A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1983)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

EC Number:

403-410-1

EC Name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Cas Number:

114565-65-0

Molecular formula:

C18 H16 N8 O3 S2 . x C7 H18 N2 . x C4 H11 N O2 . x C3 H9 N O

IUPAC Name:

(3-aminopropyl)diethylamine; 2-(methylamino)ethan-1-ol; 2-[(2-hydroxyethyl)amino]ethan-1-ol; 6-methyl-2-{4-[2-(triaminopyrimidin-5-yl)diazen-1-yl]phenyl}-1,3-benzothiazole-7-sulfonic acid

Details on test material:

- Name of test material (as cited in study report): FAT 11'184/B
- Physical state: powder, orange
- Analytical purity: No data mentioned in the report. However, in an Ames test report (CCR 124312, see IUCLID chapter 7.6.1) using the same batch of test substance the analytical purity was given as 89.6%.
- Lot/batch No.: Op. 1 BD 823/8
- Expiration date of the lot/batch: January, 1993
- Stability when pure: stable for 24 months
- Stability in vehicle: > 8 hours in H2O, ethanol, acetone, DMSO, and DMF
- Storage: at room temperature, light protected

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut für Versuchstierzucht, Hannover, Germany
- Age at study initiation: about 15 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes, the animals were distributed into the test groups at random and identified by cage number
- Housing: single, in Makrolon Type I cages with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet (ALTROMIN), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): not regulated
- Air changes (per hr): no data given
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

On the day of the experiment, the test article was suspended in 4% Carboxymethyl cellulose (CMC). The vehicle was chosen due to its nontoxicity
for the animals. All animals received a single standard dose volume of 20 ml/kg bw orally.

Details on exposure:

Pre-Experiment for Toxicity
For the purpose of dose selection, a preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity assay. From the pre-experiment, the maximum tolerated dose level was determined as the dose that caused toxic reactions without having major effects on survival within 72 hours. In the present case, 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg bw of the test article suspended in 4 % CMC. The volume administered was 20 ml/kg bw. All treated animals expressed toxic reactions consisting of reduction of spontaneous activity, eyelid closure and apathy. Thus, 5000 mg/kg bw was retained as dose level for the main test.

Duration of treatment / exposure:

Treatment
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. 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. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group.
Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 ml fetal calf serum, using a 5 ml syringe, into 1 ml fetal calf serum. The cell suspension was centrifuged at 1,000 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.

Frequency of treatment:

Single gavage

Post exposure period:

24, 48 and 72 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
5000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Six males and six females were assigned to each test group.
The test comprised a series of 3 test groups (negative control, treated group, positive control) for bone marrow sampling after 24 hours post treatment, 2 test groups (negative control and treated) for bone marrow sampling after 48 hours post treatment, and further 2 test groups (negative control and treated) for bone marrow sampling after 72 hours.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA) served for positive control substance.
The stability of CPA at room temperature is good; at 20°C only 1 % of CPA is hydrolysed per day in aqueous solution.
The substance was diluted in physiological saline at a dose level of 30 mg/kg bw/day; the solution was prepared on day of administration.
The animals were treated by single oral application (gavage), and the application volume was 10 ml/kg bw.

Examinations

Tissues and cell types examined:

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1,000 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 any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

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:

yes

Remarks:

After application of 5000 mg/kg bw of test article, the animals expressed same toxic symptoms as observed in the pre-experiment. One female died at preparation interval 48 hours.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

SUMMARY OF THE RESULTS OF THE IN VIVO MICRONUCLEUS TEST WITH NMRI MICE (mean values)
Sampling time (hr)	Test group	Test dose (mg/kg bw)	PCEs with Micronuclei (%)	Genotoxicity	PCE/NCE	Cytotoxicity	Toxic symptoms seen in the mice
24 hours	Solvent control	0	0.12	no	1000/458	no	no
	Test article	5000	0.03	no	1000/536	no	yes
48 hours	Solvent control	0	0.1		1000/577	no	no
	Test article	5000	0.11	no	1000/715	no	yes and one case of death (♀)
72 h	Solvent control	0	0.06		1000/533	no	no
	Test article	5000	0.09	no	1000/569	no	yes
24 hours	CPA	30	1.34	yes	1000/672	no	no data
PCE polychromatic erythrocytes; NCE normochromatic erythrocytes; CPA Cyclophosphamide (positive control)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative