N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

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:

11 Apr 1988 to 07 Jul 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

May 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Sep 1984

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

Sep 1987

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

EC Number:

410-690-9

EC Name:

N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Cas Number:

103055-07-8

Molecular formula:

C17 H8 Cl2 F8 N2 O3

IUPAC Name:

1-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

Test animals

Species:

mouse

Strain:

Tif:MAGf

Remarks:

(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: females 24-27 g, males 28-32 g (tolerability test); females 22-29 g, males 29-37 g (mutagenicity test, first part): females 20-26g, males 24-29 g (mutagenicity test, second part)
- Housing: The animals were individual caged.
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 -23
- Humidity (%): 46 - 52.5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 Apr 1988 To: 07 Jul 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % Carboxymethyl Cellulose (CMC)
- Amount of vehicle: 20 mL/kg body weight

Duration of treatment / exposure:

Part 1: Negative control + high dose: 16, 24 and 48 hours
Part 2: Intermediate dose + low dose + high dose+ positive control: 24 hours

Frequency of treatment:

Once

Post exposure period:

Part 1: 16, 24 or 48 hours
Part 2: 24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral 20 mL/kg body weight.
- Doses: 64 mg/kg in CMC 0.5 %

Examinations

Tissues and cell types examined:

Bone marrow, Polychromatic erythrocytes (PCEs) and the number of mature erythrocytes (RBCs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary (tolerability) test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. Three groups of 4 mice (2/sex) were treated with 3 single doses, one receiving the maximum dose of 5000 mg/kg bw, or the highest applicable dose, and the other two doses of 1/5 and 1/25 of that amount respectively. The observation period corresponded to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. The highest dose causing no death was used as the highest in the mutagenicity test. In this experiment all animals survived and the dose of 5000 mg/kg bw was determined as the highest to be applied in the mutagenicity assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Part 1: The preparation was administered orally to groups of 24 mice/sex in the treatment group (5000 mg/kg bw test substance in 20 mL/kg CMC 0.5 %) and in the negative control group (20 ml/kg bw CMC 0.5%). 16, 24 and 48 hours after treatment, 8 mice/sex/sampling time were sacrificed. Positive control was conducted on 8 mice/sex with 64 mg/kg bw cyclophosphamide in 20 mL/kg bw CMC 0.5 % which were sacrificed at 24 h.
- Part 2: The preparation was administered orally to groups of 8 mice/sex in the treatment groups (1250, 2500 and 5000 mg/kg bw, each in 20 mL/kg bw CMC 0.5%), in the negative control group (20 ml/kg bw CMC 0.5 %) and in the positive control group (64 mg/kg bw cyclophosphamide in 20 mL/kg bw CMC 0.5 %). 24 hours after treatment all animals were sacrificed.
Animals were treated once by gavage, after each sampling time, bone marrow smears were prepared from the shafts of both femurs. Prior to analysis the slides were coded. Thereafter the quality of staining was evaluated. The slides of 5 animals/sex showing the best differentiation between mature and polychromatic erythrocytes were selected for later scoring from each group and sampling tim

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs with foetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a glass slide and the preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grunwald solution for 3 min then in May-Grünwald solution / water 1/1 for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted. Giemsa solution (16.6 %) for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

METHOD OF ANALYSIS:
Prior to analysis the slides are coded. Thereafter the quality of staining is evaluated. The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes are selected for later scoring. In the first experiment the slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. In the second experiment the slides of five female and five male animals each of the negative control group, the positive control group and of the three dosage groups sacrificed at 24 hours post treatment. were examined.
1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

OTHER:
Analytical control: to prove that the animals were really exposed to the intended doses of the test substance, determination of the substance in suspension was performed by the analytical unit. This determination was performed with the highest dose of the suspension used in part 1 and in part 2 of the mutagenicity test.

Evaluation criteria:

INTERPRETATION OF RESULTS
A test substance is considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any dose and sampling time respectively.

ASSAY ACCEPTANCE CRITERIA
- The quality of the slides must allow a clear differentiation between polychromatic and normochromatic erythrocytes.
- The result obtained with the positive control has to fulfil the criteria given for a positive response.

Statistics:

The significance of difference was assessed by x2-test. A test substance was considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any dose and sampling time respectively.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

RESULTS

The analysed concentrations used were in agreement with the calculated concentrations. In the first experiment (see Table 1 in 'Any other information on results incl. tables) 2 male animals died within the treatment period of 16 hours. The bone marrow smears from the animals treated with the dose of 5000 mg/kg of lufenuron showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at the sampling times of 16, 24 and 48 hours.

The second experimental part (see Table 2 in 'Any other information on results incl. tables) was performed with dosages of 1250, 2500 and 5000 mg/kg bw test substance at the sampling time of 24 hours. Also in this second experiment the bone marrow smears at each dose levels showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the animals of the negative control group.

The respective positive control experiments with cyclophosphamide yielded an average of 0.98% (first part) and 1.06 % (second part) polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.06 % and 0.05 % respectively) treated with the vehicle (CMC 0.5 %) alone (p<0.05). The ratio of polychromatic/normochromatic erythrocytes was similar in all groups.

