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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 October 2002 - 14 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was prepared before OECD 443 became standard requirement

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Guideline No. 0378-6978

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Guideline No. 2-1-17

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:

rat

Strain:

other: Crl:CD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Mean body weight 3 days prior to test initiation: males 167.1 g; females 137.5 g
- Fasting period before study: No
- Housing: Animals were housed one per cage in stainless steel cages, except during mating (one male and one female per cage) and during the littering phase of the study. During the littering phase, dams were transferred on gestation day 19 to plastic cages provided with corn cob nesting material and remained there throughout lactation. Cages had wire-mesh floors and were suspended above catch pans. Cages contained feed containers and pressure activated, nipple-type watering systems.
- Diet: ad libitum
- Water: municipal water provided ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1°C
- Humidity: 40 - 70 % (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: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Premixes were mixed periodically throughout the study based on stability data. Diets were prepared weekly for approximately ten weeks prior to breeding of the P1 adults.
- Mixing appropriate amounts with (Type of food): Diets were prepared by serially diluting a concentrated test material- feed mixture (premix) with ground feed. The concentrations of the test material in the diets were calculated from the most recent body weight and feed consumption data. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data. To avoid potential overdosing during the breeding period, animals co-housed were provided with the lower of the two concentrations (female) for that dose group (low, middle, or high). During gestation, females from each dose group were provided with the appropriate dietary concentration given during breeding. Dietary concentrations supplied during lactation were adjusted using historical control feed consumption data for lactating females to account for the large and rapid increase in feed consumption (2 - 3x increase) typical for rats in late lactation. Until all litters were weaned, weanlings received a diet containing the same concentration of test material that was given to the females during the third week of lactation. Dams awaiting necropsy received a diet containing the same concentration that was given during the breeding period until all litters finished the lactation phase. Dietary concentrations for the P2 generation were calculated as described for P1 animals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred or two weeks elapsed
- If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. In cases where a mating partner was not available due to removal from study, the animal was paired with the next available partner.
- Proof of pregnancy: During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered day 0 of gestation.
- After successful mating each pregnant female was caged (how): The sperm- or plug-positive (presumed pregnant) females were then removed from the males’ cages and returned to their individual cages.

If available, one rat/sex/litter was randomly selected for the P2 mating to produce the F2 generation. More than one weanling may have been selected from the litters, if necessary, to achieve 30 breeding pairs/dose level for the second generation. Cohabitation of male and female littermates was avoided. In cases where a mating partner was not available due to removal from study, the animal was paired with the next available partner. A second breeding of the P2 adults was conducted to produce an F2B generation. Males and females were paired, approximately one week following weaning of the last F2A litter. The breeding procedure was the same as described above for the initial mating except that the P2 females were given new partners from within the same dose group, with females which failed to become pregnant in the first mating being paired with proven males. Also, females with evidence of mating were not removed from the males’ cage, but instead remained co-housed for the entire two week period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HOMOGENEITY
Representative samples from the test diets were evaluated to ensure homogeneous distribution of the test material at the lowest and highest concentrations in the feed four times during the study.
Analyses confirmed that the test material was homogeneously distributed in the diets.

STABILITY
Data from a thirteen-week toxicity study demonstrated that the test material was stable for at least 42 days in rodent feed at concentrations ranging from 0.0005 to 5 %. Test diets for the current study were prepared and used within this 42-day limit.

CONCENTRATION VERIFICATION
Analysis of all test diets to determine concentration of the test material was conducted four times, representing all dose levels and sexes and major study phases (note that one of the analyses was done on a breeding diet which is common to both sexes). The method used for analysing the test material in feed was solvent extraction method followed by analysis using LC-MS and solvent standards incorporating an internal standard.
The average concentrations in the diets over the study period were 95.1, 106, and 106 % of target for the male 0.5, 5, and 25 mg/kg/day dose levels, and 99.6, 105, and 106 % of target for the female 0.5, 5, and 25 mg/kg/day dose levels, respectively.

Duration of treatment / exposure:

Animals were dosed for approximately 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations.

