Disodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]amino]anthracene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Blue 19:1 is not mutagenic in the cacterial reverse mutation assay (Ames test) either with or without exogenous metabolic activation at the dose levels investigated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Reactive Blue 19:1 is not clastogenic or aneugenic in the mouse micronucleus test.

Link to relevant study records

Reference

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

Study period:

1987/1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: NMRI mouse
Strain: Hoe: NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 7 weeks
Number of animals: 70 (35 males / 35 females)
Bodyweight at start of study: males mean= 31.5 g (27 - 35 g); females: mean= 25.1 g (22 - 30 g)
Acclimatization: at least 5 days
Food: mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe) ad libitum
Water: tap water in plastic bottles, ad libitum
Housing: 5/cage
Room temperature: 22 +/- 2°C
Relative humidity: 55 +/- 10%
Lighting time: 12 h/12 h light/dark cyclus

In-life dates: 14 to 17. December 1987

Route of administration:

oral: gavage

Vehicle:

deionised water

Details on exposure:

The dose levels for micronucleus testing were selected on the basis of a preliminary study to determine the acute toxicity and the maximal applicable dose. Oral administration of 5000 mg/per kg bodyweight did not lead to a partial lethality in male and female mice. It is considered the maximal applicable dose and was selected as dose level for the main study.

The 5000 mg/kg bw dose was prepared as a 25% (w/v) solution and administerd at a volume of 20 mL/kg bw devided into two equal parts of 10 mL/kg bw administered within 2 hours

The test compound dilutions were prepared fresh each day. 6250 mg test substance were weight in a beaker, mixed with deionisized water, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.

Duration of treatment / exposure:

animals were killed after 24, 48 or 72 hours after administration of the test compound

Frequency of treatment:

The test compound was given in two equal parts within two hours on Day 1 of the study

Post exposure period:

24, 48, and 72 h

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

25% (w/v)

No. of animals per sex per dose:

5 for each killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamid (Endoxan) (Charge 105459)
- Justification for choice of positive control: positive control recommended in the guideline and regularly used by the laboratory
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw / 0.5 % (w/v)

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Staining procedure
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

Evaluation criteria:

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).

Statistics:

The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were performed using the "Diamant" computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

faeces blue coloured, urine blue coloured, diarrhea

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All animals survived after administration of 5000 mg/kg body weight test substance. The following signs of toxicity were observed: faeces blue coloured,
urine blue coloured, diarrhea. 48 hours after application all animals were free of clinical signs of toxicity.
The dissection of the animals revealed the following macroscopic findings: 24 hours after application most of the treated animals showed a blue coloured content of appendix and colon.

The incidence of micronucleated polychromatic erythrocytes in the substance treated groups was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the respective control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound. However due to a relative low ratio of polychromatic to normochromatic erythrocytes in female animals of the control group (24h and 72h killing
time), the female treatment group of 5000 mg/kg body weight at the killing time of 72 hours differed significantly. This effect is considered as of no toxicological relevance, as the values lie well within historical controls.

Cyclophosphamide induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensivity of the test system.

Conclusions:

Reactive Blue 19:1 did not lead to a substantial increase of micronucleated polychromatic erythrocytes. It is hence not genotoxic in the micronucleus test.

Executive summary:

Reactive Blue 19:1 was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg/kg body weight.

The 5000 mg/kg body weight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

The animals were treated once with the test compound (given in two equal parts of 10 mL/kg body weight within two hours) and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

Cyclophosphamide was used as positiv control substance and was administered orally at a single dose of 50 mg/kg body weight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the respective control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound. However due to a relative low ratio of polychromatic to normochromatic erythrocytes in female animals of the control group (24h and 72h killing time), the female treatment group of 5000 mg/kg body weight at the killing time of 72 hours differed significantly. This effect is considered as of no toxicological relevance, as the values lie well within historical controls.

Cyclophosphamide induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a signifi cant extend.

The results indicate that, under the conditions of the present study, Reactive Blue 19:1 is not clastogenic or aneugenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reactive Blue 19:1 was tested for mutagenicity with the bacterial strains TA100, TA1535, TA1537, TA1538, TA98 of Salmonella typhimurium and Escherichia coli WP2uvrA. The test substance is not mutagenic in these test systems either with or without exogenous metabolic activation at the dose levels investigated.

Reactive Blue 19:1 was tested for clastogenicity and aneugenicity in the in vitro chromosome aberration assay (V79 cells) and the in vivo micronucleus test in the mouse. The results indicated that, under the conditions of the present studies, the test substance is not genotoxic in these test systems.

Justification for classification or non-classification

No effect occured in in vitro and in vivo tests; hence, no classification is necessary.