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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April 1999 - 15 March 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Directive 87/302/EEC: Teratogenicity Test – Rodent and Non-Rodent Species

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF Toxicity Testing Guidelines for Teratogenicity Studies

Deviations:

no

GLP compliance:

yes

Limit test:

no

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:

- Appearance: White powder

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 to 6 months of age
- Weight at study initiation: Females weighed 2500 to 3500 g
- Fasting period before study: No
- Housing: Animals were housed one per cage in suspended stainless steel cages with flattened tube grid floors. Cages contained a stainless steel hanging feeder and a pressure activated, nipple-type watering system.
- Diet: Upon receipt, rabbits received approximately 2 oz. of pelleted feed. The amount of feed was increased incrementally by 2 oz./day up to a total of approximately 8 oz./day.
- Water: municipal water provided ad libitum
- Acclimation period: Approximately 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 23.4 °C
- Humidity: 40 - 60 % (relative)
- Air changes: Approximately 12 - 15 times/hour
- Photoperiod: A 12-hour light/dark photocycle was maintained with lights on at 06:00 and off at 18:00

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % METHOCEL (cellulose ethers) A4M

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was administered as a suspension in an aqueous vehicle of 0.5% METHOCEL (cellulose ethers) A4M. Dose volumes were adjusted daily based on individual body weights. Due to the length of the dosing period, dose suspensions were prepared periodically throughout the study.

VEHICLE
- Amount of vehicle (if gavage): A dose volume of 3 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HOMOGENEITY
The low and high dose suspensions from the first mix were initiated prior to the start of dosing to verify homogeneous distribution of the test material in vehicle.
Analyses of the dosing suspensions showed that the test material was homogeneously suspended in the vehicle.

STABILITY
The low and high-dose suspensions from the first mix were re-analysed after the period of use (16 days) to confirm stability of the test material in vehicle.
Reanalysis of the high and low dose suspensions after 16 days of use revealed mean dose concentrations for both solutions that were 97 % of the initial values, indicating that the solutions were stable during this time. Dosing suspensions were mixed periodically throughout the study based upon these stability data.

CONCENTRATION VERIFICATION
Analysis of all dosing suspensions from the first mix were initiated prior to the start of dosing using HPLC with ultraviolet detection and external standards to determine concentrations.
Analyses of the dosing suspensions showed that the mean concentrations ranged from 100 to 107 % of targeted concentrations.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- M/F ratio per cage: 1:1
- Length of cohabitation: Adult females were naturally mated with one male of the same strain at the supplier. The observed day of breeding was considered day 0 of gestation. Rabbits were shipped on day 1 of gestation and arrived in the testing laboratory on the same day.

Duration of treatment / exposure:

Days 7 - 27 of gestation

Frequency of treatment:

Once daily

Duration of test:

Animals were sacrificed on Day 28 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:
actual ingested

No. of animals per sex per dose:

25 females per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: These dose levels were selected based on the preliminary results of the probe study. Administration of 1000 mg/kg/day was expected to result in significantly increased liver weights. In addition, the high dose of 1000 mg/kg/day represented a limit dose as defined in the US EPA OPPTS 870.3700 guideline. The lower dose levels were expected to provide dose response data for any toxicity observed in the high-dose group.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once each day a cage side examination was conducted and to the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were conducted on all animals prior to the start of the study and daily throughout the study. This examination included careful, hand-held evaluations of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swelling, masses and animal behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on day 0 by the supplier, daily during the dosing period, and on Day 28 of gestation. Statistical analyses of body weights and body weight gains were performed using data collected on Days 0, 7, 10, 13, 16, 20, 24 and 28 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Feed consumption was recorded for all animals daily beginning on day 4 of gestation by weighing feeders at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) / (# of days in measurement cycle)

