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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Negative for teratogenic potential in ex vivo whole embryo cultures, Carney 1997.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Guideline No. 0378-6978

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guideline No. 2-1-17

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Dose / conc.:

0.5 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Dose / conc.:

5 mg/kg bw/day

Remarks:

Basis:
nominal in diet

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.

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see below

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.

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

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

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.

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

Reproductive effects observed:

not specified

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

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The two studies available were both conducted in standard species, under GLP conditions and largely in accordance with standardised guidelines. The quality of the database is therefore considered to be high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Two studies are available to address this endpoint, one key and one supporting. Both studies were awarded a reliability score of 1 in accordance with the criteria of Klimisch et al. (1997).

 

The key study is a two-generation dietary reproduction toxicity study which 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.

 

The supporting study was originally designed to meet two-generation test guidelines for reproduction and fertility effects, namely OECD 416, EPA OPPTS 870.3800, EEC (ISSN 0378-6978) and JMAFF No. 2-1-17; however, due to unexpected toxicity, the study design was modified to a one-generation study with a cross-fostering component. All work was carried out under GLP conditions.

The purpose of the one-generation dietary reproduction toxicity study was 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. Groups of 30 male and 30 female CD rats were fed diets supplying 0, 0.5, 5, and 100 mg /kg/day for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation. In-life parameters included clinical observations, feed consumption, body weights, oestrous cyclicity, reproductive performance, pup survival, and pup body weights. Post-mortem evaluations included gross and histopathology, and organ weights in adults, and gross pathology and organ weights in weanlings.

Treatment-related effects were limited to the high-dose animals. Aside from a few incidences of red periocular soiling, four P1 adults had tonoclonic convulsions 11 - 15 weeks after initiation of exposure. P1 males had persistent decreases in feed consumption and body weights. P1 females had decreases in gestation body weight gains and lactation body weights and body weight gains. The P1 gestation survival index (i.e., number live born) was significantly decreased, followed by pronounced decreases in pup survival and litter size at all postnatal time points examined. Tonoclonic convulsions were observed in F1 pups from 17 of 24 F1 litters. By LD 21, 23 of 24 P1 dams had lost their entire litters with only two pups surviving. F1 pup body weights also were significantly decreased. Although originally intended as a two-generation study, the study was not carried into the second generation due to excessive F1 pup mortality at 100 mg/kg/day. Instead, to determine whether the decreased pup survival from treated rats resulted from in utero or lactational exposure, in utero control and treated pups were cross-fostered to control and treated lactating dams. Regardless of in utero conditions (control or exposed), pups cross-fostered to treated lactating dams had convulsions and excessive mortality with total litter loss in seven of eight litters, whereas pups cross-fostered to control dams had normal patterns of development and survival. These data suggest that maternal exposure to the test material and subsequent lactation was responsible for decreased pup survival, whereas gestational exposure played little to no role. P1 adults and F1 weanlings in the 0, 0.5 and 5.0 mg/kg/day dose groups (10/sex/dose) were sent to necropsy to evaluate these dose levels for a subsequent two-generation study. There were no treatment-related effects in these animals.

Therefore, in this abbreviated reproductive toxicity study, the no-observed-effect- level (NOEL) for parental and reproductive toxicity was 5.0 mg/kg/day.

Short description of key information:

NOEL 0.5 mg/kg/day, male and female CD rats, OECD 416, US EPA OPPTS 870.3800, EEC Guideline No. 0.378-6978 and JMAFF Guideline No. 2-1-17, Carney et al. 2004.

Justification for selection of Effect on fertility via oral route:

This study was selected as key on the basis that it is a full two-generation reproductive toxicity study in a standard species conducted under GLP conditions.

Effects on developmental toxicity

Description of key information

NOEL 1000 mg/kg/day, female CD rats, 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, Marty & Zablotny 2000.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF Toxicity Testing Guidelines for Teratogenicity Studies

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

250 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

500 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

750 mg/kg bw/day

Remarks:

Basis:
actual ingested

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.

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.

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.

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

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.

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.

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 1999 - 14 September 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan MAFF Toxicity Testing Guidelines for Teratogenicity Studies

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: sexually mature adults
- Weight at study initiation: Females weighed 200 to 250 g
- Fasting period before study: No
- Housing: Animals were housed one per cage in stainless steel cages with wire-mesh floors 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: 4 to 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °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

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, multiple mixes of the dose suspensions were prepared periodically throughout the study period.

