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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan 1989 to 7 Feb 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 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1988

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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:

rat

Strain:

other: Crl: CD® (SD) BRVAF/Plus(tm) rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approx. 10 weeks of age at start of mating
- Housing: Rats were housed in hanging polycarbonate cages with solid bottoms containing approximately a one inch layer of hardwood chip bedding. Animals were housed in cages of approximately 62 sq. in. floor space prior to and after mating and approximately 143 sq. in. floor space during mating. Animals were caged individually except at the time of mating.
- Diet: Ad libitum, rodent chow
- Water: Ad libitum, tap water
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 23 Jan 1989 to 26 Oct 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 3% cornstarch and 0.5% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with an aqueous 3% cornstarch and 0.5% Tween 80. The dose suspensions were prepared weekly by a weight/volume method and stored refrigerated.

VEHICLE
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of all dose suspension preparations were taken for analytical determination of concentration and homogeneity. Analyses indicated that the mean measured concentrations of the test substance were within 10% of the intended concentrations for all preparations of the 20, 100 and 200 mg/mL suspensions. Analyses of dose suspensions for concentrations was based on six samples per suspension. The relative standard deviations ranged from 1.4 to 7.9% indicating adequate homogeneity.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused: One or two females with each proven male breeder
- Length of cohabitation: Overnight
- Proof of pregnancy: Presence of sperm or a copulatory plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 through 15 p.c.

Frequency of treatment:

Once daily

Duration of test:

Untill Day 21 p.c.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: Day 0, 6, 7, 9, 12, 16 and 21 of gestation

FOOD CONSUMPTION
- Time schedule for examinations: Day intervals 0-6, 6-9, 9-12, 12-16 and 16-21 of gestation

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21
- Organs examined: The oral cavity and all organs of the thoracic and abdominal cavities. The ovaries and uterus with cervix were immediately removed, trimmed, weighed intact and examined

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

Fetal examinations:

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

Statistics:

Incidence data for each dose group, such as clinical observations were analysed by the Fisher exact probability test (Siegel, 1956) with Bonferroni’s correction for multiple comparisons to a single control level (Ingelfinger et al., 1983). Enumerative data for each litter, such as the percent incidence of foetal variations and malformations and the number of corpora lutea per dam, were analysed by the nonparametric Mann-Whitney U two-sample rank test (Goldstein, 1967). Quantitative or continuous data, such as body and food consumption were analysed by one-way analysis of variance (Steel and Torrie, l960) and the Dunnett’s t-test (Dunnett, 1964). The level of significance selected for all statistical tests was p < 0.05 but values also significant at p < 0.01 are indicated

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant reductions in mean absolute body weights or mean corrected body weights occurred at any dose level. The gain in body weight at 100 mg/kg bw/day was statistically reduced before, but not after, the start of treatment. The change in corrected body weight at 100 mg/kg bw/day was also statistically reduced for gravid days 0-21 but not from the start of treatment, gravid days 6-21. The statistically significant changes in mean body weights at 100 mg/kg/day were not considered treatment-related. The mean gain in body weight was reduced at 1000 mg/kg/ day after one day of treatment and was statistically reduced for the gravid day interval 7-9.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in food consumption occurred over gravid days 6-9 at 1000 mg/kg/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant reductions in the mean number of implants/dam at 100 mg/kg/day were not considered to be adverse.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal developmental toxicity

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of live foetuses per dam was slightly but statistically reduced at 100 and 1000 mg/kg/day but considered to be non-treatment related