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
On gestation Day 28, all surviving females were euthanised by an intravenous injection of a euthanasia solution (Beuthanasia-D Special, Scherring Corporation, U.S.A., Kenilworth, N.J.) and a limited gross pathologic examination (necropsy) was performed. The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. The stomach, liver and kidneys were dissected from the carcass and incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys and gravid uterus were recorded. The ratios of liver and kidney weights to gestation Day 28 (terminal) body weight were calculated.
Representative sections of liver with gallbladder, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10 % formalin. Microscopic examination of tissues was not conducted unless deemed necessary to interpret other observations made during the study.
Any animal that died, appeared moribund or showed indications of premature delivery was submitted for a complete necropsy. This necropsy was performed as described above with the following exceptions: 1) animals submitted alive for necropsy were euthanised by the inhalation of CO₂ and subsequent decapitation, 2) the head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined, 3) all viscera were dissected from the carcass and re-examined, 4) liver, kidney and gravid uterine weights were not recorded, and 5) the number of corpora lutea and the sex and body weight of foetuses from these animals were not recorded. Development of the conceptuses was evaluated to the extent possible by external examination (as appropriate for gestational age). The conceptuses were not examined for visceral or skeletal alterations. Following external examination, these conceptuses were discarded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The uterine horns were exteriorised through an abdominal incision and the following data were recorded:
- The number and position of live foetuses in utero
- The number and position of dead foetuses
- The number, position and classification (early, late) of resorption sites
- The number of ovarian corpora lutea
- The sex and body weight of each foetus
- Any external gross alterations in the foetus

Corpora lutea counts were not conducted on females with no grossly visible evidence of pregnancy. The uteri of females without grossly visible evidence of pregnancy were stained with a 10 % aqueous solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions to verify pregnancy status.

Fetal examinations:

As part of the examination of uterine contents, the following parameters were recorded:
- The sex and body weight of each foetus
- Any external gross alterations in the foetus

- External examinations: Yes; all per litter
- Soft tissue examinations: Yes; half per litter
- Skeletal examinations: Yes; half per litter
- Head examinations: Yes; half per litter

An external examination of all live and dead foetuses was conducted. This examination included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All live foetuses were euthanised by oral administration of sodium pentobarbital solution. All foetuses were examined by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observations of the thymus, trachea, oesophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. At least one-half of the foetuses in each litter, chosen randomly, were designated for examination of the internal structures of the head. The heads of these selected foetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). All foetuses were preserved in alcohol, eviscerated, cleared, stained with alizarin red-S (Dawson, 1926) and examined for skeletal alterations. All foetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function (EPA, 1989).

Statistics:

