh top and granulated soft wood bedding.
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.).
- Water: tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf, F.R.G.).
- Acclimatization: minimum 5 days.
- Quarantine: according to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of the testing institute for one week after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 30 - 70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: aqua dest.
- Justification for choice of vehicle: the vehicle was chosen to its nontoxicity for the animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
On the day of the experiment, the test article was dissolved in aqua dest.
All animals received a single standard dose volume of 20 ml/kg body weight orally. At the beginning of the treatment, the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight.

PRE-EXPERIMENT FOR TOXICITY
A preliminary study on acute toxicity was performed with the same strain and, under identical conditions as in the mutagenicity study .

Frequency of treatment:

Once

Post exposure period:

The animals were sacrificed by cervical dislocation.

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

42 males and 42 females, 6 animals x sex x group.
10 animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: Cyclophosphamide (CPA)
- Vehicle: physiological saline.
- Dose: 40 mg/kg b.w.
- Volume Administered: 10 ml/kg b.w.
- Justification for choice of positive control: the stability of CPA at room temperature is good. At 20 °C, only 1 % of CPA is hydrolysed per day in aqueous solution.

Examinations

Tissues and cell types examined:

Bone marrow cells (polychromatic erythrocytes, PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 h.

TREATMENT AND SAMPLING TIMES
Sampling of the bone marrow was done 24, 48 and 72 h after treatment.

DETAILS OF SLIDE PREPARATION
The femora were removed, epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect, the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.

A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test (Krauth, 1971).

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with test item were in the same range as compared to the negative control groups.

40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.

PRE-EXPERIMENT FOR TOXICITY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg bw of the test substance dissolved in aqua dest. The volume administered was 20 ml/kg bw. The following animals expressed slight toxic reactions: one male and one female expressed reduction of spontaneous activity within 6 hours after administration.
Higher dosing was not attainable: appropriate solutions could be obtained only up to 250 mg/ml; application volumes higher than 20 ml/kg b.w. were not justifiable for the rodents used.

Any other information on results incl. tables

Summary of results

Test group	Dose, mg/kg b.w.	Sampling time, h	PCEs with micronuclei, %	Range	PCE / NCE
Solvent	0	24	0.02	0 - 1	1000 / 964
Test article	5000		0.06	0 - 2	1000 / 931
Cyclophosphamide	40		0.76	4 - 12	1000 / 1002
Solvent	0	48	0.04	0 - 1	1000 / 702
Test article	5000		0.05	0 - 2	1000 / 747
Solvent	0	72	0.13	0 - 3	1000 / 662
Test article	5000		0.10	0 - 2	1000 / 724

Applicant's summary and conclusion

Conclusions:

During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

The in vivo genetic toxicity potential of the test substance to induce micronuclei in polychromatic erythrocytes, PCE, in mouse bone marrow was investigated according to internationally accepted guidelines. The test article was dissolved in aqua dest; the volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article, the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article, the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w.

In a pre-experiment, this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. After treatment with the test article, the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the test article were in the same range as compared to the negative control groups. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

Conclusion

During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-clastogenic in the micronucleus assay.