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REACH

2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

EC number: 604-721-7 | CAS number: 150114-71-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Substance considered negative for teratogenic potential in this screening test, ex vivo whole embryo culture test,  Liberacki & Carney (2000)

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 November 2001 to 16 June 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

other: European Economic Community. Methods for the Determination of Toxicity. Official Journal of the European Communities, Vol. 31, No. L 133, May 30, 1988. ISSN 0378-6978, 1988.

Deviations:

Qualifier:

according to guideline

Guideline:

other: The Japan Ministry of Agriculture, Forestry and Fisheries, Notification of 12- Nohsan-8147, Guideline 2-1-17, Reproduction Study, November 24, 2000.

Deviations:

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

Purity: 94.5%

Species:

rat

Strain:

other: Crl:CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: CD (Crl: CD (SD) IGS BR)
- Age at study initiation: ca. 6 weeks
- Weight at study initiation (parental generation group means on day -2): 193.7 - 193.9 g (males); 147.6 - 147.8 g (females)
- Housing: Animals were housed individually in stainless steel cages with wire-mesh floors suspended above catch pans. Cages contained feed containers and pressure activated nipple-type watering systems. Dams were housed one per cage (with their litter) in plastic cages provided with corn cob nesting material from approximately day 19 of gestation and throughout the lactation phase of the study.
- Diet: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40 - 70 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIETARY EXPOSURE
Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Pre-mixes were prepared periodically throughout the study based on stability data. Diets were prepared weekly for approximately ten weeks prior to breeding of the P1 adults. 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 either the male or female diet, whichever is of lower concentration. During gestation, females from each dose group were provided with the appropriate dietary concentration of test material 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 (Carney et al., 1998). Until all litters were weaned, weanlings received a diet containing the same concentration of test material that was given to the P1 females during the third week of lactation. Dams awaiting necropsy received a diet containing the same concentration of test material that was given during the breeding period until all litters had finished the lactation phase. Dietary concentrations for the P2 generation were calculated as described for the P1 animals.

Details on mating procedure:

Breeding for the P1 and P2 adults commenced after approximately ten weeks of treatment.
- M/F ratio per cage: 1 male: 1 female.
- Length of cohabitation: until mating occurred or two weeks elapsed.
- Proof of pregnancy: the presence of a vaginal plug, or sperm in vaginal smear, was referred to as day 0 of pregnancy.
The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. 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 F1 male and female littermates was avoided. In cases where a mating partner had died or was otherwise not available, the animal was paired with the next available partner. A second breeding of the first or second -generation adults was not conducted.

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 at least once during the study.
Analyses confirmed that the test material was homogeneously distributed in the diets.

- Stability
A previous four-week toxicity study in mice demonstrated the test material to be stable for at least 21 days in rodent chow at concentrations ranging from 0.005 to 3 %. A two-year dietary chronic toxicity/oncogenicity and chronic neurotoxicity study has demonstrated the test material to be stable for at least 55 days at a 7 % concentration. An 18 month chronic toxicity/oncogenicity study has demonstrated the test material to be stable for at least 35 days at 0.0258 %. Test diets for the current study were prepared and used within these stability limits.

- Concentration Verification
Analyses of all test diets to determine concentration of the test material were conducted at least three times (at the start, midway, and near the end of the study). The analyses were conducted using a solvent extraction method followed by liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
One of the analyses was done on a breeding diet which is common to both sexes. The mean concentrations of test material in the test diets over the entire study period were 94.1, 100, and 102 % of target for the male 50, 250, and 1000 mg/kg/day dose levels, and 104, 108, and 106 % of target for the female 50, 250, and 1000 mg/kg/day dose levels, respectively.

Duration of treatment / exposure:

Animals were dosed for approximately ten 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 diet)

Details on study schedule:

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 F1 male and female littermates was avoided. In cases where a mating partner had died or was otherwise not available, the animal was paired with the next available partner. A second breeding of the second-generation adults was not conducted.

- Culling and Weaning
To minimise variation in pup growth due to differences in litter size, F1 and F2 litters were standardised to eight pups per litter on postnatal day 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated selection procedure, so that four males and four females (whenever possible) remained in each litter. Litters with fewer than eight pups were not culled. All litters were weaned on postnatal day 21.
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, one pup/sex/litter was selected for a gross examination with the collection of organ weights and two additional pups/sex/litter were selected for a gross examination only. Any weanlings either not held for the next generation of adult animals or not selected for necropsy were euthanised.

- Physical Maturational Landmarks
All F1 weanlings selected for mating were observed daily for vaginal opening beginning on postnatal day 28 or preputial separation beginning on day 35. The age and body weights at the time of landmark acquisition were recorded. Because there was not a treatment-related effect on the F1 sex ratio, age at vaginal opening or age at preputial separation and anogenital distance were not measured in the F2 pups.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 males and 30 females per dose.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected based on the previous 13-week dietary probe study in CD rats. Administration of test material at the high-dose (1000 mg/kg/day) was expected to produce increases in cecum weights with possible epithelial hyperplasia of the cecum (based on existing data from the reproductive toxicity probe study). The top dose level of 1000 mg/kg/day for this study was a limit dose as defined by the relevant regulatory guidelines. The middle- and low-doses were expected to provide dose response data for any treatment-related effects observed in the high-dose group and to establish a no-observed-effect level (NOEL).
- Rationale for animal assignment: Prior to test material administration, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice each day a cage-side examination was conducted. 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-study and weekly thereafter. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm- or plug-positive) females received clinical examinations on gestation day (GD) 0, 7, 14, and 21. In addition, mated females were observed for signs of parturition beginning on or about day 20 of gestation. Females that delivered litters were subsequently evaluated on lactation day (LD) 0, 1, 4, 7, 14, and 21. 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), swellings, masses, and animal behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and weekly during the pre-breeding treatment period. Body weights for males were recorded weekly throughout the course of the study. Sperm- or 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 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: Yes
- Time schedule for examinations: Feed consumption was determined pre-exposure and weekly during the ten-week pre-breeding period 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, feed consumption was measured on GD 0, 7, 14, and 21. During lactation, feed consumption was measured on days 1, 4, 7, 11, 14, 17, 19, and 21. 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) / number of days in measurement cycle

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. Additionally, on the day of scheduled necropsy, the stage within the oestrous cycle was determined for all P1 and P2 female rats.

Sperm parameters (parental animals):

Parameters examined in P1 and P2 male parental generations: testis weight, epididymis weight and seminal vesicles with coagulating glands (and seminal fluid) weight

SPERM ANALYSIS
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
Sperm motility was evaluated in all surviving P1 and P2 males at termination. 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 ca. 5 mL of SpermPrep Medium 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) 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 testis or epididymis was minced, diluted and stained with a fluorescent DNA-binding dye and spermatid or sperm count 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.
- 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). Samples with less than 200 sperm were excluded from analysis. Morphological evaluation of sperm from control and high-dose males was conducted. Sperm morphology was scored blind with respect to treatment group.

Litter observations:

LITTER DATA
Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 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 LD 1, 4 (before and after culling), 7, 14, and 21, and any visible physical abnormalities or demeanour changes in the neonates. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects. These pups were preserved in neutral, phosphate-buffered 10 % formalin.