VEHICLE
- Amount of vehicle (if gavage): A dose volume of 4 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 (12 days) to confirm stability of the test material in vehicle.
Analyses of the low and high dose suspensions indicated a stability of at least 12 days.

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 93 to 107 % of targeted concentrations.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- M/F ratio per cage: 1 male: 1 female
- Length of cohabitation: Sexually mature, adult virgin females were naturally mated with males of the same strain at the supplier. Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males’ cages.
- Proof of pregnancy: The day on which a vaginal plug was detected was considered day 0 of gestation. Rats were shipped on day 0 of gestation and arrived in the testing laboratory on day 1 or 2 of gestation.

Duration of treatment / exposure:

Days 6 to 20 of gestation

Frequency of treatment:

Once daily

Duration of test:

Animals were sacrificed on Day 21 of gestation

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

250 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

500 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

750 mg/kg bw/day

Remarks:

Basis:
actual ingested

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: Based on the results of the previous studies, a high dose of 1000 mg/kg/day was selected for this study. This dose level represents a limit dose as defined by several regulatory agencies. The lower doses were expected to provide the dose response data for any toxicity observed in the high dose rats.

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 and daily during Days 6 through 21 of gestation. Statistical analyses of body weights and body weight gains were performed using data collected on Days 0, 6, 9, 12, 15, 18, and 21 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Feed consumption was measured and statistically analysed for the following intervals: gestation days 3-6, 6-9, 9-12, 12-15, 15-18, and 18-21. Feed consumption was measured by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed crock - final weight of feed crock) / (# of days in measurement cycle)

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
On day 21 of gestation, all surviving females (non-fasted) were euthanised by carbon dioxide inhalation 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 were examined. The stomach, liver and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weights of the liver, kidneys and gravid uterus were recorded. The ratios of liver and kidney weights to day 21 (terminal) body weight were calculated. Representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10 % formalin. Microscopic examination of preserved tissues were not conducted.

Any animal found dead was submitted on that day for a complete necropsy. This necropsy was performed as described above with the following exceptions: 1) the head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined, 2) all viscera were dissected from the carcass and re-examined, 3) liver, kidney and gravid uterine weights were not recorded, 4) the number of corpora lutea and the sexes and body weights of foetuses from these animals were not recorded. Development of the conceptuses was determined to the extent possible by external examination (as appropriate for gestational age), but these embryos/foetuses 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 in utero
- 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; all 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. At least one half of all the foetuses in each litter were chosen randomly for visceral examination conducted 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. The heads of these 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). Foetuses designated for visceral examination were preserved in alcohol. The remaining foetuses designated for skeletal examination were eviscerated, skinned, preserved in alcohol, and double stained with Alcian Blue and Alizarin Red S for cartilage and bone, respectively, and cleared in glycerin (Webb and Byrd, 1994). A thorough evaluation of the foetal skeleton was conducted on the double-stained specimens. 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.

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 and resorptions per foetal population, and foetal alterations were analysed using a censored Wilcoxon test with Bonferroni's correction.
The number of corpora lutea and implantations, and litter size were 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 were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method, and excluded if justified by sound scientific reasons. Data from scratched or spilled feed were excluded from feed consumption analysis. 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.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
Examinations performed on all animals prior to the start of dosing (gestation days 1 - 6) revealed that one animal (#1623) assigned to the 1000 mg/kg/day dose group had slow, laboured respiration and was removed from study.
During the dosing interval, one rat given 500 mg/kg/day (#1599) was found dead on day 20 of gestation due to a probable gavage error. All other rats survived to the scheduled necropsy. Examinations performed during the dosing period revealed no treatment-related findings. All other observations were incidental in nature and were not related to treatment.

BODY WEIGHT
There were no effects of treatment on maternal body weights or body weight gains at any dose level compared with control animals.

FEED CONSUMPTION
There were no treatment-related effects on the amount of feed consumed by any treated groups when compared to the controls.

GROSS PATHOLOGY
Dam 1599 (500 mg/kg/day) was found dead on day 20 of gestation. At necropsy, findings recorded included dark oedematous lungs and facial soiling. All other tissues were within normal limits. The uterus contained 12 normal appearing foetuses and two early resorptions. In view of the lack of clinical observations and body weight effects prior to its death, the presence of gross pathologic alterations suggestive of gavage error, and the lack of similar effects in rats given 1000 mg/kg/day, the death was not interpreted to be treatment related. No gross pathologic alterations were noted in any of the treated dams that survived to the scheduled necropsy.

