Blue MOP 6314

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

Study period:

From 16 December 2002 to 01 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

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

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: (6-8 weeks old)
- Weight at study initiation: 27.6 -33.2 g
- Housing:Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk, Rosenberg, Gennany). Paper bedding was provided as nest material (supplied by B.M.l. Helmond, The Netherlands).
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet, ad libitum
- Water (e.g. ad libitum): Free access to tap-water, ad libitum.
- Acclimation period: Acclimatisation period was at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

suspended in corn oil

Details on exposure:

The dosing volume was 10 ml/kg body weight.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 25 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 50 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 100 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 100 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 25 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 50 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 100 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 100 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Examinations

Details of tissue and slide preparation:

All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well
stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to nomochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Statistics:

Equivocal results should be clarified by further testing using modification of experimental conditions. A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately. The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity:> = 25 mg / µg with lethargy, arched posture Decreased PCE / NCE ratio to 100 mg / µg

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Based on the results of this dose range finding study dose levels of 100, 50 and 25 mg/kg body weight were selected as appropriate doses for the Micronucleus Test.
- Solubility:
- Clinical signs of toxicity in test animals: The following clinical observations were made in the groups treated with 100 mg of test item /kg
body weight:
During the first hour after dosing all animals were lethargic, had a rough coat and a hunched posture.
Within 18 hours after dosing all animals were still lethargic, ten male and four female animals had a rough coat and two female animals also had a hunched posture. Within 42 hours after dosing four males and one female animal had a rough coat, two male and two
female animals had a hunched posture; one female animal also showed ataxia and was lethargic. One male and three female animals recovered from the treatment.
- Evidence of cytotoxicity in tissue analysed:

Applicant's summary and conclusion

Conclusions:

It is concluded that the substance is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Executive summary:

The substance was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

Two groups were dosed with 100 mg/kg body weight, one group was dosed with 50 mg/kg body weight and one group was dosed with 25 mg/kg body weight. After dosing the animals of all the dose levels showed the following toxic signs: lethargy and a hunched posture. The animals of the dose levels of 100 and 50 mg/kg body weight also showed a rough coat and one of the animals dosed with 100 mg/kg body weight showed ataxia.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with BLUE MOP 6314 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with test item. The animals of the groups treated with 100 mg/kg body weight with test item (males at the 24 and 48 hours sampling time and females at the 48 hours sampling time) showed a slight but variable decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, indicating that the substance is toxic with respect to the erythropoiesis.

The groups that were treated with cyclophosphamide showed a clear decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls. It is concluded that the substance is not mutagenic in the micronucleus test under the experimental conditions described in this report.