Frequency of treatment:

Continuous in the diet

Details on study schedule:

A second mating of the P2 adults (F2B litter) was added to the study design specifically to investigate possible effects on fertility. Given the focus on fertility evaluation, the F2B offspring were terminated on postnatal day 1 with no further data collection.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Doses / concentrations 2

Dose / conc.:

0.5 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Doses / concentrations 3

Dose / conc.:

5 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Doses / concentrations 4

Dose / conc.:

25 mg/kg bw/day

Remarks:

Basis:
nominal in diet

No. of animals per sex per dose:

30 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Considering the high pup mortality previously observed in the initial reproductive toxicity study at 100 mg/kg/day, 25 mg/kg/day was selected as the high dose level for the present study. This dose level was expected to induce parental toxicity (liver and/or adrenal gland weight and histopathological changes), but less severe effects on pup survival. A high-dose level greater than 25 mg/kg/day was not recommended, as this would likely jeopardise pup survival resulting in insufficient viable offspring to produce a second generation as required in the reproductive toxicity test guidelines. Preliminary data from other toxicity studies conducted in rodents suggested that the NOEL may be < 1 mg/kg/day; therefore, a dosing interval of 10x was selected between the middle- and low-dose levels. Overall, these doses were selected to yield parental toxicity at the high dose, a dose-response for any observed effects at the middle dose, and a NOEL at the lowest dose.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice 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 males pre-exposure and weekly thereafter. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14, and 21. Females that delivered litters were subsequently evaluated on LD 0, 1, 4, 7, 14, and 21. In addition, females were observed for signs of parturition beginning on or about day 20 of gestation. Females that failed to mate or failed to deliver litters were examined weekly. Examinations included a careful, hand-held evaluation 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: All rats were weighed during the pre-exposure period and weekly during the ten-week pre-breeding treatment. Body weights for males were recorded weekly throughout the course of the study. Sperm/plug-positive females were weighed on days 0, 7, 14, and 21 of gestation. Females that delivered litters were weighed on days 1, 4, 7, 14, and 21 of lactation. Females that delivered F1 and F2A litters were weighed on days 1, 4, 7, 14, and 21 of lactation. Females that delivered F2B litters were weighed on LD 1 only. Females that failed to mate were not weighed during the subsequent gestation and lactation segments of the study. Females that failed to deliver a litter were not weighed during the lactation phase of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Feed consumption was determined pre-exposure and weekly during the ten-week pre-breeding treatment for all animals by weighing feed crooks at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was measured weekly in males and dietary concentrations were adjusted accordingly.
During gestation of the F1 and F2A litters, feed consumption was measured at weekly intervals for sperm/plug positive females on GD 0, 7, 14, and 21. Feed consumption was not recorded for non-confirmed mated females. During lactation of the F1 and F2A litters, feed consumption was measured on day 1, 4, 7, 11, 14, 17, 19, and 21. Feed consumption was not measured in females for the F2B litters. Feed consumption was not measured in females that failed to mate or deliver a litter. Feed 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

Oestrous cyclicity (parental animals):

Vaginal lavage samples were collected daily for all P1 and P2 females for three weeks prior to mating and during cohabitation until each female was sperm or plug positive or until the two-week mating period elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring loosely adherent vaginal cells to a slide with a pipette. Vaginal lavage slides were examined to determine oestrous cycle length and pattern for the pre-mating period. Additionally, on the day of scheduled necropsy, the stage within the oestrous cycle was determined for all P1 and P2 rats.

Sperm parameters (parental animals):

Weights of the testes, epididymides and seminal vesicles with coagulating glands (and seminal fluid) were recorded.

Sperm parameters were evaluated in all P1 and P2 males surviving to the scheduled necropsy. Unless circumstances dictated otherwise, the left and right epididymides and testes were allocated as follows: right epididymis: motility and histopathology; left epididymis: counts; right testis: histopathology; left testis: counts.

- Motility
Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing ~5 mL of SpermPrep Medium (ZDL, Inc., Lexington, Kentucky) and was incubated at room temperature for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed in a chamber of the HTM Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. After sperm were released, the epididymis was placed in Bouin’s fixative and, when deemed appropriate, subjected to histological examination.

- Counts
The left testis and cauda epididymis were weighed and frozen at -80 °C for subsequent determination of the number of homogenisation-resistant spermatids and cauda epididymal sperm per testis/epididymis and per gram of testicular/epididymal tissue.
Thawed testes or caudal epididymides were minced, diluted and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and spermatid or sperm counts were determined from an aliquot loaded into the IVOS analyser as described by Stradler et al. (1996). Samples from the high-dose and control animals were evaluated. In addition, epididymal counts from the low- and mid-dose P1 rats were evaluated due to a statistically significant decrease in epididymal counts at the high-dose.

- Morphology
An aliquot of sperm suspension was also taken, placed on a slide, and a smear prepared and then air dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were evaluated and classified as normal or abnormal as described by Filler (1993).
Morphological evaluation of sperm from control and high-dose males were conducted. In addition, morphological evaluation of sperm from the low- and mid-dose P2 rats was conducted due to a statistically significant increase of the proportion of abnormal sperm at the high-dose. Sperm morphology was scored blind with respect to treatment group.

