Multi constituent substance

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: Study performed according to OECD guideline 474 in compliance to GLP.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Reference 2

Reference Type:

other: Published secondary source

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Multi constituent substance

EC Number:

916-603-5

IUPAC Name:

Multi constituent substance

Test material form:

solid: particulate/powder

Details on test material:

Test substance : Basic Red 76 (COLIPA number C008)
Batch number : 0057891101
Methylsulphate anion : 15.9%
Chloride ion : 2.7%
Water : 4.1%
Purity : 98.6% (HPLC)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Deionised water

Details on exposure:

The test item has been investigated for induction of micronuclei in the bone marrow cells of male and female mice. Test doses were based on the results of a pre-experiment on acute toxicity. Mice were treated intraperitoneally with the test item up to 200 mg/kg bw and examined for acute toxicity symptoms at 1, 2-4, 6, 24, 30 and 48 hours after treatment. In the main experiment mice were exposed orally to 0, 25, 50 and 100 mg/kg bw. The animals of the highest dose groups were examined for symptoms of acute toxicity at 1, 2-4, 6 and 24 hours after treatment. Bone marrow cells were collected 24 or 48 (high dose only) hours after dosing. Toxicity and exposure of the target cells was determined by measuring the ratio between polychromatic and total erythrocytes (PCE/TE).

Duration of treatment / exposure:

Sacrifice time : 24 and 48 (for the high dose only) hours after the treatment

Frequency of treatment:

Once

Post exposure period:

24 and 48 (for the high dose only) hours after the treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

Remarks:

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

No. of animals per sex per dose:

Five/sex/dose group

Control animals:

yes

Positive control(s):

Yes

Examinations

Tissues and cell types examined:

Bone marrow cells

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods are used as an aid in evaluating the results however the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the pre-experiment on acute toxicity 3 of the 4 animals treated with a dose of 200 mg/kg bw, 3 of the 4 animals treated with a dose of 150 mg/kg bw and one animal treated with a dose of 125 mg/kg bw died within 24 hours. Until death these animals as well as the surviving animals showed a reduction in spontaneous activity, abdominal position, eyelid closure, ruffled fur and apathy. These clinical signs were also observed in the 100 mg/kg bw group. Since no animals from this group died 100 mg/kg bw was chosen as the highest dose. In the main experiment the animals treated with 100 mg/kg bw showed a reduction in spontaneous activity, abdominal position, eyelid closure, ruffled fur and apathy. These clinical signs gradually declined at lower doses. At 25 mg/kg bw only a reduction in spontaneous activity remained. All treated animals showed red to pink coloured urine. A substantial decrease in the PCE/TE ratio was not observed indicating that the test item is not cytotoxic for bone marrow cells. The change of colour of the urineof the treated animals indicated systemic distribution of the test item and is thus considered bioavailable. At sacrifice times of 24 and 48 hours a biologically relevant increase in the number of polychromatic erythrocytes with micronuclei over the concurrent vehicle control values was not observed for any dose level. The study was considered vaild as the following criteria were met : the negative controls were in the range of the historical control data, the positive controls were in the range of the historical control data, at least 4 animals per group and sex can be evaluated, PCE to erythrocyte ratio was not less than 20% of the negative control.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the experimental conditions employed Basic Red 76 did not induce an increase in bone marrow cells with micronuclei in treated mice. The test substance was considered to be non-mutagenic in this micronucleus assay.

Executive summary:

The study was performed to investigate the potential of Basic Red 76 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water, which was also used as vehicle control. The volume administered intraperitoneally was 10mL/kg bw. At 24 and 48 hours after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.Ten animals (5 males and 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated : 24 hours preparation interval 25, 50, 100 mg/kg bw; 48 hours preparation interval : 100 mg/kg bw. As estimated by pre-experiments a dose of 100 mg/kg bw was the highest applicable dose without significant effects on the survival rates but with clear signs of toxicity. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Basic Red 76 did not exert any cytotoxic effects in the bone marrow. The urine of the treated animals had taken the colour of the test item indicating systemic distribution and thus bioavailability. In comparison to the corresponding vehicle controls there was no biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide administered intraperitoneally at a dose of 40 mg/kg bw was used as a positive control which showed a substantial increase frequency of induced micronuclei. Under the experimental conditions employed Basic Red 76 did not induce an increase in bone marrow cells with micronuclei in treated mice. The test substance was considered to be non-mutagenic in this micronucleus assay.