Maternal body weights, body weight gains, organ weights (absolute and relative), foetal body weights and feed consumption were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed, respectively.
Frequency of pre-implantation loss (number of corpora lutea minus number of implantations), resorptions per litter, resorptions per foetal population, and foetal alterations were analysed using a censored Wilcoxon test with Bonferroni's correction. The number of corpora lutea, implantations, and viable foetuses per litter was evaluated using a nonparametric ANOVA followed by the Wilcoxon Rank-Sum test with Bonferroni's correction. Pregnancy rates were analysed using the Fisher’s exact probability test with Bonferroni’s correction. Foetal sex ratios were analysed using a binomial distribution test.
Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method, and excluded if justified by sound scientific reasons. Both Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control group to keep the experiment-wise α at 0.05. Both were reported at the experiment-wise α level.
As numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal α levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
Prior to the scheduled necropsy, two rabbits died and six rabbits were euthanised due to abortion and/or their moribund condition. Most of these incidents appeared to be related to gavage error and/or inanition in the does.
Two does in the 250 mg/kg/day group were euthanised prior to the scheduled necropsy. Rabbit 1918 appeared healthy upon arrival in the laboratory (gestation day 1), but by gestation day 3 was observed to have decreased faecal output. By gestation day 4, this doe appeared thin, exhibited perineal soiling (faecal and urine), and mucoid matter was present in the faeces. The rabbit was subsequently euthanised (prior to initiation of treatment with test material). Gross pathologic observations on this rabbit included mucus in the cecum and pale cortexes of both kidneys. A second 250 mg/kg/day group rabbit (1902) had two foetuses and blood in her cage on gestation day 25 and was consequently euthanised. For several days prior to the abortion, both the amount of feed consumed and faeces excreted by this doe were decreased. At gross necropsy, this rabbit had haemolysed blood and watery contents in the gastrointestinal tract and bloody urine in the bladder. The uterus also had evidence of recent abortion.
Two rabbits in the 500 mg/kg/day group (1933 and 1941) were euthanised prior to the scheduled necropsy. Rabbit 1933 appeared normal until gestation day 10, but thereafter consumed very little feed. On gestation day 13, this rabbit had rapid/laboured respiration, perineal soiling (faecal) and had decreased amounts of faeces in the cage pan. Owing to these clinical findings, the rabbit was sacrificed on gestation day 13. Gross necropsy revealed perineal soiling (faeces and urine) congestion in the liver, slight consolidation in the lungs (failure to collapse), and froth in the trachea, suggesting gavage error as the proximate cause of death. The doe was pregnant with normally developed foetuses. Another 500 mg/kg/day rabbit (1941) aborted three foetuses on gestation day 27. Clinical observations made prior to the abortion included decreased faeces (gestation day 26 and 27) and decreased feed consumption (gestation day 24 - 27). The single gross pathologic finding for this rabbit was firm lungs, suggesting a prior gavage error as the ultimate cause for the abortion. Several normal appearing foetuses were still in the uterus along with two foetuses that appeared to have been dead for several days.
Two 1000 mg/kg/day rabbits (1954 and 1957) aborted their litters, and two others (1958 and 1960) died prior to the scheduled necropsy. Prior to the abortion of a single autolysed foetus on gestation day 22, rabbit 1954 had soft faeces on gestation day 15 and decreased faeces and/or reduced feed consumption on gestation day 13 - 22. At necropsy, blood was observed on the rabbit’s vulva and several resorbing foetuses were found within the uterus. All other tissues were normal and no cause for the abortion was apparent. Rabbit 1957 aborted five foetuses into her cage on gestation day24. Blood had been observed in the animal’s cage on gestation day 23, and decreased or absent faeces and severe inanition had been observed for several days prior.
It was noted that this animal had lost weight during each calculated body weight gain interval, including the pre-treatment (gestation day 0 - 7) period. At gross necropsy, normal postpartum involution of the uterus was apparent and all other tissues were normal. Based on these findings, particularly the pre-treatment body weight loss, this abortion was unlikely to be related to treatment. Rabbit 1958 was found dead with blood and foetuses in her cage on gestation day 25. Prior clinical observations included rapid respiration, decreased or absent faeces, and decreased feed consumption for two to three days preceding her death. Gross pathologic observations made for this doe included firm lungs (diffuse) and bloody perineal soiling. The uterus had a normal postpartum appearance. The cause of death was attributed to aspiration pneumonia. Finally, doe 1960 was found dead on gestation day 28. This rabbit was observed to have bluish skin and mucous membranes as well as soft faeces on gestation day 27. At necropsy, several foetuses (grossly normal) were found in utero. Gross pathological findings included congestion and oedema of the lungs, and perineal soiling. Although a definitive cause of death was not determined, the clinical signs of respiratory distress along with the gross findings in the lungs are suggestive of a gavage complication.
All other rabbits survived to the scheduled necropsy. Observations made upon arrival of these rabbits and prior to treatment included an enlarged vulva, lacerations, scratches, and bruises (on the ear, back, or urogenital area) which were likely inflicted during mating. In addition, some rabbits (6) were noted upon arrival to have missing digits. All other observations either occurred sporadically, were not dose related, or occurred at similar frequencies in control rabbits.

BODY WEIGHT
There were no statistically identified differences in the body weights or body weight gains in any of the treated groups when compared to their respective controls. Body weight gains during the first three days of treatment (gestation day 7 - 10) were either increased or decreased by just a few grams in all groups, including controls. Most likely, this reflected an adjustment to the gavage procedure, which is not unusual in rabbits. Body weight gain in all groups increased following the first few days of gavage and treated groups were comparable to controls by gestation days 10 - 13.

FEED CONSUMPTION
There were no significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.

GROSS PATHOLOGY
A minimal number of observations were made, all of which were considered spontaneous alterations unassociated with exposure to the test material.

ORGAN WEIGHTS
There were no significant differences in liver or kidney weights at any dose level tested. While the mean liver weights of the 1000 mg/kg/day rabbits were slightly greater than those of the controls, the values were not statistically different from controls, and were well within the historical control range. As previously mentioned, a statistically identified increase in liver weights seen in a prior probe study at 1000 mg/kg/day was only based on a sample size of three rabbits. In light of the current study findings with a much larger group size, the probe study findings are considered insignificant.