Litter observations:

LITTER DATA
Females were observed for signs of parturition beginning on or about day 20 of gestation. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of delivery was recorded as the first day the presence of the litter was noted and was designated as lactation day (LD) 0. Litters were examined as soon as possible after delivery. The following information was recorded on each F1 and F2A litter: the date of parturition, litter size on the day of parturition (day 0), the number of live and dead pups on days 0, 1, 4, 7, 14, and 21 postpartum, and the sex and the weight of each pup on days 1, 4 (before and after culling), 7, 14, and 21 of lactation. Any visible physical abnormalities or demeanour changes in the neonates were recorded as they were observed during the lactation period on LD 0, 1, 4, 7, 14, and 21.
Similar information was recorded for the F2B litters through postnatal day 1, at which time all surviving pups were euthanised with Socumb euthanasia solution (Veterinary Laboratories, Inc. Lenexa, Kansas) and discarded. Pups found dead or sacrificed in moribund condition were examined for external and visceral defects in an attempt to determine the cause of death. These pups were preserved in neutral, phosphate-buffered 10 % formalin.

CULLING AND WEANING
To reduce the variation in the growth of the pups, F1 and F2A litters with more than eight pups were culled on day 4 postpartum. At the time of weighing, each litter of pups was randomly ordered by sex. Culled litters were reduced to a total of eight pups, four males and four females, if possible. If it was not possible to have four pups/sex in each litter, partial adjustment was made so that eight pups per litter continued on the study until weaning (e.g., five males, and three females). Cull pups were selected using a computer generated randomisation procedure. Litters with eight or fewer pups were not culled.
Preferential culling of runts was not performed. All cull pups were euthanised with Socumb euthanasia solution (Veterinary Laboratories, Inc. Lenexa, Kansas) and were discarded. All litters were weaned on day 21 postpartum.
One male and one female per litter were randomly selected as P2 animals to produce the second generation. If there were fewer than 30 litters from which to select P2 animals, additional animals were randomly selected from available litters, as needed, in order to obtain the required number of animals/dose level. When possible, three pups/sex/litter were also selected for a necropsy examination. Any weanlings either not held for the next generation of adult animals or not selected for necropsy were examined grossly, euthanised by CO₂ inhalation and discarded.

PHYSICAL MATURATIONAL LANDMARKS
All F1 weanlings selected for mating were observed daily for vaginal opening beginning on postnatal day 28 (Cooper et al., 1989) or preputial separation beginning on day 35 (Korenbrot et al., 1977). On the day that vaginal opening or preputial separation was first detected, the ages and body weights of the animals were recorded. Because there was not a statistically significant effect observed on the age at vaginal opening or preputial separation, anogenital distance was not measured in the F2 pups.

Postmortem examinations (parental animals):

SACRIFICE
A complete necropsy of all P1 and P2 adults was performed as close as possible to the weaning of the last litter. Fasted adult rats submitted alive for a necropsy were anaesthetised by the inhalation of carbon dioxide. Vaginal lavage slides were prepared from all P1 and P2 females for later determination of oestrous cycle stage. While anaesthetised, their tracheas were exposed and clamped, and they were euthanised by decapitation.

GROSS PATHOLOGY
A complete necropsy was conducted on all animals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin using a hand-held syringe and blunt needle. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The uteri of all P1 females were stained with an aqueous solution of 10 % sodium sulfide stain (Kopf et al., 1964) for approximately 2 minutes and were examined for the presence and number of implantation sites. Uteri from P1 females that did not deliver a litter and had no visible implantation sites were examined for evidence of early resorption in order to verify pregnancy status. Staining of the P2 uteri was not conducted due to the fact that implantation sites from two pregnancies would be present, precluding an accurate determination of post-implantation loss. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10 % formalin. Weights of the ovaries, uterus (with oviducts and cervix), testes (paired and left side only), epididymides (paired whole epididymides and left cauda epididymal weight), seminal vesicles with coagulating glands (and seminal fluid), prostate, brain, pituitary, liver, kidneys, adrenal glands, spleen, and thyroid (post fixation) were recorded and the organ-to-body weight ratios calculated.
Representative samples of tissues listed below were collected and preserved in neutral, phosphate-buffered 10 % formalin, except that the ovaries, right testis and right epididymis were preserved by immersion in Bouin’s fixative. Similar necropsy procedures were followed for animals found dead or moribund, except organ weights were not collected and both testes and epididymides were preserved.
The following tissues were collected and preserved: adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland - females only, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues nasal tissues, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve -tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

