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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to internationally accepted testing guidelines and performed according to GLP. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to Section 13 of this dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
rodent dominant lethal assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate]
EC Number:
240-521-2
EC Name:
Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate]
Cas Number:
16470-24-9
Molecular formula:
C40H44N12Na4O16S4
IUPAC Name:
tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[bis(2-hydroxyethyl)amino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany.
- Age at study initiation: 6 - 12 weeks.
- Weight at study initiation: 36 to 46 g males, 26 - 34 g females.
- Assigned to test groups randomly: yes.
- Housing: during mating one male and one female per cage; after mating one female per cage.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: at least one week.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 0.5 °C
- Humidity: 45 - 55 %
- Air changes: 10 air changes per hour
- Photoperiod: 12/12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water.
- Concentration of test material in vehicle: 250 or 125 mg/ml.
- Amount of vehicle: 20 ml/kg.
Duration of treatment / exposure:
Sinlge treatment.
Frequency of treatment:
Single dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
2500, 5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
50 males per group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Historical data.

Examinations

Tissues and cell types examined:
Number of corpora lutea, implantations, alive and dead embryo.
Evaluation criteria:
1. Living implantations: reddish color, in addition reflexes and heartbeat my be found.
2. Dead implantations: dark color, no reflexes, no heartbeat. Dead implants are subdivided by their size and color into those embryos which died early and those which died late.
3. Total implantations: living and dead implantations.
4. Corpora lutea: Ovaries were examined using a dissection microscope. Corpora lutea can be seen as sickle-shaped, light-yellow bodies on the surface of ovaries.

If no implantations were found, uteri were stained using 10 % ammoniumsulfide solution in water. After staining, implantation sites can be seen as black dots or lines.
Statistics:
The frequency distribution of the various parameters (dead and living implants, total implants, and pre-impllantation losses) of the control and treatment groups was compared by the non-parametric KOLMOGOROV-SMIRNOV test.

The number of dead implants and total implants (square root transformation), the ratio of dad implants to total implants and the preimplantation loss based on corpora lutea (angular transformation) were checked with the bifactorial ANALYSIS OF VARIANCE.

Analysis of contrasts will be performed for the respective parameter, if its interaction factor (differences between the groups) is significantly altered (p < 0.05)

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
FERTILIZATION RATE
No effect on the treated males ferilization rates were recorded.

PRE-IMPLANTATION LOSSES
The pre-implantation losses are based on the implantation rates and corpora lutea per fertilized female. The test substance had no effect on the pre-implantation losses. The frequency distribution comparisons by the Kolmogorov-Smirnov test, in respect to total implants and pre-implantation losses based on the corpora lutea, did not detect any significant differences, neither for single periods nor for the total test.
The analysis of variance of the females implantation counts also did not reveal any indication of relevant variations. This also applied to the analysis of variance of the pre-implantation losses based on corpora lutea.
The test substance did not therefore result in an increase in pre-implantation losses.

POST-IMPLANTATION LOSSES
The post-implantation losses are based on the rates of live and dead implants per fertilized female. The results do not reveal that the test substance produced an effect. The comparison of frequency distribution by Kolmogorov-Smirnov test for live and dead implants did not reveal significant variations for the individual periods or for the total test. The analysis of variance of dead implants likewise did not detect a significant variation.
This also applied to the analysis of variance of the ratio of dead implants to total implants.

Any other information on results incl. tables

Toleration by the Male Mice

After acute oral treatment with 2500 mg/kg or 5000 mg/kg the treated males showed no symptoms. The animals appeared normal. Their external appearance and physical activity then remained unaffected. There were no substance-induced mortalities.

Observations and Findings

Until the end of the test one male of the group dosed at 2500 mg/kg showed roughened fur and emaciation from day 20 to day 23 after treatment. Furthermore one male of the group treated with 5000 mg/kg of the test item died at 48 days (at the end of the last mating interval) after treatment.

These findings were judged as not treatment-related

Applicant's summary and conclusion

Conclusions:
There was no indication of a mutagenic effect of the test item at the acute oral doses of 2500 and 5000 mg/kg bw in the dominant lethal test on the male mouse.
Executive summary:

Method

The compound was administered orally in single doses to male albino mice (NMRI) which were then mated to untreated females. Doses of 2500 and 5000 mg/kg bw were given each to 50 males.

Number of corpora lutea, implantations, alive and dead embryo were examinated.

Results

No effect on the treated males ferilization rates were recorded. The test substance did not therefore result in an increase in pre-implantation losses. Furthermore the results do not reveal that the test substance produced an effect on post-implantation losses.