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations were observed in 19 foetuses. All of these malformations were considered to have occurred spontaneously. At 0 mg/kg/day, 6 foetuses from 5 litters were considered to be malformed. The most severely affected was a foetus (202/17 F; litter/foetus-sex) with absence of a lumbar vertebral centrum, absence of lumbar vertebrae and sacral vertebrae, and absence of the tail. Two foetuses from two litters had absence of a lumbar vertebra (213/2 F; 214/7 F). Two foetuses from one litter (223/14 and 16 F) had moderate dilation of a kidney pelvis. The sixth foetus (212/8 F) had moderate dilation of a kidney pelvis and extreme dilation of an ureter. At 100 mg/kg/day, six foetuses from four litters were considered malformed. The most severely affected was a foetus (258/4 F) that had incomplete situs inversus of the abdomen and thorax with only one lung lobe on the left side, moderate dilation of both kidney pelvis, and extreme convolution of both ureters. A second foetus in the same litter (258/16 M) had moderate dilation of both kidney pelvis. One foetus (254/5 F) had no anus and a rudimentary tail. One foetus from litter 257 (12 F) had umbilical eventration. A second foetus from litter 257 (8 M) had moderate dilation of a kidney pelvis. The sixth foetus (250/15M) had umbilical eventration. At 500 mg/kg/day, three foetuses from two litters were considered malformed. One foetus (283/2 H) had an absence of a lumbar vertebra. In litter 286 one foetus (1 F) had moderate dilation of both kidney pelvis and one foetus (9 F) had moderate dilation of one kidney pelvis. At 1000 mg/kg bw/day four foetuses from four litters were considered malformed. One foetus (322/3 F) had situs inversus of the abdomen and thorax, two foetuses (325/11 F; 335/1 F) had extreme dilation and convolution of both ureters and a fourth foetus (342/4 F) had moderate dilation of a kidney pelvis

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The reduction at 100 mg/kg/day reflected a slight reduction in the number of corpora lutea (developed before treatment) and at 1000 mg/kg/day the <4% reduction in mean number of live foetuses compared to concurrent controls exceeded the mean values for controls from the previous five studies.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Analytical verification:

Table 1. Analysis of the dosing suspensions

Date prepared and analysed	Concentration expected (mg/mL)	Number of samples	Mean concentration (mg/mL)	Range (mg/mL)	Standard deviation	Relative standard deviation (%)
1-20-89	20	6	18.4	16.9-19.6	1.3	7.0
	100	6	97	93-101	3	3.1
	200	6	198	189-205	5	2.7
1-27-89	20	6	18.1	16.0-19.9	1.4	7.9
	100	6	98	93-104	5	4.7
	200	6	194	166-206	14	7.4
2-3-89	20	6	18.5	17.5-19.2	0.7	3.9
	100	6		89-102	5	5.2
	200	6	202	199-207	3	1.4

Table 2. Intergroup comparison of maternal body weight gain (g)

Days	Dose level of mg test substance technical/kg/day
	0	100	500	1000
7-9	11	9.4	13	3.4*
Number#	22	23	19	25

#Number of females with live fetuses in utero at termination.

*Statistically significant difference from control group mean, p<0.05 (Dunnett’s t-test)

Table 3. Intergroup comparison of maternal food consumption (g/day)

Interval (days)	Dose level of mg test substace technical/kg/day
	0	100	500	1000
6-9	24	23	24	21*
Number#	22	23	19	25

#Number of females with live fetuses in utero at termination.

*Statistically significant difference from control group mean, p<0.05 (Dunnett’s t-test)

Table 4. Intergroup comparison of selected intrauterine data

Parameter	Dose level of mg test substance technical/kg/day
	0	100	500	1000
Mean number of implants per dam	16.5	15.4*	16.4	16.2
Mean number of implants per dam	15.9	15.0*	15.7	15.3*

*Statistically significant difference from control group mean, p<0.05 (two-tailed)

Table 5. Historical control data of selected intrauterine data

Parameter	Study number (start date)
	12 (11/4/88)	11 (22/6/87)	10 (16/3/87)	9 (22/7/85)	8a(14/5/85)
Mean number of implants per dam	15.8	14.4	14.2	14.7	15.6
Mean number of live fetuses per dam	15.0	13.8	12.8	14.2	14.2

Table 6. Intrauterine data for gravid intergroup comparison of maternal food consumption (g/day).