HISTOPATHOLOGY
Histologic examination of the P1 and P2 adult rats included the reproductive tissues (testis, epididymis, seminal vesicles, coagulating glands, prostate, ovaries, oviducts, uterus, cervix and vagina), pituitary, mammary gland (females), adrenals, kidneys, liver, and relevant gross lesions of all control and high-dose rats as listed above. Examination of tissues from the remaining groups was limited to the liver, relevant gross lesions, and the reproductive organs of animals with signs of reduced fertility. A qualitative evaluation of the testis (PAS-stained sections) included examination for retained spermatids, missing germ cell layers or types, multinucleated giant cells, and sloughing of spermatogenic cells into the lumen. The right epididymis, from which sperm were obtained for motility assessment, was also examined including evaluation of the caput, corpus and cauda. Examination of the ovaries included enumeration of primordial follicles of 15 randomly selected control and 15 high-dose level of the P2 females using a method similar to Bucci et al. (1977). Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with haematoxylin and eosin and examined using a light microscope. Tissues from weanlings were not examined microscopically.
For rats found dead or euthanised moribund, all tissues listed above were collected and preserved. A complete set of tissues was examined microscopically to establish cause of death.
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25 % involvement of the parenchyma. This type of change would neither be expected to significantly affect the function of the specific organ/tissue nor would it have a significant effect on the overall health of the animal. A moderate grade would have been used for conditions that were of sufficient severity and/or extent (up to 50 % of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not be life threatening. A severe grade would have been used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may have been life threatening.

Postmortem examinations (offspring):

SACRIFICE
When litter size permitted, three pups/sex/litter from the F1 and F2A litters were randomly selected at the time of weaning for a complete necropsy. In order to control for variations in body and organ weights, pups selected for a complete necropsy were euthanised at the same age (postnatal day 22). The pups were anaesthetised with carbon dioxide, weighed and euthanised by decapitation.

GROSS PATHOLOGY
For the F1 and F2A pups examined macroscopically, one pup/sex/litter was randomly selected for the collection of brain, spleen, uterus, and thymus weights. Organ-to-body weight ratios were calculated. Gross pathological examination was performed as described for adults, except that weanlings were not fasted overnight.
Representative samples of grossly abnormal tissues were collected from all weanlings at the scheduled necropsy. In addition, the brain, spleen, uterus and thymus were saved for the weanlings selected for organ weight measurements in the event that an effect on organ weight was observed and future evaluation was determined to be necessary. Tissues were fixed in neutral phosphate-buffered 10 % formalin. There were no treatment-related gross lesions or organ weight changes in the F1 or F2A weanlings; therefore, histological examinations were not conducted.

Statistics:

Body weights, gestation and lactation body weight gains, litter mean body weights, feed consumption, sperm count, follicle count, percent total and progressively motile sperm, mean oestrous cycle length, and organ weights were first evaluated by Bartlett's test (α = 0.01) for equality of variances. Based upon the outcome, either a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant at α = 0.05, a Dunnett's test (α = 0.05) or the Wilcoxon Rank-Sum (α = 0.05) test with Bonferroni's correction was performed.
Gestation length, age at vaginal opening, age at preputial separation, average time to mating, and litter size were analysed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Sperm morphology data were arcsine transformed and analysed by a parametric ANOVA. If the ANOVA was significant, the Dunnett’s test was performed.
Statistical outliers (α = 0.02) were identified by the sequential method of Grubbs and were routinely excluded from feed consumption only. The mating, conception, fertility and gestation indices were analysed by the Fisher exact probability test (α = 0.05) with Bonferroni's correction. Evaluation of the neonatal sex ratio on postnatal day 1 was performed by the binomial distribution test (α = 0.05).
Gender was determined for pups found dead on postnatal day 0 and these data were included in sex ratio calculations. Survival indices, post-implantation loss, and other incidence data among neonates were analysed using the litter as the experimental unit by the censored Wilcoxon test (α = 0.05) modified by Haseman and Hoel (1974) with Bonferroni’s correction. Non-pregnant females were excluded from the appropriate analyses. Both the Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control to keep the experiment-wise error rate at 0.05. Both were reported at the experiment-wise α level.

