1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

16 Aug 1999 to 18 Nov 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 July 1992

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MITI, Ministry of International Trade and Industry, Japan, 62 Notification of Basic Industries Bureau, No. 303, (Sho-62)

Version / remarks:

31 March 1987

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

EC Number:

262-104-4

EC Name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Cas Number:

60207-90-1

Molecular formula:

C15H17Cl2N3O2

IUPAC Name:

1-{[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl}-1H-1,2,4-triazole

Test animals

Species:

mouse

Strain:

other: Ico:CD1(CRL)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adults, 7 - 8 weeks old
- Weight at study initiation: 33-39 g males, 24-29 g females
- Housing: 5 animals per cage
- Diet: Certified standard diet, ad libitum
- Water: Tap water ad libitum
- Acclimation period: The animals were kept on location for at least five days prior to being used in the study. Shortly before use the health status of the animals was checked by the laboratory personnel according to veterinary/scientific standards.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.5-22.0°C
- Humidity: 50-79%
- Air changes (per hr): Not reported
- Photoperiod: 12 hours dark/12 hours light

IN-LIFE DATES: 16 Aug 1999 to 18 Nov 1999

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: arachis oil
- Concentration of test material in vehicle: 16, 32, 64 mg/mL
- Amount of vehicle: 5 mL/kg bw

Frequency of treatment:

Single dose

Post exposure period:

24 hours (all groups) and 48 hours (high dose group and negative control group only)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

80 mg/kg bw (total dose)

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

160 mg/kg bw (total dose)

Remarks:

Mid dose group

Doses / concentrations 3

Dose / conc.:

320 mg/kg bw (total dose)

Remarks:

High dose group

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance: cyclophosphamide (CPA)
Route of administration: Oral gavage
Doses: 64 mg/kg bw
Dosing volume: 10 mL/kg bw

Examinations

Tissues and cell types examined:

Bone marrow, Polychromatic erythrocytes (PCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary toxicity testing

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by CO2 asphyxiation. Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Smears prepared from this suspension were stained with May-Grunwald/Giemsa solution and mounted.

METHOD OF ANALYSIS: The slides of five animals/sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were scored by a laboratory technician. The incidence of micronucleated polychromatic erythrocytes (MNPCE) among at least 2000 polychromatic erythrocytes (PCE) and the ratio of PCE to normochromatic erythrocytes (NCE) among a total of at least 1000 erythrocytes were determined for each slide.

Evaluation criteria:

Please, see "Any other information on results incl. tables" section

Statistics:

The significance of differences between control and treated groups was assessed by the Chi-Squared-Contingency-Test (F=1, p<0.05).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

the high dose group

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the first step of the tolerability test, the dose of 2000 mg/kg of the test substance, was administered to one female and one male animal. One to two hours after dosing, both mice exhibited recumbence and died within two to three hours after administration. In the second step, a pair of mice was treated with 800 mg/kg of the test substance. Both animals exhibited recumbence and decreased muscle tone. They were found dead three hours after dosing. In the third step, one female and one male were dosed with 320 mg/kg of the test substance. No symptoms of toxicity occurred during the three days observation period . In the confirmatory step, again one male and one female received 320 mg/kg of the test substance. No symptoms of toxicity were observed. Based on these results, the dose of 320 mg/kg for females and males was chosen as the highest one to be administered in the micronucleus test.

RESULTS OF DEFINITIVE STUDY
- In the high dose group (24 hours sampling time), two female and four male animals exhibited creeping movements, recumbence and decreased muscle tone on day of sacrifice. No symptoms of toxicity were noted in the intermediate and low dose groups.
- In the group of 48 hours sampling time dosed with 320 mg/kg test substance, one female and one male showed creeping movements, recumbence and decreased muscle tone on day of sacrifice. One male was found dead on day of sacrifice and was replaced by a reserve animal. No symptoms of toxicity occurred in the other animals.
- At both sampling times (24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the respective doses of the test substance, as compared with the negative control animals.
- At the 24 hours sampling time after treatment with the test substance the mean percentage of micronucleated PCEs was 0.07 (80 mg/kg), 0.10 (160 mg/kg) and 0.08 (320 mg/kg ) respectively compared to a negative control value of 0.08.
- At the 48 hours sampling time after treatment with 320 mg/kg of the test substance the mean percentage of micronucleated PCEs was 0.12 compared to a negative control value of 0.10
- In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was significantly increased. The mean percentage of micronucleated PCEs evaluated was 1.80. In comparison with the negative control of 0.08% this value is highly significant (p<0.05).

Any other information on results incl. tables

Table 1. Overall mean percentage of micronucleated PCEs

Treatment time	320 mg/kg	160 mg/kg	80 mg/kg	Positive control	Negative control
24 hours	0.08	0.10	0.07	1.80	0.08
48 hours	0.12				0.10

Analytical results

The test material in suspension was analysed by HPLC with UV detection to confirm the intended concentrations to be used in the micronucleus test and the stability of the test substance in the vehicle used.

Nominal concentrations		Actual concentrations
Experiment	pg/mL	pg/mL	% of nominal value*
24 + 48 h	64000	62250/62899	97.3/98.3
24 h	16000	18700/18804	117/118

* acceptable range: 100 ± 20 %

The values found by analysis of all samples were in agreement with the intended concentrations, demonstrating sufficient stability of the test substance in the vehicle.

Applicant's summary and conclusion

Conclusions:

In a mouse bone marrow micronucleus assay performed in compliance with GLP and following an OECD 474 guideline, there was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. There was no evidence for clastogenic or aneugenic effects in mice treated with the test substance.

Executive summary:

In a mouse bone marrow micronucleus assay performed in compliance with GLP and following an OECD 474 guideline, groups of 5 male and 5 female young adult, Ico:CD1 [CRL] mice were dosed orally, by gavage, with the test substance at doses of 0, 0, 80, 160, 320 and 320 mg/kg/day. An additional group was dosed with the positive control, cyclophosphamide at 64 mg/kg/day. One group of mice at each test substance dose level, plus one negative control group and the positive control group were killed after 24 hours. The other negative control group and 320 mg/kg/day group were killed after 48 hours. Femoral bone marrow cells were harvested and polychromatic erythrocytes scored for micronuclei.

In the 320 mg/kg dosage group (24 hours sampling time), four males and two females exhibited creeping movement, recumbence and decreased muscle tone the day after administration. No symptoms of toxicity were noted in the 160 and 80 mg/kg groups. In the 320 mg/kg group (48 hours sampling time) one male was found dead two days after administration and was replaced by a reserve animal. Creeping movement, recumbence and decreased muscle tone were occasionally observed shortly before sacrifice. In the reserve group one female was found dead two days after administration. No statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed in any group when compared with the respective negative control group. The positive control induced the appropriate response.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. There was no evidence for clastogenic or aneugenic effects in mice treated with the test substance.