CULLING AND WEANING
To minimise variation in pup growth due to differences in litter size, F1 and F2 litters were standardised to eight pups per litter on postnatal day 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated selection procedure, so that four males and four females (whenever possible) remained in each litter. Litters with fewer than eight pups were not culled. Culled pups were euthanised by administration of Socumb euthanasia solution into the buccal cavity, and then discarded. All litters were weaned on postnatal day 21.
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, one pup/sex/litter was selected for a gross examination with the collection of organ weights and two additional pups/sex/litter were selected for a gross examination only. Any weanlings either not held for the next generation of adult animals or not selected for necropsy were 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). The age and body weights at the time of landmark acquisition were recorded. As there was not a treatment-related effect on the F1 sex ratio, age at vaginal opening or age at preputial separation and anogenital distance were 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 weighed and 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 NECROPSY
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 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 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. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10 % formalin.

ORGAN WEIGHTS
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides (total weights for both epididymides and cauda weight for the left side only), seminal vesicles with coagulating glands (and seminal fluid), prostate, brain, pituitary, liver, kidneys, adrenal glands, spleen, cecum (empty and full) and thyroid (after fixation) were recorded. Organ-to-body weight ratios were calculated for all weighed organs with the exception of the left testis and left cauda epidymidis.

HISTOPATHOLOGY
Representative samples of the following tissues 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 (paired testes and epididymides were preserved for dead males).
- 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/pharynx, 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, vagina.
Histologic examination of the adult rats included the reproductive tissues, target organs, and relevant gross lesions of all control and high-dose rats as listed above (including thyroid gland due to weight increases). Reproductive organs of low-and mid-dose animals with signs of reduced fertility also were examined histologically. A qualitative evaluation of the testis 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, also was examined including evaluation of the caput, corpus and cauda. Examination of the ovaries included enumeration of primordial follicles of the P2 females using a method similar to Bucci et al. (1977). A subset of 15 randomly selected control and high-dose P2 females were chosen for primordial follicle enumeration, based on a power analysis. As no treatment-related effects were observed at the high dose, primordial follicle enumeration from the lower dose levels was not conducted. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with haematoxylin and eosin and examined using a light microscope. Examinations of tissues from the remaining groups were limited to those tissues that demonstrated treatment-related histologic effects at the high-dose and relevant gross lesions. Rats found dead or moribund were histologically examined in a similar manner.

Postmortem examinations (offspring):

SACRIFICE
When litter size permitted, three pups/sex/litter from the F1 and F2 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 NECROPSY
For the F1 and F2 pups that were 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 above for adults, except that the 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, thymus, cecum and ileum were saved for the weanlings selected for organ weight measurements in the event that an effect on organ weight was observed and/or future evaluation was desirable. Tissues were fixed in neutral phosphate-buffered 10 % formalin. There were no treatment-related gross lesions in the F1 or F2 weanlings; therefore, histological examination was not conducted.

Statistics:

See "Any other information on materials and methods incl. tables" for information.

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:
- 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 delivering a litter / No. mated) x 100
- Male conception index = (No. males siring a litter / No. mated) x 100
- Female fertility index = (No. females with evidence of delivering a litter / 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. live after culling) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

Overall, no treatment-related effects on behaviour or demeanour were observed in any phase of the study at any dose level in the P1-generation animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the first generation two males and one female died prior to scheduled termination, however on investigation these were not attributed to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Aside from one isolated value, there were no statistically identified differences in the body weights of the P1 or P2 males or the body weights and body weight gains of the P1 and P2 females during their respective pre-mating, mating, gestation, or lactation periods. The exception was an isolated finding of decreased body weight gain in the low-dose P1 females over the entire lactation period (LD 1-21), which was considered spurious due to the lack of dose-response and lack of repeatability in the P2 generation. Low-dose P2 females had the largest gain over the entire lactation period in the P2 generation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Several statistically identified increases and decreases in feed consumption were noted at different periods throughout the study. However these were slight and there was no consistent pattern of change nor any corroborating findings on paternal or maternal body weight or body weight gains during these time periods. These findings were not considered to be biologically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic changes due to exposure to the test material in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to the test material. The cecal organ weight and gross pathologic changes were unassociated with microscopic changes indicative of a significant toxicologic effect. The normally formed faeces in the descending colon and rectum further supports the conclusion of a physiologic effect localised to the cecum and of no toxicological significance. The absence of microscopic changes in the cecum, as well as the other tissues examined, demonstrates the test material and/or its metabolite(s) were not significantly toxic, even at the highest dose tested.
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 control females as compared to females given 1000 mg/kg/day.

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no evidence of an effect on oestrous cyclicity at any dose level of test material.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects of the test material on any sperm analysis parameter at any dose level.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating, gestation length, pup survival or pup sex ratio in either generation. The only parameter that was significantly altered was pup survival, which was statistically increased on LD 4 in the 1000 mg/kg/day dose group of the P1 generation. This finding is likely spurious and is not considered adverse.

MORTALITY AND CLINICAL SIGNS
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.
In the first generation two males and one female died prior to scheduled termination, however on investigation these were not attributed to treatment.
In the second generation one adult male died prior to the start of the pre-breeding phase, other incidental observations were noted in other P2 animals. None of these were attributed to treatment with the test material.

FEED CONSUMPTION
Several statistically identified increases and decreases in feed consumption were noted at different periods throughout the study. However these were slight and there was no consistent pattern of change nor any corroborating findings on paternal or maternal body weight or body weight gains during these time periods. These findings were not considered to be biologically significant.

BODY WEIGHTS
Aside from one isolated value, there were no statistically identified differences in the body weights of the P1 or P2 males or the body weights and body weight gains of the P1 and P2 females during their respective pre-mating, mating, gestation, or lactation periods. The exception was an isolated finding of decreased body weight gain in the low-dose P1 females over the entire lactation period (LD 1-21), which was considered spurious due to the lack of dose-response and lack of repeatability in the P2 generation. Low-dose P2 females had the largest gain over the entire lactation period in the P2 generation.

ANATOMIC PATHOLOGY
- Organ Weights: There were statistically identified differences in cecal weights of middle- and high-dose P1 males and females when compared to their respective controls. Other organ weights and terminal body weights were not altered by treatment with the test material in either sex of P1 adult rats. Similar to the P1 males, there were statistically identified differences in cecal weights of middle- and high-dose P2 males. However, the low-dose P2 males also had significantly increased relative empty cecal weights, contrary to the P1 low-dose males. In the P2 females, cecum weights were altered only at the highest dose level of test material. Other organ weights and terminal body weights were not altered by treatment with the test material in either sex of P2 adult rats.
- Gross Pathology: All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material, except those in the cecum of male and female rats. In the 250 mg/kg/day dose group, a few animals (P1 and P2 females, P2 males) had grossly observed increases in cecum size, but this cecal observation was noted most frequently in high-dose males and females in both the P1 and P2 generations, where the incidence of animals affected was 33-70 %. The cecum of P1 and P2 adults of both sexes in the 50 mg/kg/day group were grossly unaffected by treatment.
- Histopathology: There were no treatment-related microscopic changes due to exposure to the test material in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to the test material. The cecal organ weight and gross pathologic changes were unassociated with microscopic changes indicative of a significant toxicologic effect. The normally formed faeces in the descending colon and rectum further supports the conclusion of a physiologic effect localised to the cecum and of no toxicological significance. The absence of microscopic changes in the cecum, as well as the other tissues examined, demonstrates the test material and/or its metabolite(s) were not significantly toxic, even at the highest dose tested.
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 control females as compared to females given 1000 mg/kg/day.