Reproductive indices:

- Female mating index = (No. females with evidence of mating / No. paired) x 100
- Male mating index = (No. males with evidence of mating / No. paired) x 100
- Female conception index = (No. females with evidence of pregnancy / No. mated) x 100
- Male conception index = (No. males siring a litter / No. mated) x 100
- Female fertility index = (No. females with evidence of pregnancy / No. paired) x 100
- Male fertility index = (No. males siring a litter / No. paired) x 100
- Gestation index = (No. females delivering a viable litter / No. females delivering a litter) x 100
- Gestation survival index = Percentage of delivered pups alive at birth
- Post-implantation loss = ((No. implants - No. viable offspring) / No. implants) x 100

Offspring viability indices:

- Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4 / No. born live) x 100
- Day 7, 14, or 21 pup survival index = (No. viable pups on day 7, 14 or 21 / No. viable after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see below

Details on results (P0)

MORTALITY AND CLINICAL SIGNS
Examinations performed on all animals prior to the study start revealed that all animals were in good health for study purposes. All P1 adults survived the dosing regimen and were necropsied on the scheduled termination dates. Observations noted in the P1 adults were limited to a small number of incidental findings. There were no treatment-related effects on behaviour or demeanour observed in the P1 adults at any dose level.
Three P2 animals died or were euthanised moribund. Male 8629 from the low-dose group was found dead on test day 90. This animal did not exhibit any clinical signs prior to its death. Necropsy findings did not establish a cause of death nor was one apparent following microscopic examination of a complete set of tissues. Another P2 male (8668) from the 5 mg/kg/day group was noted with an observation of absent activity on test day 5 and died shortly thereafter. Necropsy findings indicated hydrocephalus as the cause of death. Lastly, a P2 adult female (8800) from the high-dose group was euthanised moribund on GD 15 of the P2A mating (test day 91). Through GD13 this animal had no remarkable observations, but on GD 14 it exhibited red perioral soiling. On GD 15 this rat was noted with decreased quantity of faeces, red, vulvar discharge, blood in its cage, pale skin and mucous membranes, peri-nasal soiling and was cold to the touch. Subsequent necropsy findings revealed a pale animal with blood soiling of the face and perineum, a pale liver, a gastric erosion of the glandular mucosa, a haemorrhage within the lumen of the vagina and a pregnant uterus with eight normal appearing foetuses and four foetuses with haemorrhage into the conceptus (amniotic fluid). Following microscopic examination of tissues, the moribund condition of this rat was attributed to centrilobular hepatic necrosis and cardiac inflammation. This appeared to be an isolated occurrence not seen in any other animals, nor in other studies in which CD rats were given higher dose levels of the test material. Thus, all three deaths were considered to be spontaneous occurrences unrelated to treatment.
All other clinical observations noted among the P2 adults were considered incidental and/or were common findings in CD rats (e.g., maloccluded incisors and associated periocular soiling). Overall, no treatment-related effects on behaviour or demeanour were observed in any phase of the study at any dose level in either the P1 or P2 generation animals.

BODY WEIGHT
There were no treatment-related effects on parental body weights or body weight gains at any dose level. Although there were isolated instances of statistically-identified increases or decreases in body weight or body weight gain values in various treatment groups relative to controls, there was no consistent pattern of change, nor any clear dose-response relationship for these changes. Therefore, none of these differences were considered to be treatment related.

FEED CONSUMPTION
There were no treatment-related effects on feed consumption at any dose level. Although there were isolated instances of statistically-identified increases or decreases in feed consumption in various treatment groups relative to controls, there was no consistent pattern of change, no clear dose-response relationship, nor any corroborating findings on maternal body weight or body weight gains during similar time periods. Therefore, none of these differences in feed consumption were considered to be treatment-related.

GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material.

ORGAN WEIGHTS
Treatment-related increases in absolute and relative liver weights were evident in P1 and P2 males and females of the 25 mg/kg/day group, and in the P1 females of the 5 mg/kg/day group. Absolute and relative liver weights of the 5 mg/kg/day group P2 females were elevated slightly, but the differences were not statistically identified, and changes in relative weights were not dose-related.
Several other statistically significant changes in organ weights occurred in this study, but these appeared to be isolated findings, were very minor in nature, and/or lacked a clear dose response. Thus, they were considered unrelated to treatment with the test material.

HISTOPATHOLOGY
There were no treatment-related microscopic changes in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to the test material.
Histopathologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects considered due to test material administration.
There were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females given 25 mg/kg/day as compared to control females.

SPERM PARAMETERS
There were no effects of treatment on overall sperm motility or progressive sperm motility at any dose level. Determination of epididymal sperm counts of the P1 control and high-dose males revealed a slight, but statistically significant decrease in the high-dose males, thus triggering an analysis of the 0.5 and 5 mg/kg/day dose groups.
Subsequent analysis of the combined data from all groups indicated that the decrease at the high-dose was no longer significant. Furthermore, the epididymal count values for the high-dose P1 males were within the range of historical controls and sperm concentration per gram of tissue was actually greater in high-dose P1 males than in P2 controls. There were no effects on P1 or P2 testicular sperm counts, nor any effects on P2 male epididymal sperm counts. This combined weight of evidence indicated a lack of an effect on sperm counts.
There were no significant effects on sperm morphology in the P1 males. The proportion of abnormal sperm was statistically increased in the high-dose P2 male rats relative to controls. However, this value was less than the proportion of abnormal sperm in the P1 controls, and also was well within the range of historical control values. Therefore, this change was not considered treatment related.