ORGAN WEIGHTS
Test material treatment, at doses up to and including 1000 mg/kg/day, had no effect on any of these endpoints when compared with control animals.

REPRODUCTIVE AND FOETAL PARAMETERS
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 rats given up to and including 1000 mg/kg/day, the highest dose level tested. Although there was a very slight increase in the percentage of implantations resorbed and the percentage of litters with resorptions at the high dose level (Table 1), these values are well within the range of historical control values for CD rats used in the testing laboratory.

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

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no statistically identified differences in the incidence of any foetal alteration in any of the treated groups when compared to the controls. There were no malformed foetuses among the controls. Four foetuses, from four different litters were malformed in the 250 mg/kg/day dose level. The first foetus had hindlimb rotation; the second had an omphalocele and the third had impatent nares. The fourth foetus exhibited multiple skeletal malformations consisting of fused cervical and lumbar vertebrae along with a hemivertebra in the cervical and lumbar regions. Fused and missing ribs were also noted. Three foetuses from two different litters were malformed in the 500 mg/kg/day dose level. One mid-dose female had a missing lumbar vertebra and the remaining two foetuses had class II wavy ribs. Among the high-dose, two foetuses from two different litters were malformed. The first foetus had a cleft lip and palate and the second had a missing rib. These malformations were observed as isolated occurrences, found only in single foetuses, and therefore were not attributed to treatment. With regard to variations, all variations observed in foetuses from dams administered the test material either occurred at low frequencies and/or were not dose related.

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: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Selected Reproductive Indices

Parameter	Dose Group (mg/kg/day)
	0	250	500	1000
% Implantations resorbed	3.2	2.3	3.1	4.9
% Litters with resorptions	34.8	25.0	39.1	45.8

Conclusions:

Under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity and embryonal/foetal effects 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 administered to CD rats 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 CD rats were administered the test material (suspended in 0.5 % methylcellulose) by gavage at targeted dose levels of 0, 250, 500 and 1000 mg/kg/day on days 6 through 20 of gestation. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain and feed consumption. On day 21 of gestation, all surviving rats were euthanised and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions and live/dead foetuses.

All foetuses were weighed, sexed and examined for external alterations. Visceral or skeletal examinations, each involving approximately one half of the foetuses were conducted. 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 rats given up to and including 1000 mg/kg/day, the highest dose level tested.

Gavage administration of the test material up to and including the limit dose of 1000 mg/kg/day resulted in no maternal toxicity, nor were there any indications of embryonal/foetal toxicity. There was no evidence of teratogenicity with test material treatment. Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity and embryonal/foetal effects was 1000 mg/kg/day, the highest dose level tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There were several studies conducted to standardised guidelines under GLP conditions in two species. The quality of the database is therefore considered to be high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Four studies are available to address this endpoint, two key studies in two species and two supporting studies (probe studies in both species). All studies were awarded a reliability score of 1 in accordance with the criteria of Klimisch et al. (1997).

 

Key Studies: Pre-Natal Developmental Toxicity (Rat and Rabbit)

The key study was conducted to evaluate the maternal and developmental toxicity potential of the test material administered to CD rats 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.

During the study groups of twenty-five time-mated female CD rats were administered the test material (suspended in 0.5 % methylcellulose) by gavage at targeted dose levels of 0, 250, 500 and 1000 mg/kg/day on days 6 through 20 of gestation. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain and feed consumption. On day 21 of gestation, all surviving rats were euthanised and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions and live/dead foetuses.

All foetuses were weighed, sexed and examined for external alterations. Visceral or skeletal examinations, each involving approximately one half of the foetuses were conducted. 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 rats given up to and including 1000 mg/kg/day, the highest dose level tested.

Gavage administration of the test material up to and including the limit dose of 1000 mg/kg/day resulted in no maternal toxicity, nor were there any indications of embryonal/foetal toxicity. There was no evidence of teratogenicity with test material treatment. Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity and embryonal/foetal effects was 1000 mg/kg/day, the highest dose level tested.

 

The second key 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.

During the study 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.

