H112323

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 Jul. 1, 1992 to Nov. 11, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD Guideline 474 in compliance with GLP

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Substance H112323
- Physical state: Dark blue powder
- Storage condition of test material: Ambient temperature in the dark

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK
- Age at study initiation: 7-9 wk in Phase I and 6-20 wk in Phase II
- Diet (e.g. ad libitum): Porton Combined Diet , ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum
- Acclimation period: at least 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 °C
- Humidity (%): 55±15 %
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Soluble in corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were prepared in corn oil.
A solution of cyclophosphamide was prepared in physiological saline. All dosing preparations were administered at a volume of 20 mL/kg bw.

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

Twice at an interval of 24 h

Post exposure period:

24 h after last dosing

Doses / concentrations

Remarks:

Doses / Concentrations:
5600 mg/kg (equivalent to 5000mg/kg corrected for a water content of 10.5 % w/w)
Basis:
nominal conc.

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 65 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on patterns of lethalities or severe toxicity observed over a 4-d observation period following a single oral dose in a maximum tolerated dose (MTD)-Phase I

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow smears were prepared 24 and 48 h after dosing for the vehicle control and treated animals and 24 h after dosing for the cyclophosphamide treated animals.

DETAILS OF SLIDE PREPARATION: The preparations were stained with polychrome methylene blue and eosin to visualise the various cell types.

METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
Number of polychromatic erythrocytes (PCE) per slide: 1000 PCE
Percentage of polychromatic erythrocytes in the total erythrocyte population: 1000 Erythrocytes

Evaluation criteria:

A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group

Statistics:

- The incidence of micronucleated PCE and percentage PCE in the erythrocyte sample, were considered by ANOVA.
- All analyses were carried out using the GLM procedure in SAS.
- One-sided Student's t-test:

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No significant adverse reactions to treatment

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 5600 mg/kg (5000 mg/kg corrected for a water content of 10.5 % w/w in the test sample)
- Clinical signs of toxicity in test animals: No significant reactions to treatment were recorded during the 4 d observation period. All animals excreted blue/black urine and faeces and to have staining of the genital area, tail and coat.
- Evidence of cytotoxicity in tissue analyzed: No


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Nil
- Induction of micronuclei (for Micronucleus assay): No significant difference as compared to controls
- Ratio of PCE/NCE (for Micronucleus assay): No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes. No statistically significant decreases in the percentage of polychromatic erythrocytes, compared to the vehicle control values were observed.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

Others:
No significant adverse reactions to treatment were recorded for the majority of animals dosed with test substance, the only clinical signs observed being black colouration of the faeces, blue colouration of the urine and skin (subcutaneous fat) and blue staining of the genital area and coat. In addition, colouration of the internal organs and skin (subcutaneous fat) was also observed when the animals were dissected following termination

Any other information on results incl. tables

Table 1. Mean incidence of micronucleated PCE/1000 polychromatic erythrocytes ± SD (SD) at two sampling times

			Mean Incidence of MPE/1000 PE ± SD Males		Mean Incidence of MPE/1000 PE ± SD Females
Group	Compound	Dose	24 h	48 h	24 h	48 h
11	Vehicle Control (corn oil)	20ml/kg	0.8±1.1	0.0±0.0 (4)	0.2±0.5	0.4±0.6
12	Cyclophosphamide	65 mg/kg	29.0±3.7	-	21.8±13.2	-
13	Test substance	5600 ±	0.8±1.0 (4)	0.3±0.6 (3)	1.0±1.7	0.4±0.9

** 'Statistically significant increase in micronucleated polychromatic erythrocytes at p<0.01 in the Student's 't' test (one-sided) on transformed data.

All means based on 5 animals except where indicated in parentheses

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.

Executive summary:

A study was conducted to assess the potential of the test substance to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice performed according to OECD Guideline 474 in compliance with GLP.

A single oral dose was given to groups of 5 male and 5 female mice at a dose level of 5600 mg/kg (equivalent to 5000 mg/kg corrected for a water content of 10.5 % w/w), this being the limit dose level for the assay. Bone marrow samples were taken 24 and 48 h after dosing.

No statistically or biologically significant, increases in the incidence of micronucleated PCE, over the vehicle control values, were seen in either sex at either of the sampling times investigated. Comparison of the percentage of PCE showed, no statistically significant decreases, compared to the vehicle control values, in either sex at either of the sampling times. However, colouration of the urine, internal tissues and skin (subcutaneous fat) was observed in the animals treated with test substance, indicating that the test substance was absorbed following administration via the oral route.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.