OESTROUS CYCLICITY
Although there some small, but statistically significant increases in oestrous cycle length in the low- and middle-dose P1 females, these changes were not seen in the high-dose P1 animals, nor in any of the P2 groups. Thus, there was no evidence of an effect on oestrous cyclicity at any dose level.

REPRODUCTIVE INDICES AND SEX RATIO
There were no effects of treatment on the P1 adults at any dose level on mating, conception, and fertility indices.
However, decreases in mating (mid- and high-dose only), conception and fertility indices (all treated groups) were apparent in the P2/F2A litter. While none of these changes were statistically-identified, all of the aforementioned indices were outside or very close to the lower bounds of recent historical control values. In order to further evaluate the fertility of the P2 adults, they were re-mated to produce an F2B litter. Among the high-dose animals, the F2B mating and fertility indices remained decreased and outside historical control values, while conception indices were comparable to concurrent control values and within historical control ranges. A similar pattern was seen in the middle-dose group, with mating indices remaining outside historical control values, and fertility indices decreased as well (values were just slightly above the lower bound of historical control values). Thus, decreases in mating and fertility indices at the 5 and 25 mg/kg/day dose levels were considered to be treatment-related effects. In contrast, none of the apparent decreases in mating, conception or fertility indices of the first mating of the 0.5 mg/kg/day animals were repeatable in their second mating, and thus, were not considered to be treatment-related effects.
Inspection of the individual animal data in order to further evaluate the apparent decrease in P2 generation fertility was conducted by comparing the number of animals that were infertile in both the F2A and F2B matings. Among males of the 0, 0.5, 5 and 25 mg/kg/day groups, there were 0, 1, 0 and 2 animals, respectively, that failed to sire a litter in either mating. Among the P2 females, the number failing to produce a litter was 1, 1, 5 and 7, respectively.
This analysis suggests that male fertility was not affected by exposure to the test material, a conclusion further supported by the lack of effects on sperm parameters, male reproductive organ weights and histopathology. Instead, the increase in the number of infertile P2 females at the mid- and high-dose level indicates that females were the affected sex. However, a mechanism for such effects is not apparent, as oestrous cyclicity, female reproductive organ weights and histopathology were unaffected by treatment.
Gestation index, gestation survival index, time to mating, sex ratio and postimplantation loss (P1 females) were normal in all dose groups in all matings. There was a very small, but statistically significant increase in gestation length of the high-dose P1 females. This finding was not considered to be toxicologically significant, as the value (21.9 days) was within the laboratory’s historical control range. Also, the finding was not repeatable in the P2 generation.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on any reproductive or neonatal parameters at 0.5 mg/kg/day

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see blow

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

MORTALITY AND CLINICAL SIGNS
Treatment-related clinical observations were noted in the F1, F2A, and F2B litter pups during their respective lactation periods. Convulsions, characterised as tonoclonic, were observed in five high-dose F1 litters. In all these F1 pups, only single episodes of convulsions were observed, and these occurred between postnatal days 9 - 16. No pup convulsions were noted in the F1 generation control, low- or middle-dose levels. In addition, increased incidences of decreased activity, total litter loss, dead, cannibalised, autolysed, and moribund pups were observed in the high-dose F1 litters. One high-dose pup exhibited the malformation of unilateral anophthalmia. Due to the isolated nature of this malformation, it was most likely an incidental finding which was unrelated to treatment.
Similar clinical observations as seen in the F1 high-dose litters also were observed in the F2A and F2B high-dose litters. These included four high-dose F2A litters with pups exhibiting convulsions between lactation days 7 - 16. Multiple occurrences of convulsions were observed in three of these litters, and three of the four dams with convulsing pups subsequently lost their entire litters between lactation days 8 - 14. Although convulsions were not observed in the F2B litter, the observation period was limited in this litter (pups terminated on lactation day 1).
In the 5 mg/kg/day group, there were no treatment-related clinical observations noted for the F1 or F2B litters. However, convulsions were noted in a single F2A litter on lactation day 14, and this was the only treatment-related clinical observation in pups at this dose level. A single occurrence of anasarca in a middle-dose group F2A pup was considered spurious. This was the only malformed pup found amongst all dose levels of the F2A and F2B litters. There were no treatment-related clinical observations reported in pups from the 0.5 mg/kg/day group.