SPERM PARAMETERS
There were no effects of the test material on any sperm analysis parameter at any dose level.

OESTROUS CYCLICITY
There was no evidence of an effect on oestrous cyclicity at any dose level of test material.

REPRODUCTIVE INDICES, PUP SURVIVAL AND SEX RATIO
There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating, gestation length, pup survival or pup sex ratio in either generation. The only parameter that was significantly altered was pup survival, which was statistically increased on LD 4 in the 1000 mg/kg/day dose group of the P1 generation. This finding is likely spurious and is not considered adverse.

Key result

Dose descriptor:

NOAEL

Remarks:

(parental toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically adverse effects at highest dose tested.

Key result

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive effects at the highest dose tested

Clinical signs:

no effects observed

Description (incidence and severity):

Overall, no treatment-related effects on behaviour or demeanour were observed in any phase of the study at any dose level in the P2-generation animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the second generation one adult male died prior to the start of the pre-breeding phase, other incidental observations were noted in other P2 animals. None of these were attributed to treatment with the test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Similar to the P1 males, there were statistically identified differences in cecal weights of middle- and high-dose P2 males. However, the low-dose P2 males also had significantly increased relative empty cecal weights, contrary to the P1 low-dose males. In the P2 females, cecum weights were altered only at the highest dose level of test material. Other organ weights and terminal body weights were not altered by treatment with the test material in either sex of P2 adult rats.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material, except those in the cecum of male and female rats. In the 250 mg/kg/day dose group, a few animals (P1 and P2 females, P2 males) had grossly observed increases in cecum size, but this cecal observation was noted most frequently in high-dose males and females in both the P1 and P2 generations, where the incidence of animals affected was 33-70 %. The cecum of P1 and P2 adults of both sexes in the 50 mg/kg/day group were grossly unaffected by treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no evidence of an effect on oestrous cyclicity at any dose level of test material.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects of the test material on any sperm analysis parameter at any dose level.

Reproductive performance:

no effects observed

Description (incidence and severity):

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically adverse systemic effects at the highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on reproduction at the highest dose tested

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pup survival was statistically increased on LD 4 in the 1000 mg/kg/day dose group of the P1 generation. This finding is likely spurious and is not considered adverse. There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size at any time in any dose group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on pup body weights at any time in any dose group. Pup weights were significantly higher than the controls on LD 14 in the F1 litters of the 250 and 1000 mg/kg/day dose groups. This increases were considered spurious because this response lacked of a dose-response relationship, occurred at isolated time points, and was not accompanied by other reproductive or developmental effects.

Sexual maturation:

no effects observed

Description (incidence and severity):

Age at vaginal opening or preputial separation was similar in all groups, indicating no influence of treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on terminal body weights or organ weights for the male and female F1 weanlings. F1 female weanling in the 250 mg/kg/day dose group exhibited a significant decrease in absolute uterine weight, but this effect was considered spurious based on the absence of a dose-response relationship and the lack of reproducibility in the F2 weanlings.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings:

not examined

Observations made on the F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings were noted. However, no malformed pups were observed in either generation at any dose level tested.

Key result

Dose descriptor:

NOAEL

Generation:

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects at the highest dose tested

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size at any time in any dose group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on pup body weights at any time in any dose group. Pup weights were significantly higher than the controls on LD 7 in the F2 litters of the 1000 mg/kg/day dose group. These increases were considered spurious because this response lacked of a dose-response relationship, occurred at isolated time points, and was not accompanied by other reproductive or developmental effects.

Sexual maturation:

no effects observed

Description (incidence and severity):

Age at vaginal opening or preputial separation was similar in all groups, indicating no influence of treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The final body weight and organ weight data for the male and female F2 weanlings were unaffected by treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Key result

Dose descriptor:

NOAEL

Generation:

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest dose tested

Key result

Reproductive effects observed:

Table 1: Significant Organ Weight Effects

P1 Male rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	525.2	518.6	523.2	520.8
Cecum full (g)	5.223	4.951	6.493*	11.534*
Cecum full (g/100)	1.001	0.961	1.251*	2.230*
Cecum empty (g)	2.206	2.172	2.418	3.022#
Cecum empty (g/100)	0.420	0.420	0.462#	0.580#
P1 Female rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	278.0	275.0	277.1	268.6
Cecum full (g)	3.913	3.701	4.723*	6.653*
Cecum full (g/100)	1.405	1.345	1.694*	2.469*
Cecum empty (g)	1.874	1.876	2.036	2.412*
Cecum empty (g/100)	0.675	0.682	0.739*	0.898*
P2 Male rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	580.8	584.5	587.9	564.7
Cecum full (g)	4.614	5.211	6.065*	8.700*
Cecum full (g/100)	0.795	0.899	1.036*	1.546*
Cecum empty (g)	1.994	2.283	2.561*	2.878*
Cecum empty (g/100)	0.343	0.390*	0.439*	0.513*
P2 Female rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	296.3	308.2	307.5	293.3
Cecum full (g)	3.493	3.753	4.093	5.050#
Cecum full (g/100)	1.183	1.234	1.344	1.757#
Cecum empty (g)	1.590	1.580	1.744	2.059*
Cecum empty (g/100)	0.546	0.517	0.572	0.720*

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

#Statistically different from control mean by Dunnett's test, alpha = 0.05

Bold indicates effects considered treatment-related

Table 2: Incidence of Gross Pathologic Findings

P1 Male rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	0/30	21/30
P1 Female rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	2/30	20/30
P2 Male rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	2/30	17/30
P2 Female rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	4/30	10/30

Table 3: Mean Pup Weights

F1 litters	Dose level (mg/kg/day)
Mean pup weights (g)	0	50	250	1000
14 days after culling - Female	30.6	31.4	32.8#	32.3
14 days after culling - Male	31.1	32.1	33.6#	33.3#
21 days after culling - Female	49.6	50.1	49.4	50.9
21 days after culling - Male	50.8	51.2	50.7	53.0
F2 litters	Dose level (mg/kg/day)
Mean pup weights (g)	0	50	250	1000
7 days after culling - Female	15.3	16.1	16.0	16.5#
7 days after culling - Male	16.0	16.9	16.6	17.4
14 days after culling - Female	32.2	32.0	33.0	33.2
14 days after culling - Male	33.6	33.4	33.9	34.7

#Statistically different from control mean by Dunnett's test, alpha = 0.05

Table 4: F1 Weanling Uterine Weights

F1 weanlings	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Uterus (g)	0.047	0.046	0.039*	0.043
Uterus (g/100 g)	0.091	0.091	0.079	0.084

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

Conclusions:

There were no adverse effects of the test material on any parameter of reproductive function, pup survival, growth or development. Based upon the absence of any significant adverse effects, the No-Observed-Adverse-Effect-Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day, while 1000 mg/kg/day (the highest dose level tested) was considered a No-Observed-Effect-Level (NOEL) for reproductive toxicity.

Executive summary:

The toxicity of the test material to reproduction was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.3800, OECD 416, EEC 0378 -6978 and JMAFF 2 -1 -17.

The purpose of the two-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.

