long chain alkyl thio carbamide metal complex

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May to 2 June 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

only one sampling time, a second sampling time is recommended where two or more daily doses are given

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

liquid

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 Laboratories
- Age at study initiation: about 9 weeks
- Weight at study initiation: males: 30.1-35.3 g; females: 22.6-28.3 g
- Assigned to test groups randomly: yes, using computer-generated body weight sorting programme
- Fasting period before study: no data
- Housing: singly in suspended stainless steel cages
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Peanut oil
- Vehicle(s)/solvent(s) used: peanut oil
- Justification for choice of solvent/vehicle: test substance soluble in peanut oil
- Concentration of test material in vehicle: 50, 100 and 200 mg/ml
- Amount of vehicle (if gavage or dermal): 10.0 ml/kg bw
- Lot/batch no. (if required): 116H1037
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dosing solutions prepared once and used for all 3 days of dosing

Duration of treatment / exposure:

3 days

Frequency of treatment:

daily for 3 days

Post exposure period:

18-24 hr after the last dose

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 472, 939 or 1640-1930 mg/kg bw/day
Basis:
actual ingested
analytical dose

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control with peanut oil only

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: cyclophosphamide
- Justification for choice of positive control(s): included in list of recommended substances
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg bw/day

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on range-finding study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treated daily for 3 days with 0, 500, 1000 or 2000 mg/kg bw/day. Samples taken 18-24 hr after the last dose.

DETAILS OF SLIDE PREPARATION: bone marrow aspirated from the femurs, washed, centrifuged and the erythrocytes resuspended in any remaining supernatant. Bone marrow smears were prepared (2/animal) and the slides stained with acridine orange.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. The percentage of PCEs were determined by counting 1000 erythrocytes per animal.

Evaluation criteria:

The test substance was considered positive if the mean incidence of micronucleated PCEs showed a dose-related increase, including at least one dose that was statistically different from the mean incidence in the vehicle control groups and outside the normal mean range of the vehicle controls (ie. greater than 4), or at least two dose levels are statistically different from the normal range for the vehicle control (again, greater than 4).

Statistics:

Standard one-way analysis of variance (ANOVA) and, if this was significant, Duncan's multiple range test was used to compare vehicle and treated groups. A standard regression analysis was performed to test for dose-response.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: Evidence of cytotoxicity was observed at 1000 mg/kg bw/day (males and females) and 2000 mg/kg bw/day (males only) as indicated by a significant decrease in PCE/NCE ratio compared with controls.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0, 500, 1000 or 2000 mg/kg bw/day
- Solubility: yes
- Clinical signs of toxicity in test animals: none observed, and body weights did not change during the study.
- Evidence of cytotoxicity in tissue analyzed: none observed
- Rationale for exposure: no data
- Harvest times: 18-24 hr after the last dose


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction in the treated groups
- Ratio of PCE/NCE (for Micronucleus assay): see Table 1 (below)
- Statistical evaluation: not applicable, no induction of micronuclei

Any other information on results incl. tables

Table 1. Mean number of micronuclei and PCE:NCE ratios per treatment

	Males (mean of 5 animals)		Females (mean of 5 animals)
	%PCE (ratio PCE:NCE)	Micronuclei/100PCEs	%PCE(ratio PCE:NCE)	Micronuclei/100PCEs
0 mg/kg bw/day (vehicle control)	54.46 ± 3.34 (1.18)	0.8 ± 0.8	53.14 ± 1.63 (1.13)	1.2 ± 0.7
500 mg/kg bw/day	49.68 ± 4.28 (0.99)	1.2 ± 0.4	51.32 ± 4.41 (1.05)	0.4 ± 0.2
1000 mg/kg bw/day	47.88 ± 5.64** (0.92)	0.4 ± 0.2	45.64 ± 5.56* (0.84)	0.7 ± 0.8
2000 mg/kg bw/day	44.54 ± 2.87** (0.80)	1.3 ± 0.6	47.40 ± 5.11 (0.90)	1.4 ± 0.7
20 mg/kg bw/day cyclophosphamide	36.78 ± 3.90** (0.58)	9.9 ± 2.2**	36.66 ± 6.65** (0.60)	11.3 ± 1.6**

* p < 0.05; ** p < 0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
In a GLP study conducted according to OECD guideline 474, test substance MRD-98-158 (EC#434-650-5) showed no genotoxic potential in an in vivo bone marrow micronucleus assay when given orally to male and female mice at doses of up to 2000 mg/kg bw/day for three consecutive days.

Executive summary:

In a GLP study conducted according to OECD guideline 474, the genotoxic potential of test substance MRD-98-158 (EC# 434-650-5) was assessed in an in vivo bone marrow micronucleus assay in mice.

Groups of five CD-1 mice of each sex were dosed via gavage with 0, 500, 1000 or 2000 mg/kg bw/day for three consecutive days with the test substance in peanut oil. Between 18-24 hr after the final dose, bone marrow erythrocytes were recovered from the femurs of the animals and used to prepare two slides per animal for examination. After staining with acridine orange, 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. To determine cytotoxicity the number of polychromatic erythrocytes in the total erythrocyte population were counted.

No increase in micronucleated cells was evident in the treated groups compared to the vehicle controls. Cytotoxicity, indicated by a reduction in the ratio of polychromatic erythrocytes to normochromatic erythrocytes, was detected at the mid- and high-dose levels in males and the mid-dose in females. The positive controls gave the expected increase in micronuclei demonstrating the effective performance of the assay.

In conclusion, EC# 434-650-5 showed no genotoxic potential in an in vivo bone marrow micronucleus assay when given orally to male and female mice at doses of up to 2000 mg/kg bw/day for three consecutive days.

In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to EC# 457-320-2.