PUP SURVIVAL
In the high-dose group F1 litter, pup survival was normal through postnatal day 7, but was decreased on postnatal days 14 and 21. These differences were very close to being statistically-identified (P<0.06), were outside of historical control ranges, and thus, were considered treatment-related. This decreased pup survival was partly attributable to two complete litter losses occurring on postnatal days 10 and 21, as described previously. Pup survival also was decreased in the F2A high-dose litters on postnatal days 7, 14 and 21. Again, some of the decreases in pup survival were attributable to several total litter losses. A few statistically-identified increases or decreases in pup survival were seen at the low- dose level, but due to their isolated nature and lack of a dose-response, they were considered spurious and unrelated to treatment. Evaluation of pup survival in the F2B litter was limited due to the study design, but there were no effects on F2B pup survival noted through postnatal day 1. A slight, but statistically identified decrease in day 1 F2B pup survival at the low dose was considered spurious, due to the lack of a dose-response.

LITTER SIZE
There were no statistically- identified effects on litter sizes of the F1 or F2B litters at any dose level. A slight, numerical decrease (not statistically significant) in high-dose F1 litter sizes on postnatal days 14 and 21 corresponded with previously mentioned decreases in pup survival in these same litters.
Similar effects on litter size occurred in the F2A litters, although in the latter case the decreased litter sizes on postnatal days 14 and 21 were statistically-identified.
There were no treatment-related effects on litter size in the 0.5 or 5 mg/kg/day dose level groups at any time in the study.

PUP BODY WEIGHTS
Mean body weights of the male and female F1 and F2A pups were decreased on postnatal days 14 and 21, with most of these changes being statistically-identified. These effects temporally correspond with previously mentioned effects on pup clinical observations and survival during this same period. A few other statistically-identified increases in pup body weights did not correspond with any other toxicologic effects and thus were considered unrelated to treatment.
There were no treatment-related effects on pup body weights at the 0.5 and 5 mg/kg/day dose levels. Lactation day 1 pup body weights were not affected at any dose level in the F2B litters.

PUBERTY ONSET
Ages at vaginal opening or preputial separation were similar in all groups, indicating no influence of treatment.

GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material.

ORGAN WEIGHTS
There were no treatment-related alterations in organ weights of male and female F1 weanlings. Males given 25 mg/kg/day had treatment-related statistically-identified decreases in final body weights and absolute spleen weights, and a statistically-identified increase in relative brain weight.
The alterations in brain and spleen weights were interpreted to be reflective of the decreased body weights of males given 25 mg/kg/day. There were no treatment-related alterations in organ weights of female F2 weanlings at any dose level.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on any reproductive or neonatal parameters at 0.5 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: P2 Reproduction Indices

Parameter (mean %)	Dose Group (mg/kg/day)
	0	0.5	5.0	25
P2A Adults
Male Mating Index	93.3	93.3	89.7	76.7
Female Mating Index	93.3	93.3	90.0	76.7
Male Conception Index	96.4	78.6	80.8	82.6
Female Conception Index	96.4	78.6	81.5	82.6
Male Fertility Index	90.0	73.3*	72.4	63.3
Female Fertility Index	90.0	73.3*	73.3	63.3
P2B Adults
Male Mating Index	96.7	96.6	86.2	79.3
Female Mating Index	96.7	96.7	86.7	79.3
Male Conception Index	96.6	92.9	92.0	87.0
Female Conception Index	96.6	93.1	92.3	87.0
Male Fertility Index	93.3	89.7	79.3	69.0
Female Fertility Index	93.3	90.0	80.0	69.0

Bold indicates effects considered to be toxicologically significant

*Not considered toxicologically significant due to lack of repeatability in P2B mating

 

Table 2: Significant Effects on Survival Index

Parameter (mean %)	Dose Group (mg/kg/day)
	0	0.5	5.0	25
F1 Survival
Day 1 Survival	97.7	99.7*	98.3	98.2
Day 4 Survival	96.7	98.9*	97.5	96.2
Day 14 Survival	98.2	99.5	99.1	89.2
Day 21 Survival	98.2	99.5	98.6	88.8
F2A Survival
Day 7 Survival	100.0	98.2	99.4	90.0*
Day 14 Survival	100.0	97.1*	99.4	72.9*
Day 21 Survival	100.0	97.1*	99.4	69.3*
F2B Survival
Day 1 Survival	99.5	96.9*	98.9	98.4