During the study groups of 30 male and 30 female rats were fed diets supplying 0, 50, 250, and 1000 mg test material/kg/day for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. In-life parameters included clinical observations, feed consumption, body weights, oestrous cyclicity, reproductive performance, puberty onset, pup survival, and pup body weights. 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.

Body weights of both adult P1 and P2 animals and weanling F1 and F2 pups were unaffected by treatment. Furthermore, there were no treatment-related effects on organ weights with the exception of the cecum. Statistically significant increases in absolute and relative full and empty cecal weights were identified in the 1000 mg/kg/day P1 and P2 adults of both sexes. Similar effects were seen at 250 mg/kg/day, although of lesser severity. At 50 mg/kg/day, the only significant parental effect was a statistically significant increase in relative empty cecal weights in P2 males. Increased cecal size also was noted at gross necropsy of the 250 and 1000 mg/kg/day adults. There were no treatment-related histopathologic changes in any tissue examined, including the cecum. Thus, cecal enlargement was interpreted to represent an adaptive process to a physiological effect localised to the cecum and was not considered toxicologically significant. There were no adverse effects of the test material on any parameter of reproductive function, pup survival, growth or development.

Based upon the absence of any significant adverse effects, the No-Observed-Adverse-Effect-Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day, while 1000 mg/kg/day (the highest dose level tested) was considered a No-Observed-Effect-Level (NOEL) for reproductive toxicity.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: chronic
Species:: rat
Quality of whole database:: A probe study was conducted to inform on dose selection for the 2-generation study. Both studies were conducted under GLP conditions. The probe study was conducted to sound scientific principles and was assigned a reliability score of 2 in line with the criteria of Klimisch et al. (1997). The 2-generation reproductive toxicity study was conducted in accordance with standardised guidelines and was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The overall quality of the database is 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

In a probe study conducted in the rat, the toxicity of the test material, following repeated dietary exposure to groups of rats, was assessed to inform on dose selection for a prospective two-generation reproductive toxicity study. The study was conducted under GLP conditions.

During the study groups of 10 male and 10 female CD (Sprague-Dawley strain) rats were fed diets providing 0, 100, 500, or 1000 mg test material/kg body weight/day for 13 weeks. In-life observations included daily cage-side and weekly clinical examinations, body weights, and feed consumption. After 13 weeks, a complete necropsy was conducted. The cecum, liver, and kidney weights were recorded, and these organs, along with the ileum, were examined histopathologically.

Males and females administered 500 or 1000 mg test material/kg/day had treatment-related effects in the cecum and ileum. The absolute and relative weights of ceca (full and empty) were significantly increased in males and females given 500 or 1000 mg/kg/day. Treatment-related gross pathologic findings in males and females given 500 and 1000 mg/kg/day were limited to increased size of the cecum. Histopathologic alterations in animals given 1000 mg/kg/day were confined to the cecum (males and females) and ileum (males only) which was characterised by very slight diffuse hyperplasia of the crypt epithelium of the respective mucosa. The cecal hyperplasia correlated with statistically identified increased mean cecum weights (full and empty) of males and females given 1000 mg/kg/day. There was no histopathologic correlate to the statistically identified increased cecum weights of males and females given 500 mg/kg/day. No treatment-related effects were noted in either sex administered 100 mg/kg/day.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for male and female rats was 100 mg/kg/day.

Following on from the probe study, the toxicity of the test material to reproduction was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.3800, OECD 416, EEC 0378 -6978 and JMAFF 2 -1 -17. The purpose of the two-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.

Short description of key information:
ORAL
NOAEL = 1000 mg/kg bw/day (parental and reproductive toxicity), rat (male/female), EPA OPPTS 870.3800, OECD 416, EEC 0378-6978, JMAFF 2-1-17, Marty et al. (2003)

Justification for selection of Effect on fertility via oral route:
Only one guideline study is available to assess the reproductive toxicity of the substance.

Effects on developmental toxicity

Description of key information

ORAL
NOEL (maternal toxicity, embryonal/foetal toxicity, teratogenicity) = 1000 mg/kg bw/day, rat, OECD 414, EPA OPPTS 870.3700, EEC 0378-6978, JMAFF 4200, Carney & Tornesi (2001)
NOEL (maternal toxicity) = 250 mg/kg bw/day, NOEL (developmental) = 500 mg/kg bw/day, rabbit, OECD 414, EPA OPPTS 870.3700, EEC 0378-6978 and JMAFF 2-1-18, Marty et al. (2002)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2001 to 13 March 2002

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:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

other: European Economic Community. Methods for the Determination of Toxicity. Official Journal of the European Communities, Vol. 31, No. L 133, May 30, 1988. ISSN 0378-6978, 1988.

Deviations:

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Agriculture, Forestry and Fisheries, Notification of 12-Nohsan-8147, Guideline 2-1-18, Teratogenicity Study, November 24, 2000.

Deviations:

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

Purity: 94.5%

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Sexually mature adults, 5 - 6 months of age
- Weight at study initiation: 2.5 - 3.5 kg
- Housing: Animals were housed individually 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: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: ca. 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 40 - 60 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % METHOCEL® A4M

Details on exposure:

DOSE PREPARATION
The test material was administered as a suspension in an aqueous vehicle of 0.5 % METHOCEL® A4M such that a dose volume of 4 mL/kg body weight yielded the targeted dose. 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.

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 analysed prior to the start of dosing to verify homogeneous distribution of the test material in vehicle.
Phase I: Analysis of aliquots from the low- and high-dose suspensions indicated that the test material was homogeneously distributed.
Phase II: These suspensions were also found to be homogeneously distributed.

- Concentration Verification
Analysis of all dosing suspensions from the first mix for both Phase I and Phase II were initiated prior to the start of dosing using HPLC with ultraviolet detection and external standards to determine concentrations.
Phase I: Analyses of all dosing suspensions from the first mix revealed mean concentrations of test material ranging from 103 to 106 % of the targeted concentrations.
Phase II: The mean concentrations for both dosing suspensions (500 mg/kg/day and 750 mg/kg/day) from the first mix were found to be 102 % of the targeted concentrations.

- Stability
The test material was previously found to be stable in 0.5% METHOCEL® A4M at concentrations ranging from 62.5 mg/mL to 250 mg/mL for at least 32 days. Since the low concentration in the present study was 6.25 mg/mL, stability of the test material in the vehicle for the low-and high-dose levels was confirmed for the period of use (i.e., 14 days). Stability analyses were conducted concurrent with the conduct of the study.
Phase I: Reanalysis of the high- and low-dose suspensions after 14 days of use revealed mean dose concentrations for the low- and high-dose suspensions were 90.1 and 106.4 % of the initial values, respectively, indicating that the solutions were stable during this time. Thereafter, dosing suspensions were mixed periodically throughout the study based upon these stability data.
Phase II: Subsequent dose suspensions were mixed periodically based upon stability data generated during Phase I of this study.

Details on mating procedure:

Sexually mature, adult virgin females, weighing approximately 2500-3500 g were naturally mated with males of the same strain at the test animal supply laboratory. The observed day of breeding was considered day 0 of gestation. Day 0 body weights and records of mating pairs were provided by the animal supplier and maintained in the study record. Rabbits were shipped on day 0 or 1 of gestation and arrived in the test laboratory on the same day.

