N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-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:

29 May 2001 - 17 June 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

601-779-5

Cas Number:

121451-02-3

Molecular formula:

C17H7Cl2F9N2O3

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: White powder
- Storage condition of test material: Ambient temperature

Test animals

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Range-finding study = 8 weeks old. Definitive test = 9 weeks old.
- Weight at study initiation: Range-finding study = 32.8 - 38.8 g (males) and 26.0 - 30.9 g (females). Definitive test = 32.1 - 37.3 g (males).
- Assigned to test groups randomly: yes, using a computer program.
- Housing: in groups by sex.
- Diet: commercial rodent feed, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: at least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64 - 79 °F
- Humidity (%): 30 - 70 %
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prior to dosing, the dosing stocks of the test material were prepared on each day of dosing by mixing the appropriate volume of the vehicle, 0.5 % methylcellulose, with pre-weighed quantities of the test article, forming homogenous suspensions.

Duration of treatment / exposure:

Two consecutive days

Frequency of treatment:

Daily

Post exposure period:

24 hours after administration of the last dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Basis:
actual ingested

No. of animals per sex per dose:

Range finding test: 4 per sex per dose
Definitive test: 7 males per dose (6 animals/group were required for micronucleus analysis)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide
- Route of administration: the positive control was dissolved in sterile deionized water at approximately 12 mg/mL and administered once, approximately 24 hours prior to harvest, via oral gavage at approximately 10 mL/kg to achieve the final dosing concentration.
- Doses / concentrations: 120 mg/kg

Examinations

Tissues and cell types examined:

The PCE:NCE cell ratio was examined in bone marrow (erythrocytes).

Details of tissue and slide preparation:

EXTRACTION OF BONE MARROW
At the appropriate harvest time point, the animals were euthanized by CO2 inhalation followed by incision of the diaphragm. The hind limb bones (tibias) were removed for marrow extraction from six surviving animals. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL foetal bovine serum (one tube per animal). Animals not needed for bone marrow collection were euthanised at the completion of the assay.

PREPARATION OF SLIDES
Following centrifugation to pellet the bone marrow tissue, the supernatants were removed by aspiration and portions of the pellets were spread on slides and air dried. The slides were fixed in methanol, stained with May-Grünwald solution and Giemsa, protected by mounting with coverslips, and analysed by fluorescence microscopy. For control of scoring bias, all slides were coded prior to analysis.

SLIDE ANALYSIS
Slides prepared from the bone marrow collected from six animals per group were scored for micronuclei and the PCE:NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analysing the number of micronucleated PCEs from 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 200 erythrocytes on a slide.
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occurred.
Micronuclei had sharp borders and were generally between one-twentieth and one-fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.
The staining procedure permitted the differentiation by colour of PCEs and NCEs (bluish-grey and red, respectively).

Evaluation criteria:

The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.

Statistics:

The raw data on the counts of micronucleated PCEs for each animal were first transformed by adding 1 to each count and then taking the natural log of the adjusted number. The transformed micronucleated PCE data and the data on percent PCE were analysed by a one-way analysis of variance (Winer, 1971) when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was significant (p # 0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were significantly different from the vehicle control. Additionally, parametric or nonparametric tests for trend may have been employed to identify any dose-related response. The alpha level at which all tests were conducted was 0.05.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING STUDY
- Toxicity:
> 500 mg/kg/day dose group: Clinical signs of toxicity were observed in one female (# 6086), approximately 5 hours after administration of the first dose, until the individual was found dead prior to administration of the second dose. Signs included: hypoactive, cold to touch, hunched posture, rough haircoat, flattened posture, squinted eyes.
> 1000 mg/kg/day: Clinical signs of toxicity were observed in one male (# 6078), approximately 5 hours after administration of the first dose, until the individual was found dead immediately prior administration of the second dose. Signs included: hypoactive, hunched posture, rough haircoat, irregular respiration, flattened posture and squinted eyes.
> 2000 mg/kg/day: Clinical signs of toxicity were observed in three individuals.
Slight hypoactivity was observed in one male (#6070), one day after administration of the first dose, before being found dead immediately prior to administration of the second dose.
A second male (# 6081) displayed slight hypoactivity with the left eye sealed shut one day after administration of the first dose. Both eyes were sealed shut one hour after administration of the second dose. One day after administration of the second dose this individual was cold to the touch, both eyes were sealed shut and the animal was recumbent. This individual was found dead four to five hours later.
One female (# 6088) displayed clinical signs of toxicity approximately 5 hours after administration of the first dose, until the individual was found dead one day after administration of the sceond dose. Signs included: slightly hypoactive, hunched posture, rough haircoat, hypoactive, squinted eyes, cold to touch and flattened posture.
- Mortality:
> 500 mg/kg/day dose group: One female died (# 6086).
> 1000 mg/kg/day dose group: One male died (# 6078).
> 2000 mg/kg/day dose group: Two males (#6070 and # 6081), and 1 female died (# 6088).
All mortalities were necropsied to ascertain whether the condition(s) were related to dosing mishap. None of the animals exhibited evidence of dosing error. However, all five mortalities in the dose range-finding study were attributed to colon/rectal perforations which could be evidence of body temperature errors and thus they are noted to be accidental deaths.
- Body temperature: No relevant variances in the body temperature were observed in any of the dose groups in the dose range-finding study.
- Conclusion: Based on these results, the maximum dose selected was 2000 mg/kg/day, the limit dose for this assay.