Bold indicates effects considered to be toxicologically significant

*Statistically different from control mean by censored Wilcoxon’s test, alpha = 0.05

 

Table 3: F2A Mean Litter Size

Parameter (mean size)	Dose Group (mg/kg/day)
	0	0.5	5.0	25
F1 Survival
Day 14	7.9	7.5	7.9	5.7*
Day 21	7.9	7.5	7.9	5.4*

Bold indicates effects considered to be toxicologically significant

*Statistically different from control mean by Wilcoxon’s test, alpha = 0.05

 

Table 4: Significant Effects on Mean pup Weights

Parameter (g)	Dose Group (mg/kg/day)
	0	0.5	5.0	25
F1 Mean Pup Weights
Day 1 (BC) Female Day 1 (BC) Male	6.5 6.9	6.7 7.1	6.7 7.1	7.0* 7.4*
Day 14 (AC) Female Day 14 (AC) Male	32.5 33.1	31.8 32.8	31.8 33.1	30.5* 31.3*
Day 21 (AC) Female Day 21 (AC) Male	52.8 54.7	52.8 55.2	52.4 55.4	49.9* 52.4
F2A Mean Pup Weights
Day 1 (BC) Female Day 1 (BC) Male	6.8 7.1	6.7 7.1	6.7 7.0	7.2 7.7*
Day 14 (AC) Female Day 14 (AC) Male	33.4 34.4	30.9* 32.8	33.1 33.8	31.8 32.5
Day 21 (AC) Female Day 21 (AC) Male	54.1 56.7	51.2 54.5	53.5 55.0	49.6* 50.8*

 

Table 5: Significant Body and Organ Weight Effects in F2 Male Weanlings

Parameter (mean values)	Dose Group (mg/kg/day)
	0	0.5	5.0	25
Final Body Weight (g)	59.8	57.8	58.5	53.7*
Brain (g)	1.525	1.517	1.525	1.482
Brain (g/100)	2.558	2.665	2.614	2.786*
Spleen (g)	0.276	0.263	0.266	0.222**
Spleen (g/100)	0.465	0.450	0.452	0.413

Bold indicates effects considered to be toxicologically significant

Italics indicate effects considered treatment-related, reflective of decreased final body weights

*Statistically different from control mean by Wilcoxon’s test, alpha = 0.05

**Statistically different from control mean by Dunnett’s test, alpha = 0.05

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observable-effect level (NOEL) for parental and reproductive toxicity was 0.5 mg/kg/day.

Executive summary:

A two-generation dietary reproduction toxicity study was conducted to evaluate the potential effects of the test material on male and female reproductive function, as well as the survival, growth and development of the offspring. The study was conducted in accordance with the standardised guidelines OECD 416, US EPA OPPTS 870.3800, EEC Guideline No. 0.378-6978 and JMAFF Guideline No. 2-1-17 under GLP conditions.

Groups of 30 male and 30 female CD rats were fed diets supplying 0, 0.5, 5, and 25 mg/kg/day for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations (including two matings of the P2 generation). In-life parameters included clinical observations, feed consumption, body weights, oestrous cyclicity, reproductive performance, puberty onset, pup survival, pup body weights, and anogenital distance. In addition, post- mortem evaluations included gross and histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Parental toxicity was evident at 25 mg/kg/day, based on increased absolute and relative liver weights in the P1 and P2 males and females. Absolute and relative liver weights also were increased at 5 mg/kg/day (P1 females only). No other effects on general parental toxicity were noted at these dose levels, and there were no effects on any parameter of parental toxicity at 0.5 mg/kg/day. There were no effects on reproductive performance of the P1 adults, but mating and fertility indices were decreased in the P2 rats at dose levels of 5 and 25 mg/kg/day. Females were suspected as the affected sex based on an increased number of females (but not males) which were infertile in both of the F2A and F2B matings. However, there were no effects observed on any other parameters of reproductive function, reproductive organ weights or histopathology in either sex.

In neonates from the 25 mg/kg/day group, pup survival, litter size and body weight all were decreased in both generations. Tonoclonic convulsions also were observed in several high-dose litters in each generation, as well as in one F2 litter from the middle-dose group. These effects on neonates generally occurred between lactation days 7 - 21. Minor effects on relative brain weight and absolute spleen weight of the F2 male weanlings were considered secondary to decreased body weights.

There were no other effects on any neonatal or developmental parameters at these dose levels, and no effects on any reproductive or neonatal parameter at 0.5 mg/kg/day. Based on these data, the no-observable-effect level (NOEL) for parental and reproductive toxicity was 0.5 mg/kg/day.