Duration of treatment / exposure:

Animals were administered on days 7-27 of gestation.

Frequency of treatment:

Daily

Duration of test:

Animals were necropsied on gestation day 28.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Phase I

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Phase I

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Phase I

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Phase I

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Phase II

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Phase II

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Phase II

No. of animals per sex per dose:

26 females per dose.

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels for Phase I were selected based on the preliminary results of the probe study. The high-dose (250 mg/kg/day) was expected to induce overt signs of maternal toxicity (i.e., decreased maternal weight gain). The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high-dose group rabbits. However, rabbits at 250 mg/kg/day in the full study did not exhibit the significant body weight gain reductions that were observed at 250 mg/kg/day in the probe study. Consequently, a second dosing phase was added to this study. Dose levels for Phase II were chosen based upon body weight gain decreases and decreased food consumption observed at the next highest doses (500 and 750 mg/kg/day) in the probe study.
- Rationale for animal assignment: Randomisation of time-mated rabbits into dose groups was performed using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice each day cage-side examinations were conducted and to the extent possible, the following 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. Morning cage-side examinations were conducted approximately one hour after the completion of dosing on gestation days 7-27.
Moribund animals that were not expected to survive until the next observation period, and any animals found dead, were necropsied on that day. However, animals found dead after routine working hours or on weekends, and holidays were refrigerated until the next scheduled workday, at which time they were necropsied. For animals showing indications of premature delivery, the delivered foetuses were counted and examined to the extent possible. Dosing was discontinued, and animals were euthanised and necropsied on that day or, if found after regular working hours, the next scheduled workday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were conducted daily throughout the study period. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), unusual swelling or masses and animal behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on day 0 by the supplier, day 4, and daily during the dosing period, and at necropsy (day 28 of gestation). Statistical analyses of body weights and body weight gains were performed using data collected on gestation days 0, 4, 7, 10, 13, 16, 20, 24, and 28.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed consumption was recorded for all animals, daily beginning on day 4 by weighing feed containers 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 container - final weight of feed container) / number of days in measurement cycle

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- All surviving females were euthanised by an I.V. injection of a euthanasia solution (Beuthanasia-D Special) and a limited gross pathologic examination (necropsy) was performed. Given the rapid growth and ossification of the late gestation foetuses, the timing of the maternal sacrifice was counterbalanced across groups (e.g., control, high, middle and low).
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 weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to 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.

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, 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 determined 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. Near term foetuses were euthanised.

Ovaries and uterine content:

The uterine horns were exteriorised through an abdominal incision and the following data were recorded:
1) The number and position of live foetuses in utero.
2) The number and position of dead foetuses.
3) The number, position and classification (early, late) of resorption sites - resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognisable embryonic/foetal form.
4) The number of ovarian corpora lutea.
5) The sex and body weight of each foetus.
6) Any external gross alterations in the foetus.
Corpora lutea were not counted for non-pregnant females or for females with totally resorbed litters. The uteri of females without grossly visible evidence of pregnancy were stained with a 10 % aqueous solution of sodium sulphide (Kopf et al., 1964) and examined for evidence of early resorptions to determine pregnancy status.

Fetal examinations:

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, gallbladder, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. At least one-half of the foetuses in each litter, chosen randomly via computer, 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 and/or which occurs at a relatively low incidence in the specific species/strain.

Statistics:

Maternal body weights, maternal body weight gains, organ weights (absolute and relative), foetal body weights and feed consumption were evaluated by Bartlett’s test (alpha=0,01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha=0.05, analysis by Dunnett's test (alpha=0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha=0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966) was performed, respectively.
Frequency of pre-implantation loss, post-implantation loss, resorptions per litter and resorptions per foetal population and foetal alterations were analysed using a censored Wilcoxon test with Bonferroni’s correction (Haseman and Hoel, 1974). The number of corpora lutea and implantations and litter size was evaluated using a nonparametric ANOVA (alpha=0.05) followed by the Wilcoxon Rank-Sum test (alpha=0.05) with Bonferroni's correction. Pregnancy rates were analysed using the Fisher exact probability test (alpha=0.05; Siegel, 1956) 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 (alpha=0.02; Grubbs, 1969), and excluded if justified.
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 alpha levels. Therefore, the final interpretation of the data took into consideration 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.

Indices:

- Pre-implantation loss = [(No. corpora lutea - implantations) / No. corpora lutea] x 100
- Post-implantation loss = [(No. implantations - live born pups) / No. implantations] x 100
Percent pre- and post-implantation loss was determined for each litter, followed by calculation of the mean and standard deviation of these litter values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on maternal effects below

Mortality:

mortality observed, treatment-related

Description (incidence):