	Dose level of mg test substance technical/kg/day
	0		100		500		1000
Dams with implants	22		23		19		25
Dams with live fetuses	22		23		19		25
Dams with resorptions	10		8		10		17
Dams with affected implants^a	14		11		12		17
	Mean^b	SD	Mean	SD	Mean	SD	Mean	SD
Corpora lutea/dam Implants/dam	17.5 16.5	3.2 2.6	16.5 15.4*	1.9 1.9	18.5 16.4	3.1 2.6	18.1 16.2	3.1 1.3
Implantation index^c(%) Pre implantation loss^d(%)	95.3 4.7	13.1 13.1	93.8 6.2	10.2 10.2	89.3 10.7	12.0 12.0	91.1 8.9	11.6 11.6
Implant viability index^e(%) Post implantation loss^f(%)	95.7 4.3	8.1 8.1	97.5 2.5	3.7 3.7	95.8 4.2	4.9 4.9	94.4 5.6	5.3 5.3
Live fetuses/dam Dead fetuses/dam	15.9 0.0	2.9 0.0	15.0* 0.0	1.8 0.0	15.7 0.0	2.6 0.0	15.3* 0.0	1.4 0.0
Resorptions/dam - early - mid - late	0.5 0.1 0.0	0.6 0.5 0.0	0.3 0.1 0.0	0.5 0.3 0.0	0.3 0.2 0.2	0.5 0.4 0.5	0.5 0.4 0.0	0.7 0.7 0.0
Malformed live foetuses^g(%) Affected implants^h(%)	1.6 5.9	3.3 8.0	1.7 4.1	3.9 4.9	1.1 5.3	3.9 5.1	1.1 6.6	2.6 6.3
Sex ratio (%)^i	50.2	15.2	45.7	12.4	45.6	13.1	49.3	12.2
Mean body weights (grams)/live foetus Reproductive tract weight (grams)/dam	5.2 108.03	0.2 18.0	5.4 105.61	0.4 11.25	5.2 107.66	0.3 17.79	5.4 108.00	0.3 10.70

a Affected implants includes dead fetuses, resorptions and malformed live fetuses

b Values determined on a per litter basis

c (implants/corpora lutea)x 100

d 100 – implants index

e (viable implants/total implants) x 100

f 100 – implant viability index

g (fetuses with observations classified as malformations/total live fetuses) x 100

h [ (dead fetuses + resorptions + malformed live fetuses) /total implants] x 100

i (live female fetuses/total live fetuses) x 100

* Significantly different from 0 mg/kg /day dose level, p< 0.05, two-tailed

Applicant's summary and conclusion

Conclusions:

Minimal maternal toxicity was evident at 1000 mg/kg bw/day and consisted of transient reductions in body weight gained and food consumed. No embryofoetal toxicity or teratogenic effects were apparent at any of the dose levels tested. The NOAEL in this study for maternal toxicity was 500 mg/kg bw/day and for developmental toxicity was 1000 mg/kg bw/day.

Executive summary:

The test substance was tested for embryotoxic and teratogenic action in accordance with OECD 414 and performed under GLP principles. The test substance was administered to Crl: CD® (SD) BRVAF/Plus™ rats by oral gavage as a suspension in aqueous 3% corn starch and 0.5% Tween 80 to groups of 25 mated females at dosages of 0, 100, 500 or 1000 mg/kg/day. The females were treated for 10 consecutive days from gestation day 6 through 15. The dose was based on each female’s gravid day 6 body weight. The females were monitored daily for clinical signs. Body weights and food consumption were recorded for selected intervals during gestation. The dams were sacrificed on gravid day 21; the status of the reproductive tract determined; and the foetuses examined for external, soft and tissue skeletal variations and malformations.

Results showed that treatment with the test substance caused minimal maternal toxicity at 1000 mg/kg/day indicated by a short period (gravid days 6-9) of slightly reduced body weight gain and food consumption. No embryofoetal and teratogenic toxicity was apparent at any dose level.

In conclusion, minimal maternal toxicity was evident at 1000 mg test substance/kg/day and consisted of transient reductions in body weight gained and food consumed. No embryofoetal toxicity or teratogenic effects were apparent at any of the dose levels tested. The NOAEL in this study for maternal toxicity was 500 mg/kg bw/day and for developmental toxicity was 1000 mg/kg bw/day.