Cuprate(4-), [2-[[[[2-hydroxy-3-sulfo-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]phenylmethyl]azo]-4-sulfobenzoato(6-)]-, sodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

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:

1992 / 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to recent EU & OECD test guidance in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

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:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony (own breed)
- Strain: Hoe: NMRKf (SPF71)
- Age at study initiation: 7 weeks
- Weight at study initiation (mean): males: 29.3 g; females: 22.5 g
- Assigned to test groups randomly: yes, under following basis: computer assisted randomization plan
- Fasting period before study: No data
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): Altromin 1324, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water

Details on exposure:

The test compound dilutions were freshly prepared at the day of administration. 12500 mg Remazol-Brillantblau BB Neu were weight in a beaker, mixed with deionized water, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.

For the EndoxanR stock solution, 5 ml distilled water were added to 100 mg EndoxanR in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2 %stock solution were mixed with 6 ml distilled water.

Duration of treatment / exposure:

24, 48 and 72 hours

Frequency of treatment:

Once

Post exposure period:

None

Remarks:

Doses / Concentrations:
5000 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females per dose group and killing time-point

Control animals:

yes

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):proven cytostatic agent and known clastogen with bifunctional alkylation action
- Route of administration: oral, gavage
- Doses / concentrations: 50 mg/kg body weight

Tissues and cell types examined:

chromosomes of bone-marrow erythroblasts

Details of tissue and slide preparation:

Rationale for dose selection
Oral administration of 5000 mg Remazol-Brillantblau BB Neu per kg bodyweight did not lead to a partial lethality in male and female mice. Consequently, the maximum applicable dose and was selected as dose level for the main study.

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 approx. 1000 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
- 5 minutes in May-Grünwalds solution
- 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 which they belonged to 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.

Both biological and statistical significances are considered together in evaluation:
A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

A Wilcoxon-Test (one-sided) was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.
A Wilcoxon-Test (one-sided) was calculated for each measurement group (24h, 48h, 72h) and for polychromatic and normochromatic erythrocytes. These tests were performed sequentially with a multiple level of significance of 5%. Tests on lower dose groups are only performed if the higher dose group is significantly different from the control.

The presupposition to make any of the following comparisons is a difference between the positive control and the negative control (24h). This is tested with a Wilcoxon-Test (two-sided) with 5%-level of significance. Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each measurement group (24h, 48h, 72h) at a multiple level of significance of 5%. Lower dose groups are only tested if the higher dose group is significantly different from the control. Actual data were also compared with historical controls.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after application of 5000 mg per kg bodyweight. The following signs of toxicity were observed: faeces blue coloured and diarrhea. 24 hours after application all animals were free of clinical signs of toxicity.
The dissection of the animals revealed no test substance related findings.
The bone marrow smears were examined for the occurance of micronuclei in red blood cells.
The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of Remazol-Brillantblau BB Neu 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 from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

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

Conclusions:

Interpretation of results (migrated information): negative
Administration of Remazol-Brillantblau BB Neu at a limit dose of 5000 mg/kg bw did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the mouse micronucleus test.

Executive summary:

Remazol-Brillantblau BB Neu was tested in the micronucleus test. The test compound was suspended in deionized water and dosed once orally at 5000 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Endoxan was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

All animals survived after application of 5000 mg per kg bodyweight. The following signs of toxicity were observed: faeces blue coloured and diarrhea. 24 hours after application all animals were free of clinical signs of toxicity.

The dissection of the animals revealed no test substance related findings.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with

Remazol-Brillantblau BB Neu and was statistically not different from the control values.

Endoxan 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 significant extent.

The results indicate that, under the conditions of the present study, Remazol-Brillantblau BB Neu is not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Remazolbrillantblau BB was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2 uvrA. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A doserange of different doses from 4 µg/plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positiv control compounds gave the expected increase in the number of revertant colonies.

Toxicity:

The test compound proved to be not toxic to the bacteria. 5000 µg/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity:

In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of metabolic activation system, treatment of the cells with Remazolbrillantblau BB did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Remazolbrillantblau BB is not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.