Table 1 Number of polychromatic erythrocytes with micronuclei and ratio of polychromatic to normochromatic (p/n) erythrocytes, arithmetic mean per sex and group. Part 1

Sacrifice	Dose	sex	PCE	NCE	Ratio of PCE/NCE	n.0 of PCE with micronuclei	% of PCE with micronuclei
16 h	Control 0.5 % CMC	F	426	574	0.7	0.2	0.02
		M	469	531	0.9	0.4	0.04
	Test substance 5000 mg/kg	F	544	456	1.2	0.4	0.04
		M	482	518	0.9	0.4	0.04
24 h	Control 0.5 % CMC	F	470	530	0.9	0.8	0.08
		M	444	556	0.8	0.4	0.04
	Test substance 5000 mg/kg	F	484	516	0.9	0.2	0.02
		M	447	553	0.8	0.2	0.02
48 h	Control 0.5 % CMC	F	433	567	0.8	0.6	0.06
		M	462	538	0.9	0.2	0.02
	Test substance 5000 mg/kg	F	477	523	0.9	0.2	0.02
		M	452	548	0.8	0.2	0.02
Positive control
24 h	Control 0.5 % CMC	F	470	530	0.9	0.8	0.08
		M	444	556	0.8	0.4	0.04
	CP 64 mg/kg	F	399	601	0.7	8.2	0.82
		M	383	617	0.6	11.4	1.14

PCE : polychromatic erythrocytes

NCE : normochromatic erythrocytes

Table 2 Number of polychromatic erythrocytes with micronuclei and ratio of polychromatic to normochromatic (p/n) erythrocytes, arithmetic mean per sex and group. Part 2

Sacrifice	Dose	sex	PCE	NCE	Ratio of PCE/NCE	n.0 of PCE with micronuclei	% of PCE with micronuclei
24 h	Control 0.5 % CMC	F	424	576	0.7	0.6	0.06
		M	415	585	0.7	0.4	0.04
	Test substance 1250 mg/kg	F	460	540	0.9	2	0.2
		M	484	516	0.9	0.2	0.02
	Test substance 2500 mg/kg	F	474	526	0.9	1	0.1
		M	478	522	0.9	0.8	0.08
	Test substance 5000 mg/kg	F	440	560	0.8	1	0.1
		M	454	546	0.8	1.4	0.14
	CP 64 mg/kg	F	467	533	0.9	7.8	0.78
		M	470	530	0.9	13.4	1.34

PCE : polychromatic erythrocytes

NCE : normochromatic erythrocytes

Applicant's summary and conclusion

Conclusions:

The results indicate that under the given experimental conditions no evidence of mutagenic effects was obtained in mice treated with test substance.

Executive summary:

The test substance was evaluated for its ability to induce micronuclei in polychromatic erythrocytes (PCE’s) of male and female Tif: MAGF(SPF) mouse bone marrow in a study following GLP principles and performed similar to OECD TG 474. A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. Three groups of 4 mice (2/sex) were treated with 3 single doses, one receiving the maximum dose of 5000 mg/kg bw, or the highest applicable dose, and the other two doses of 1/5 and 1/25 of that amount respectively. The highest dose causing no death was used as the highest in the mutagenicity test. In this experiment all animals survived and the dose of 5000 mg/kg bw was determined as the highest to be applied in the mutagenicity assay. In the subsequent mutagenicity the test substance was administered orally to groups of 24 mice/sex in the treatment group (5000 mg/kg bw test substance in 20 mL/kg CMC 0.5%) and in the negative control group (20 mL/kg bw CMC 0.5%). 16, 24 and 48 hours after treatment, 8 mice/sex/sampling time were sacrificed. Positive control was conducted on 8 mice/sex with 64 mg/kg bw cyclophosphamide in 20 mL/kg bw CMC 0.5% which were sacrificed at 24 h.

In the first experiment, 2 male animals died within the treatment period of 16 hours. The bone marrow smears from the animals treated with the dose of 5000 mg/kg of test substance showed no statistically significant increase (p>0.05) in the number of micronucleated PCE’s in comparison with the negative control animals at the sampling times of 16, 24 and 48 hours. The second experimental part was performed with dosages of 1250, 2500 and 5000 mg/kg bw test substance at the sampling time of 24 hours. Also in this second experiment the bone marrow smears at each dose levels showed no statistically significant increase (p>0.05) in the number of micronucleated PCE’s in comparison with the animals of the negative control group. The respective positive control experiments with cyclophosphamide yielded an average of 0.98 % (first part) and 1.06 % (second part)PCE’s with micronuclei. This is significantly different from the controls (0.06 % and 0.05 % respectively) treated with the vehicle (CMC 0.5 %) alone (p<0.05). The ratio of polychromatic/normochromatic erythrocytes was similar in all groups. It was concluded that, under the given experimental conditions, no evidence of mutagenic effects was obtained in mice treated with treated.