 

Supporting Studies: Probe Studies for Dose Selection in Key Studies

The first supporting study, a probe study, was conducted to evaluate the test material for potential developmental toxicity effects in rats. This study was not conducted to a standardised guideline as its purpose was to act as a probe in order to allow the setting of dose levels for a subsequent study; experimental work was carried out under GLP conditions.

Groups of eight time-mated female CD rats were administered the test material by gavage at targeted dose levels of 0, 250, 500, 750 or 1000 mg/kg/day on days 6 through 20 of gestation in a vehicle of 0.5 % METHOCEL (cellulose ethers) A4M. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain and feed consumption. On day 21 of gestation, all surviving rats were euthanised and examined for gross pathologic alterations and changes in liver and kidney weights. The number of uterine implantations and resorptions and the number of corpora lutea were also determined.

Oral administration up to and including the limit dose of 1000 mg/kg/day resulted in no maternal toxicity, nor were there any adverse effects on embryonal/foetal survival. Therefore, under the conditions of this study the no-observed-effect level (NOEL) for maternal toxicity and embryonal/foetal effects was 1000 mg/kg/day, the highest dose level tested.

 

The second supporting study, a probe study, was conducted to evaluate the test material for potential developmental toxicity effects in the rabbit. This study was not conducted to a standardised guideline as its purpose was to act as a probe in order to allow the setting of dose levels for a subsequent study; experimental work was carried out under GLP conditions.

The dosing schedule was based on the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3700 Prenatal Developmental Toxicity Study).

Groups of seven time-mated female New Zealand White rabbits were administered the test material by gavage at targeted dose levels of 0, 250, 500, 750 and 1000 mg/kg/day on days 7 through 27 of gestation in a vehicle of 0.5 % METHOCEL (cellulose ethers) A4M. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain and feed consumption. On day 28 of gestation, all surviving rabbits were euthanised and examined for gross pathologic alterations and changes in liver and kidney weights. The number of uterine implantations and resorptions and the number of corpora lutea were also determined.

Oral administration of 1000 mg /kg/day to time-mated New Zealand White rabbits resulted in a treatment-related statistical increase (25 %) in relative liver weight. Although not statistically identified, absolute liver weight of the 1000 mg/kg/day rabbits was also increased 20 % when compared to controls. No other treatment-related maternal effects were noted for rabbits administered 1000 mg/kg/day. No significant maternal effects were observed among rabbits administered 250, 500 or 750 mg/kg/day and no evidence of embryo/foetal lethality attributable to treatment was noted at any dose level.

Therefore, under the conditions of this study, the no-observed-effect-level (NOEL) for maternal effects was 750 mg/kg/day and the NOEL for embryo/foetal lethality was 1000 mg/kg/day, the highest dose level tested.

Justification for selection of Effect on developmental toxicity: via oral route:

Two guideline, GLP studies in standard species were available. The study in the rat was selected on the basis that this is usually the first-choice species for toxicity studies.

Toxicity to reproduction: other studies

Additional information

In a supporting study, the test material was screened for teratogenic potential in ex vivo whole embryo cultures. It was awarded a reliability score of 2 in accordance with the criteria of Klimisch et al. (1997).

Seven non-pregnant female rats received 1000 mg/kg/day of the test material for 3 days via oral gavage. Four hours after the last dose, the rats were exsanguinated and their blood centrifuged to obtain serum. Six control rats were similarly dosed with 0.5 % Methocel vehicle and their serum collected. Rat conceptuses from a group of untreated, time-mated female rats were explanted on the afternoon of gestation day 9 and cultured in the sera (100 %) from the respective donors. After approximately 48 hours in culture, the embryos were evaluated for viability, growth and morphology.

No effects on body weight or clinical observations were noted in the serum donors dosed with the test material. Embryo viability and growth parameters were either comparable or slightly higher than those of the control group. There were no treatment-related morphological abnormalities in the embryos. Under the conditions of the screening test, the test material was considered negative for teratogenic potential.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I (Classification and Labelling Requirements for Hazardous Substances and Mixtures), Regulation (EC) No. 1272/2008 (CLP), the substance requires classification for reproductive toxicity as follows:

In the multigeneration studies (Carney 2004 and Marty 2004) based on decreased P2 female fertility, decreased litter size/ pup survival, lactation effects (mortality of untreated pups cross fostered to treated dams); neurotoxicity effects in parents (convulsions) and pups (convulsions), Reproduction Cat. 2 (H361, H362) is required.