REPRODUCTIVE PARAMETERS
No treatment related differences between control and exposed rabbits were observed for pregnancy rates, number of corpora lutea, number of implantations, number of viable foetuses, or resorption rates at any dose level. A statistically identified increase in pre-implantation loss at 250 mg/kg/day was considered spurious for the following reasons: 1) increases were not observed at higher dose levels in this study, 2) no increases were noted at the same dose or higher dose levels in a previously conducted probe study and 3) the value was well within the range of historical control data.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment-related differences in the incidence of any foetal alteration in any of the treated groups when compared to controls. A single statistically identified decrease relative to controls in the incidence of missing caudal lobe of lung was identified at 500 mg/kg/day. This finding was not considered biologically significant as toxicity would be reflected as an increased incidence relative to controls rather than a decrease, and effects were not seen at higher doses in the study. All other alterations observed in foetuses from dams administered the test material either occurred at low frequencies and/or were not dose-related.
A low incidence of various malformations was observed scattered among all dose groups. A total of nine foetuses from six control litters were malformed. Litter 1873 had three foetuses with malformations as follows: 1) one foetus with a hemivertebra accompanied by a missing rib and fused thoracic vertebrae, 2) another foetus with fused ribs, a missing rib and a hemivertebra in the thoracic region, and 3) a third foetus with fused ribs and a hemivertebra. Another control foetus (1876) had fused lung lobes (superior to diaphragmatic) and a retro-oesophageal right subclavian artery. Two more control foetuses from litters 1880 and 1882 had hypoplastic atria (bilateral) and a retro-oesophageal right subclavian artery, respectively. Two foetuses from control litter 1886 were malformed. The first foetus had agenesis of the aortic arch, while the second foetus had a retro-oesophageal right subclavian artery. A single malformed foetus from litter 1888 had a missing gallbladder.
Three foetuses from three 250 mg/kg/day litters were malformed. One foetus (1899) had generalised anasarca, microtia, and multiple hyperplastic lung lobes. Another 250 mg/kg/day foetus (1917) had a forked rib, hemivertebra, and fused thoracic centra. A third 250 mg/kg/day foetus (1922) had truncus arteriosus with a ventricular septal defect.
No 500 mg/kg/day foetuses were malformed.
Finally, two 1000 mg/kg/day foetuses had malformations. One foetus (1962) had fused thoracic centra and a hemivertebra, while the other foetus (1966) had fused lung lobes.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no treatment-related differences in the incidence of any foetal alteration in any of the treated groups when compared to controls.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-effect-level (NOEL) for maternal and developmental toxicity was 1000 mg/kg/day, the highest dose level tested.

Executive summary:

A study was conducted to evaluate the maternal and developmental toxicity potential of the test material when administered to New Zealand White rabbits via oral gavage. The study was conducted in accordance with the standardised guidelines OECD 414, US EPA OPPTS 870.3700, Directive 87/302/EEC: Teratogenicity Test - Rodent and Non-Rodent Species and Japan MAFF Toxicity Testing Guidelines for Teratogenicity Studies under GLP conditions.

Groups of twenty-five time-mated female New Zealand White rabbits were administered the test material (suspended in 0.5 % methylcellulose) by gavage at targeted dose levels of 0 (vehicle control), 250, 500 or 1000 mg/kg/day on days 7 through 27 of gestation. In-life maternal parameters included clinical observations, body weight, body weight gain and feed consumption. On gestation day 28, all surviving rabbits were euthanised and examined for gross pathologic alterations and changes in liver, kidney and gravid uterine weights. The number of corpora lutea, uterine implantations, resorptions and live/dead foetuses also were determined. All foetuses were weighed, sexed and examined for external, visceral and skeletal alterations. In addition, the internal structures of the head were examined by serial sectioning for approximately one-half of the foetuses in each litter.

Examinations performed during the dosing period revealed no treatment-related findings. There were no effects of treatment on maternal body weights or body weight gains at any dose level compared with control animals, and there were no treatment-related effects on the amount of feed consumed by any treated groups when compared to the controls. No gross pathologic alterations or effects on organ weights were noted in any of the treated dams.

No significant differences were observed for pregnancy rates, numbers of corpora lutea, implantations, resorptions, viable foetuses per litter, percent pre-implantation loss, resorption rates, foetal sex ratios, foetal body weights or gravid uterine weight in rabbits given up to and including 1000 mg/kg/day, the highest dose level tested.

Under the conditions of this study, the no-observed-effect-level (NOEL) for maternal and developmental toxicity was 1000 mg/kg/day.