Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines, are well documented and scientifically acceptable.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1973

Report date:

1973

Reference 2

Reference Type:

publication

Title:

Testing Mutagenic Properties with the Dominant Lethal Test on the Male Mouse.

Author:

Lorke D. and Machemer L.

Year:

1975

Bibliographic source:

Environmental Quality and Safety, Suppl. Vol. 4, 239-246

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

EC Number:

224-073-5

EC Name:

Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Cas Number:

4193-55-9

Molecular formula:

C40H44N12O10S2.2Na

IUPAC Name:

disodium 2-[(1E)-2-[4-({4-[bis(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]ethenyl]-5-({4-[bis(2-hydroxyethyl)amino]-6-[(cyclohexa-1,3-dien-1-yl)amino]-1,3,5-triazin-2-yl}amino)benzene-1-sulfonate

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S. Ivanovas GmbH, Kisslegg, Germany.
- Age at study initiation: about 10 weeks.
- Weight at study initiation: mean: male: 30-35 g, female: 25-30 g.
- Housing: singly housed in Makrolon Type I, cages with wire mesh top.
- Diet: ad libitum, Altromin pelleted.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 24 - 26 °C.
- Humidity: about 60 %.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Tragacanth.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- Test solution: 20 % solution in demineralized water with 1 % tragacanth (corresponding to a volume of 25 ml/kg body weight).

Duration of treatment / exposure:

Single dosage.

Frequency of treatment:

Once.

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

20 males and 480 females.

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethanesulfonate (MMS) and trimethyl phosphate (TMPO)
- Route of administration: MMS: ip and TMPO: oral - gavage
- Doses / concentrations: MMS = 100 mg/kg and TMPO = 1000 mg/kg

Examinations

Tissues and cell types examined:

Number of fertile matings, implantation, resorption, live foetuses, and corpora lutea.

Statistics:

The χ2-test, the t-test or the distribution-free rank sum test (Wilcoxon) were used to assess the results.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The male mice treated with the test substance showed slightly somnolent after the application for about 3 hours. Then they showed a normal activity again, leaving no evidence of adverse effect of the substance seen.
All treated and untreated males survived to the end of the experiment.

FERTILIZATION
There were no significant differences between the untreated controls and the groups treated with the test substance.
The fertilization rates of the control and the treated group differed in all pairing weeks not significant from each other. The values ​​of the treated group were in all weeks slightly under the control; the effect of a substance can not be concluded, since the fertilization rate in general in line with the standard of the tribe.
The results also showed that TMPO treatment had no effect. MMS, on the other hand, considerably reduced the ability of the male to fertilize the female in the first two weeks of mating.

PRE IMPLANTATION LOSSES
Treatment with the test substance did not cause a biologically significant increase in preimplantation losses. This is also shown by comparing the implantation rates, from which pre-implantation losses can be estimated. Similarly TMPO did not increase pre-implantation losses. MMS, however clearly increased pre-implantation losses during the first week and - as can be seen from the number of implantations per female - even caused pre-implantation losses of embryos during the second and third week of mating.

POST IMPLANTATION LOSSES
The results did not differ significantly from those of the controls for any of the mating weeks. TMPO, however, caused a marked increase in post-implantation losses in the second mating week, and MMS in the first and second weeks. This led to a decrease in the number of live foetuses during these weeks.

DOMINANT LETHAL ASSAY
The test substance did not cause dominant lethal mutations. TMPO was effective in the second mating week. MMS had a marked effect in the first two weeks and it was probably also effective to a lesser extent in the third week.

Any other information on results incl. tables

Fertilization

	Number of femals				Fertilization rate
	Used		Per female fertilized
Pairing week	Control	TS	Control	TS	Control	TS
1	60	60	53	49	88.3	81.7
2	60	60	49	45	81.7	75.0
3	60	60	51	48	85.0	80.0
4	60	60	42	40	70.0	66.7
5	60	60	41	37	68.3	61.7
6	60	60	44	37	73.3	61.7
7	60	60	43	35	71.7	58.3
8	60	60	42	32	70.0	53.3
Total	480	480	365	323	76.0	67.3

TS: treated group

Post-implantation loss

	Living implants				Dead implants
	Total		Used		Total		Used
Pairing week	Control	TS	Control	TS	Control	TS	Control	TS
1	565	537	10.7	11.0	23	35	0.4	0.7
2	485	475	9.9*	10.6	29	25	0.6	0.6
3	579	529	11.4	11.0	20	16	0.4	0.3
4	479	418	11.4	10.5*	18	17	0.4	0.4
5	441	412	10.8	11.1	17	15	0.4	0.4
6	534	425	12.1	11.5	19	14	0.4	0.4
7	491	373	11.4	10.7	17	22	0.4	0.6
8	454	334	10.8	10.4	18	10	0.4	0.3
Total	1028	3503	11.0	10.8	161	154	0.4	0.5

TS: treated group; *: significant difference between control and test group.

Pre-implantation loss

	Corpora lutea				Implantation*				Pre implantation losses
	Total		Used		Total		Used		Total		Used
Pairing week	Control	TS	Control	TS	Control	TS	Control	TS	Control	TS	Control	TS
1	605	582	11.4	11.9	587	570	11.1	11.6	18	12	0.3	0.2
2	544	513	11.1	11.4	511	499	10.4	11.1	33	14	0.7**	0.3
3	608	559	11.9	11.6	598	543	11.7	11.3	10	16	0.2	0.3
4	503	450	12.0	11.2	495	435	11.8	10.9**	8	15	0.2	0.4
5	475	437	11.6	11.8	457	425	11.1	11.5	18	12	0.4	0.3
6	557	446	12.7	12.1	552	438	12.5	11.8	5	8	0.1	0.2
7	517	407	12.0	11.6	505	393	11.7	11.2	12	14	0.3	0.4
8	483	357	11.5	11.2	472	344	11.2	10.8	11	13	0.3	0.4
Total	4292	3751	11.8	11.6	4177	3647	11.4	11.3	115	104	0.3	0.3

TS: treated group; *: since it occurs that a placenta with 2 seedlings found at an implantation site, the number of implantations may be less than the sum of live and dead implants. **: significant difference between control and treated group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The dominant lethal test investigation did not shown any evidence of mutagenicity caused by the test substance.

Executive summary:

Method

The compound was administered orally in single dose to NMRI mice by gavage at the concentration of 5000 mg/kg. Methylmethanesulfonate (MMS) and trimethyl phosphate (TMPO) were used as positive controls.

Each of the 20 male mice in each group was mated with three untreated females directly after treatment. After insemination (determined by vaginal smear), or after a week, the females were isolated. This procedure was repeated weekly for eight weeks.

On the fourteenth day of gestation the females were sacrificed and the numbers of fertile matings, implantations, resorptions, live foetuses, and corpora lutea were determined.

Results

There were no significant differences between the untreated controls and the groups treated with the test substance. Treatment with the test substance did not cause a biologically significant increase in preimplantation losses and the results did not differ significantly from those of the controls for any of the mating weeks.

The test substance did not cause dominant lethal mutations.