see details on maternal effects below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Phase I: There were no statistically identified differences in the body weights or body weight gains for any treated groups when compared to their respective controls.
Phase II: The body weight of rabbits administered 750 mg/kg/day was significantly decreased by 6 % relative to controls on gestation day 10. Mean body weights of the 500 mg/kg/day rabbits were comparable to controls throughout the study. Body weight gains of rabbits administered 500 or 750 mg/kg/day were significantly decreased relative to controls on gestation days 7-10. In the controls (0 mg/kg/day), 4 out of 24 rabbits lost weight with a mean weight gain of 25 g reported for the group during gestation days 7-10. At 500 and 750 mg/kg/day, weight losses were seen during this interval in 12 out of 25 and 18 out of 25 rabbits, respectively. Mean weight losses of 11.8 g for the 500 mg/kg/day group and 70.0 g for the 750 mg/kg/day group were reported. During subsequent periods (gestation days 10-28), body weight gain at 500 mg/kg/day were comparable to or higher than controls. At 750 mg/kg/day weight gain remained slightly decreased on gestation days 13-16. The decreases in weight gain observed in the 750 mg/kg/day rabbits were accompanied by significant decreases in feed consumption during the same period. Due to the magnitude of the body weight gain decrease seen on gestation days 7-10 and the severity of clinical signs observed in several 750 mg/kg/day rabbits, this entire dose group was removed from study early without further data collection.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Phase I: There were no significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.
Phase II: Feed consumption of rabbits administered 750 mg/kg/day was decreased for several days (gestation days 7-17) following the initiation of dosing. Statistically identified decreases were noted on 8 of 10 days and the magnitude of the decreased feed consumption ranged from 20 to 45 % of the control values. The 750 mg/kg/day group was terminated early as previously mentioned due to the severity of maternal toxicity observed. Rabbits administered 500 mg/kg/day had an 18 % decrease in feed consumption relative to controls on GD 7-8 that was statistically identified.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Phase I: There were no statistically identified differences in any of the measured parameters for any treated groups when compared to their respective controls.
Phase II: There were no statistically identified differences in any of the measured parameters for the 500 mg/kg/day group compared to the controls. Organ weights were not evaluated for the 750 mg/kg/day group which was terminated early due to excessive toxicity.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Phase I: 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.
Phase II: Treatment-related gross pathologic observations were noted in three rabbits given 750 mg/kg/day which were terminated early due to excessive toxicity. These observations included, pale kidneys (cortex) and an increased incidence of alterations in the glandular mucosa of the stomach (erosion-ulcer, hyperaemia or mottled appearance). A single rabbit given 500 mg/kg/day had ulcers/erosions in the glandular mucosa of the stomach which may have been treatment-related. All other gross pathologic observations in rabbits given 500 mg/kg/day were considered to be spontaneous alterations, unassociated with exposure to the test material.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean percent pre-implantation loss was significantly increased at 25 mg/kg/day; however, this result was considered spurious based on the lack of a dose-response and because treatment is initiated when implantation is complete.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
> Phase I: Examinations performed on all animals prior to the study and at termination revealed no treatment-related findings. Health evaluations performed on all animals upon arrival revealed various lacerations and scratches which were likely inflicted during breeding at the supplier’s facility.
During the dosing period, soft faeces and absent or decreased amounts of faeces were noted in various rabbits in the treated and untreated (control) groups. In the absence of any significant decreases in body weight or body weight gain, these incidences were considered within the normal range for rabbits. One rabbit (2085) was removed from study on gestation day 3 (prior to initiation of treatment). This rabbit had been assigned to the 250 mg/kg/day group, and was apparently not eating after arrival and had decreased activity. In addition, this doe had soft/watery faeces with an abnormal odour which contained a mucoid matter. A single 250 mg/kg/day rabbit (2088) had blood in her cage and aborted one resorbing foetus on gestation day 19 with no prior clinical observations. This rabbit was subsequently euthanized due to the abortion and examined grossly at necropsy. Gross findings for this rabbit included pale kidneys (bilateral) with evidence of recent abortion in the uterus. The cause of this abortion was not determined. Another 250 mg/kg/day rabbit (2095) was found dead on gestation day 15. This rabbit had exhibited a reflux of the test material at the time of dosing (gestation day 15) and subsequently had noisy and laboured respiration along with bluish skin and mucous membranes. At necropsy, this rabbit was found to be pregnant with 14 normally developed foetuses and one early resorption within the uterus. This rabbit also had mottled lungs with oedema, froth in the trachea and bronchi, and external soiling (blood tinged froth) on the nares and muzzle. All of these finding supported a cause of death due to a gavage complication. Finally, one 25 mg/kg/day rabbit (2037) had blood in her cage and aborted four foetuses on gestation day 26. In the days preceding (gestation day 14-26), this doe was noted as having decreased and mucoid faeces in her cage pan. Upon gross examination following euthanasia, this rabbit was found to have seven normally developed foetuses and one late resorption still within the uterus. Other observations for this rabbit included perineal soiling and dark ingesta/watery contents in the cecum. The cause of the abortion was not determined. All other Phase I rabbits survived to the scheduled necropsy.
> Phase II: As in Phase I, health evaluations performed on all animals upon arrival revealed several lacerations, scratches and bruises which were likely inflicted during breeding at the supplier’s facility. Two 750 mg/kg/day rabbits were sacrificed early on gestation day 17 (moribund) with prior clinical signs that included inco-ordinated gait, perineal urine soiling, decreased faeces, and decreased activity. Additionally, these two does showed significant decreases in body weight gains and feed consumption (gestation days 7-16). At gross necropsy examination, both rabbits were pregnant and observed to have pale kidneys (cortex), watery, dark cecal contents, erosions/ulcers in the glandular mucosa of the stomach and hairballs. The moribund condition of these rabbits was considered due to treatment. Another 750 mg/kg/day rabbit died on gestation day 8. Just prior to death, this rabbit was observed to have perineal urine and faecal soiling, laboured respiration, and an absence of faeces in its cage pan. Gross findings at necropsy included perineal soiling, congestion in the kidneys and liver, watery contents in the cecum, lung consolidation (anterior ventral portions), abnormal lung distension (dorsal portions), exudate in the bronchus, mucoid exudate and froth in the trachea, and petechia in the stomach. The cause of death was attributed to a gavage complication. One doe in the 500 mg/kg/day dose group died on gestation day 27. Observations at gross necropsy included a frothy red discharge around the external nares and muzzle, froth in the trachea and bronchi (bilateral), and dark consolidation in both lungs with accumulation of serosanguinous fluid and abnormal lung distension (bilateral and diffuse). Death was attributed to the diffuse, oedematous state of the lungs, which suggests a gavage complication. All other rabbits survived their respective test periods. Treatment-related clinical signs were noted in rabbits administered 750 mg/kg/day and included increased incidences of decreased amounts of faeces (associated with significant reductions in body weight and body weight gain), and inco-ordinated movement shortly following dosing.
The incoordination observed at 750 mg/kg/day was characterised by a reluctance to move unless manually stimulated by the observer. Significant stiffening or dragging of the limbs was evident when movement was attempted and on various occasions several rabbits tipped onto their sides. In most cases, the inco-ordination was transient, with complete resolution of the inco-ordination within two hours post-dosing. Also, the degree of inco-ordination observed did not appear to progressively worsen on subsequent days. A similar incidence of inco-ordination was also observed among the rabbits in the 500 mg/kg/day dose group. However, the degree of inco-ordination observed was much less pronounced than that observed in the 750 mg/kg/day rabbits. In general, when stimulated to move, the 500 mg/kg/day rabbits exhibited cautious placement of their paws with a slight stiffening or dragging of the forelimbs. Unlike the 750 mg/kg/day rabbits, none of the 500 mg/kg/day rabbits tipped over when moving. The inco-ordination observed at 500 mg/kg/day resolved within two hours of dosing. Due to the severity of clinical signs and corresponding significant decreases in body weight gain on gestation days 7-10 observed in the majority of the 750 mg/kg/day rabbits, this entire dose group was removed from study on October 8, 2001.

BODY WEIGHTS
> Phase I: There were no statistically identified differences in the body weights or body weight gains for any treated groups when compared to their respective controls.
> Phase II: The body weight of rabbits administered 750 mg/kg/day was significantly decreased by 6 % relative to controls on gestation day 10. Mean body weights of the 500 mg/kg/day rabbits were comparable to controls throughout the study. Body weight gains of rabbits administered 500 or 750 mg/kg/day were significantly decreased relative to controls on gestation days 7-10. In the controls (0 mg/kg/day), 4 out of 24 rabbits lost weight with a mean weight gain of 25 g reported for the group during gestation days 7-10. At 500 and 750 mg/kg/day, weight losses were seen during this interval in 12 out of 25 and 18 out of 25 rabbits, respectively. Mean weight losses of 11.8 g for the 500 mg/kg/day group and 70.0 g for the 750 mg/kg/day group were reported. During subsequent periods (gestation days 10-28), body weight gain at 500 mg/kg/day were comparable to or higher than controls. At 750 mg/kg/day weight gain remained slightly decreased on gestation days 13-16. The decreases in weight gain observed in the 750 mg/kg/day rabbits were accompanied by significant decreases in feed consumption during the same period. Due to the magnitude of the body weight gain decrease seen on gestation days 7-10 and the severity of clinical signs observed in several 750 mg/kg/day rabbits, this entire dose group was removed from study early without further data collection.

FEED CONSUMPTION
> Phase I: There were no significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.
> Phase II: Feed consumption of rabbits administered 750 mg/kg/day was decreased for several days (gestation days 7-17) following the initiation of dosing. Statistically identified decreases were noted on 8 of 10 days and the magnitude of the decreased feed consumption ranged from 20 to 45 % of the control values. The 750 mg/kg/day group was terminated early as previously mentioned due to the severity of maternal toxicity observed. Rabbits administered 500 mg/kg/day had an 18 % decrease in feed consumption relative to controls on GD 7-8 that was statistically identified.