DEFINITIVE TEST
- Toxicity: All animals in the vehicle and positive control groups, and all animals in all dosage groups appeared normal after dosing and remained healthy until harvest.
- Mortality: No unscheduled mortality was observed during the definitive test.
- Cytotoixicty: No cytotoxicity was observed in the bone marrow, the test material did not induce a statistically significant decrease in the PCE:NCE ratio. A statistically significant increase in micronucleated PCEs was not observed at any dose level.
- Positive control: Cyclophosphamide, induced statistically significant increases in micronucleated PCEs as compared to that of the vehicle controls, with a mean and standard error of 1.52 ± 0.18 %.

Any other information on results incl. tables

Table 3: Micronucleus Data Summary

Treatment	Dose (mg/kg/day)	Harvest Time** (hours)	% Micronucleaded PCEs		Ratio PCE:NCE
			Mean	S.E.	Mean	S.E.
Vehicle control	0.5 % MC	24	0.08	0.02	0.63	0.07
Positive control	CP 120	24	1.52	0.18*	0.87	0.06
Test material	500	24	0.07	0.04	0.79	0.02
	1000	24	0.04	0.02	0.58	0.06
	2000	24	0.04	0.02	1.08	0.08

* Significantly greater that the corresponding vehicle control, p 0.01.

**Post second dose

Mean values determined from 2000 PCE’s per animal.

MC = Methyl cellulose; CP = Cyclophophamide; PCE = Polychromatic erythrocyte; NCE = Normochromactic erythrocyte

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Under the conditions of the test, the test material induced no signs of clinical toxicity in the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). A statistically significant increase in micronucleated PCEs was not observed at any dose level. Therefore the test material was considered to be negative in the mouse bone marrow micronucleus assay.

Executive summary:

The clastogenicity of the test material was determined in an in vivo mouse micronucleus assay, performed under GLP conditions and in accordance with the following standardised guidelines OECD 474, EPA OPPTS 870.5395 and EU Method B.12.

In the dose range-finding study, the test material was suspended in 0.5 % methylcellulose. Four animals per sex were dosed once daily by oral gavage for 2 consecutive days with the test article at dose levels of 500, 1000, or 2000 mg/kg.

Based on results from the dose range-finding study, dose levels of 500, 1000, or 2000 mg/kg/day were selected for testing in male animals only in the micronucleus study. The test article was suspended in 0.5 % methylcellulose. Seven animals were dosed once daily by oral gavage for 2 consecutive days with either the test article at dose levels of 500, 1000, or 2000 mg/kg, or the vehicle. Approximately 24 hours prior to harvest, seven animals were dosed one-time only by oral gavage with the positive control article. Six animals per group were euthanized approximately 24 hours after the last dose for extraction of the bone marrow. At least 2000 PCEs per animal were analysed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal.

Under the conditions of the test, the test material induced no signs of clinical toxicity in the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). A statistically significant increase in micronucleated PCEs was not observed at any dose level. Therefore the test material was considered negative in the mouse bone marrow micronucleus assay.