Remazol-Brillantblau BB neu was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified pre incubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive con­trol compounds gave the expected increase in the number of revertant colonies. Toxicity: The test compound proved to be not toxic to the bacterial strains. 5000 microgram/plate was chosen as top dose level for the mutagenicitystudy.

a) Ames Test:

In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10%), treatment of the cells with Remazol-Brillantblau BB neu did not result in relevant increases in the number of revertant colonies.

b) Prival Test:

In the presence of rat liver S-9Mix (30%) and hamster liver S-9Mix (30%) using the pre incubation method according to Prival Remazol-Brillantblau BB neu did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Summarizing, it can be stated that Remazol-Brillantblau BB neu is not mutagenic in the standard plate test (Ames Test) and in the pre incubation method according to Prival

The test substance Remazol-Brillantblau BB FWTR was examined for clastogenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced no significant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 25 µg/ml up to the limit of solubility (2000 µg/ml). No cytotoxic effect was also observed with metabolic activation up to the limit of solubility. For mutagenicity testing two independent cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 µg/ml) were used.

For main experiment dose levels of 200, 600 and 2000 µg/ml were used, in the absence and in the presence of S9-mix metabolic activation.

The test compound did induce a slight but not statistical increase of the aberration rate exclusive Gaps at 6 h 2000 µg/ml (without S9-mix), at 18 h 600 and 2000 µg/ml (with and without S9-mix) and inclusive Gaps at 28 h 2000 µg/ml (with S9-mix). Additionally a statistical increase exclusive Gaps was observed at the preparation time 28 h in the presence of metabolic activation. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

A second Chromosome Aberration Assay was performed to investigate the potential of Remazol-Brillantblau BB neu to induce chromosome aberrations in V 79 cells of the Chinese hamster in vitro at even higher concentrations than the former assay. The assay was performed in two parallel cultures, using identical procedures, both with and without rat liver microsomal activation.

The test article was tested with the following concentrations:

without S9-mix:                                                  with S9-mix:

7 h: 5000 µg/ml                                                  7 h: 5000 µg/ml

18 h: 500, 2500 and 5000 µg/ml                     18 h: 500, 2500 and 5000 µg/ml

28 h: 5000 µg/ml                                              28 h: 5000 µg/ml

According to the preliminary experiment for solubility and toxicity the concentration ranges were selected. The highest concentration produced a slight decrease of the mitotic index without S9-mix at 7 and 18 h. Up to the highest investigated dose the test compound Remazol-Brillantblau BB neu induced no significant increase in the number of chromosome aberrations. Appropriate reference mutagens were used as positive controls and showed a distinct increase in the chromosome aberrations indicating the sensitivity of the assay.

Remazol-Brillantblau BB neu does not induce chromosome aberrations in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described. Therefore Remazol-Brillantblau BB neu is considered to be not clastogenic in this chromosome aberration assay. Hence, the positive result in the first assay was either due to an impurity, or more likely, the assay produces a false positive outcome which is commonly observed when using V79 cells in this assay.

Remazol-Brillantblau BB Neu was tested in the micronucleus test. The test compound was suspended in deionized water and dosed once orally at 5000 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Endoxan was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

All animals survived after application of 5000 mg per kg bodyweight. The following signs of toxicity were observed: faeces blue coloured and diarrhea. 24 hours after application all animals were free of clinical signs of toxicity.

The dissection of the animals revealed no test substance related findings.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with

Remazol-Brillantblau BB Neu and was statistically not different from the control values.

Endoxan 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 significant extent.

The results indicate that, under the conditions of the present study, Remazol-Brillantblau BB Neu is not mutagenic in the micronucleus test. This result confirms the negative outcome of the second Chromosome Aberration Assay in vitro performed withthe test substance.

It can therefore be concluded that Reactive Blue 220 is neither genotoxic, not does it form genotoxic metabolites in vitro or in vivo.