ANATOMIC PATHOLOGY
Organ Weights
> Phase I: There were no statistically identified differences in any of the measured parameters for any treated groups when compared to their respective controls.
> Phase II: There were no statistically identified differences in any of the measured parameters for the 500 mg/kg/day group compared to the controls. Organ weights were not evaluated for the 750 mg/kg/day group which was terminated early due to excessive toxicity.

Gross Pathology
> Phase I: 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.
> Phase II: Treatment-related gross pathologic observations were noted in three rabbits given 750 mg/kg/day which were terminated early due to excessive toxicity. These observations included, pale kidneys (cortex) and an increased incidence of alterations in the glandular mucosa of the stomach (erosion-ulcer, hyperaemia or mottled appearance). A single rabbit given 500 mg/kg/day had ulcers/erosions in the glandular mucosa of the stomach which may have been treatment-related. All other gross pathologic observations in rabbits given 500 mg/kg/day were considered to be spontaneous alterations, unassociated with exposure to the test material.

REPRODUCTIVE PARAMETERS
> Phase I: Mean percent pre-implantation loss was significantly increased at 25 mg/kg/day; however, this result was considered spurious based on the lack of a dose-response and because treatment is initiated when implantation is complete. Thus, there were no significant treatment related effects on pregnancy rates, numbers of corpora lutea, implantations, or viable foetuses per litter, percent pre-implantation loss, percent post-implantation loss, resorption rates, foetal body weights, foetal sex ratios or gravid uterine weights at any dose level.
> Phase II: There were no significant treatment related effects on pregnancy rates, numbers of corpora lutea, implantations, or viable foetuses per litter, percent pre-implantation loss, percent post-implantation loss, resorption rates, foetal body weights, foetal sex ratios or gravid uterine weights at 500 mg/kg/day.

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

other: maternal toxicity

Key result

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse test substance related effects at the highest dose level with fetuses

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details on fetal effects below

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details on fetal effects below

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FOETAL ALTERATIONS
> Phase I: There were no treatment-related differences in the incidence of any foetal malformations in any of the dose groups. There were two malformed control foetuses from two separate litters. One control foetus had a bifurcated renal artery, while the other exhibited skeletal malformations in the thoracic region which included fused vertebrae and centra coupled with a hemivertebra. A total of four 25 mg/kg/day foetuses from three litters had malformations. Three foetuses presented with bifurcated renal arteries. A fourth foetus had a missing gallbladder. Among the eight 100 mg/kg/day foetuses (six affected litters) that were malformed, a single foetus had a bifurcated renal artery. A second foetus had a persistent truncus arteriosus. Two other foetuses had hemivertebra in the cervical region (atlas) with one also having a missing gallbladder. Another foetus had visceral malformations which included multiple missing lung lobes in association with a diaphragmatic hernia and a missing gallbladder. The sixth foetus had malformed ribs (fused and forked) with a hemivertebra. Another foetus had multiple skeletal malformations which included misshapen short and long bones of the fore- and hind-limbs along with class II wavy ribs. The final 100 mg/kg/day foetus had fused ribs and centra along with a hemivertebra in the thoracic region. Finally, three foetuses from three separate 250 mg/kg/day litters were malformed. One foetus had a bifurcated renal artery. Another had a displaced aortic arch (right-sided) and missing innominate artery. The third foetus had persistent truncus arteriosus. With regard to foetal variations, the only statistically identified difference was a decrease in the incidence of fused sternebrae at 100 mg/kg/day when compared to controls. There were no fused sternebrae at either 25 or 250 mg/kg/day. These decreases in fused sternebrae were considered spurious as a decrease rather than an increase in incidence was identified. Other foetal variations observed during Phase I of this study occurred at low frequencies and/or did not follow a dose-response relationship in litter incidence. Therefore, there were no differences in the incidence of foetal alterations related to treatment with the test material.
> Phase II: There were no treatment-related differences in the incidences of any foetal malformations or variations at 500 mg/kg/day when compared to controls. In total, there were ten malformed foetuses from 5 litters in the control group. Of these, six foetuses had bifurcated renal arteries. One foetus had a cardiac malformation consisting of transposition of the great vessels, a misshapen aortic semilunar valve and right ventricle, and a ventricular septal defect. Other control foetuses had dilated cerebral ventricles, missing cervical vertebrae, and fused ribs. At 500 mg/kg/day, three foetuses from a single litter were malformed. One foetus had a missing testis, one had fused thoracic centra, and another had fused thoracic centra associated with a hemivertebra.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse test substance related effects at the highest dose level

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

Table 1: Phase II Clinical Observations

Clinical observations*	Dose level (mg/kg/day)
Clinical observations*	0	500	750
Faeces, abnormal quantity, decreased	1	3	6
Gait, incoordinated	0	23	23

*n = 26 animals per treatment group

Table 2: Phase II Select Mean Body Weights and Body Weight Gains

Endpoint	Dose level (mg/kg/day)
Endpoint	0	500	750
Body weight on GD 10 (g)	3265.0	3246.7	3068.7*
Body weight gain on GD 7-10 (g)	25.0	-11.8#	-70.0#
Body weight gain on GD 13-16 (g)	97.3	91.8	33.2
Body weight gain on GD 0-28 (g)	359.7	374.5	no data

*Statistically difference from control mean by Dunnett's test, alpha = 0.05

#Statistically different from control mean by Wilcoxon's test, alpha = 0.05

Conclusions:

Under the conditions of the study the no-observed-effect-levels (NOEL) for maternal and developmental toxicity were 250 mg/kg/day and 500 mg/kg/day, respectively.

Executive summary:

The developmental toxicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414, EPA OPPTS 870.3700, EEC 0378-6978 and JMAFF 2-1-18.

During the study groups of 26 time-mated female New Zealand White rabbits were administered test material, via gavage, at targeted dose levels of 0, 25, 100, or 250 mg/kg/day (Phase I) or 0, 500 or 750 mg/kg/day (Phase II) on gestation days 7 through 27. 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 numbers of corpora lutea, uterine implantations, resorptions and live/dead foetuses were determined. All foetuses were weighed, sexed and examined for external, visceral and skeletal alterations. Also, the internal structures of the head were examined by serial sectioning for approximately one-half of the foetuses in each litter.

Phase I rabbits given ≤ 250 mg/kg/day test material did not exhibit any treatment-related maternal or developmental toxicity, thus prompting the evaluation of higher doses in Phase II. At 500 mg/kg/day, maternal toxicity was evidenced by transient uncoordinated gait (23/26 does) and decreased body weight gains (gestation days 7-10), but there was no evidence of embryonal/foetal effects. At 750 mg/kg/day, uncoordinated gait was accompanied by decreased amounts of faeces, significant reductions in body weight, body weight losses, decreased body weight gains, and decreased feed consumption. Also, two 750 mg/kg/day does were euthanised in moribund condition (gestation day 17). At necropsy, these does had pale kidneys, watery, dark cecal contents, erosions/ulcers of the stomach (glandular mucosa) and hairballs. As 750 mg/kg/day exceeded the maximum tolerated dose, all surviving rabbits in the dose group were removed from study.

Based upon these results, the no-observed- effect-levels (NOEL) for maternal and developmental toxicity were 250 mg/kg/day and 500 mg/kg/day, respectively.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 May 2001 to 20 November 2001

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:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

other: European Economic Community. Methods for the Determination of Toxicity. Official Journal of the European Communities, Vol. 31, No. L 133, May 30, 1988. ISSN 0378-6978, 1988. Directive 87/302/EEC: Teratogenicity Test – Rodent and Non-Rodent Species.

Deviations:

Qualifier:

according to guideline

Guideline:

other: Ministry of Agriculture, Forestry and Fisheries, Requirements for Safety Evaluation of Agricultural Chemicals, 59 NohSan, Notification No. 4200, Agricultural Production Bureau, Japan, 1985.

Deviations:

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

Purity: 94.5%

Species:

rat

Strain:

other: Crl:CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: CD (Crl: CD (SD) IGS BR)
- Age at study initiation: Sexually mature adults, 10 - 11 weeks of age
- Weight at study initiation: 200 - 250 g
- Housing: Animals were housed individually 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, ad libitum
- Acclimation period: approximately four days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 49 - 51 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % METHOCEL® A4M

Details on exposure:

DOSE PREPARATION
Test material suspensions were prepared in an aqueous vehicle of 0.5 % METHOCEL® A4M at concentrations of 25, 75 and 250 mg/mL with a dose volume of 4 mL/kg body weight in order to yield the targeted dose levels. Dose volumes were adjusted daily based on individual body weights.

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 analysed prior to the start of dosing to verify homogeneous distribution of the test material in vehicle.

- Stability
The stability of test material in 0.5 % METHOCEL® A4M was established to be at least 17 days in a concurrent probe study in rabbits. The samples analysed included a low concentration of 6.25 mg/mL and a high concentration of 250 mg/mL. The stability of the low- and high-dose suspensions was determined concurrent with the study using HPLC with ultraviolet detection and external standards.

- Concentration Verification
Analysis of all dosing suspensions from the first mix was initiated prior to the start of dosing using HPLC with ultraviolet detection and external standards to determine concentrations.
Analysis of all dosing solutions from the first mix revealed mean concentrations of test material ranging from 97 to 98 % of targeted concentrations. Analysis of aliquots for the low- and high-dose suspensions indicated that the test material was homogeneously distributed.

Details on mating procedure:

Sexually mature, adult virgin females, were naturally mated with males of the same strain (1:1) at the test animal supplier laboratory. Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males’ cages. The day on which a vaginal plug was detected was considered day 0 of gestation. Day 0 body weights were provided by the animal supplier laboratory. Rats arrived at the test laboratory on gestation day 1 or 2.

Duration of treatment / exposure:

Animals were administered on days 6-20 of gestation.

Frequency of treatment:

Daily

Duration of test:

Animals were necropsied on gestation day 21.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females per group.

Control animals:

yes

Details on study design:

- Dose selection rationale: These dose levels were selected based on the preliminary results of the existing probe study. The high-dose of 1000 mg/kg/day represented a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3700 Prenatal Developmental Toxicity Study). The lower dose levels were selected to provide dose response data for any potential toxicity among the high-dose group rats.
- Rationale for animal assignment: Animals were stratified based upon gestation day 0 body weights and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of dosing.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once each day, a cage-side examination was conducted and to the extent possible, the following was evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.
Moribund animals that were not expected to survive until the next observation period, and any animals found dead, were necropsied on that day. However, animals found dead after routine working hours or on weekends, or on holidays were refrigerated until the next scheduled workday, at which time they were necropsied. For animals showing indications of premature delivery, the delivered foetuses were counted and examined to the extent possible. Dosing was discontinued, and animals were euthanised and necropsied on that day or, if found after regular working hours, the next scheduled workday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were conducted daily throughout the study period. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), unusual swelling or masses and animal behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on day 0 (by the supplier), day 3 and daily during the dosing period, and at necropsy (day 21 of gestation). Statistical analyses of body weights were performed using data collected on gestational days 0, 3, 6, 9, 12, 15, 18, 21 and body weight gains were performed using data collected for the following intervals 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed consumption was recorded for all animals for gestation days 3-6, 6-9, 9-12, 12-15, 15-18, and 18-21 by weighing feed containers at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container) / number of days in measurement cycle

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- All surviving females (not fasted) were euthanised by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) was performed. Given the rapid growth and ossification of the late gestation foetuses, the timing of the maternal sacrifice was counterbalanced across groups (e.g., control, high, middle, low).
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 weight 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 tissues was not conducted.

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, 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 determined to the extent possible by external examination (as appropriate for gestational age), but they were not examined for visceral or skeletal alterations. Following external examination, these conceptuses were discarded. Near term foetuses were euthanised.

Ovaries and uterine content:

Fetal examinations:

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 via computer 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, pancreas, 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).
The remaining foetuses not selected for visceral examination were skinned, eviscerated, preserved in alcohol, and double stained with Alcian Blue and Alizarin Red S for cartilage and bone according to methods derived from McLeod (1980), Kimmel and Trammell (1981) and Webb and Byrd (1994).
After staining, skeletons were macerated and cleared. A thorough evaluation of the foetal skeleton was conducted on the remaining foetuses not selected for visceral examination. All foetal alterations were classified as a variation or malformation. A variation was defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation was defined as a permanent structural change that may adversely affect survival, development, or function and/or which occurs at a relatively low incidence in the specific species/strain.

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 (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with Bonferroni's correction was performed, respectively.
Frequency of pre- and post-implantation loss 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 non-parametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analysed using the Fisher’s exact probability test (alpha = 0.05; Siegel, 1956) 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 (alpha = 0.02; Grubbs, 1969), and excluded if justified. Both Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both were reported at the experiment-wise alpha 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 alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationship.

Indices:

- Pre-implantation loss = [(No. corpora lutea - implantations) / No. corpora lutea] x 100
- Post-implantation loss = [(No. implantations - Live born pups) / No. implantations] x 100
Percent pre- and post-implantation loss was determined for each litter, followed by calculation of the mean and standard deviation of these litter values.

Clinical signs:

no effects observed

Description (incidence and severity):

Examinations performed on all animals prior to the study, during dosing, and at termination revealed no treatment-related findings.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the middle dose group, one animal was found dead on gestation day 10. There were no gross lesions found in the animal, and therefore, the cause of death was not determined. This dam was pregnant and all foetuses appeared normal.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically identified differences in the body weights or body weight gains of any treated groups when compared to their respective control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in the amount of feed consumed by any treated groups with the exception of a slight (8 %), but statistically significant, decrease in feed consumption noted on gestation days 6–9 for the 1000 mg/kg/day group. This decrease did not correspond to any body weight effect and therefore was considered not toxicologically significant.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically identified differences in any of the measured parameters for any treated groups when compared to their respective control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations at any dose level. All observations were considered to be spontaneous alterations, unassociated with exposure to the test material.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

REPRODUCTIVE PARAMETERS
There were no significant treatment related effects on pregnancy rates, numbers of corpora lutea, implantations, viable foetuses per litter, percent pre-implantation loss, percent post-implantation loss, resorption rates, foetal sex ratios, foetal body weights or gravid uterine weights resorptions or litter size at any dose level.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse treatment related effects at the highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse treatment related effects at the highest dose tested

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in the incidence of any foetal alteration in any of the treated groups compared to controls. The small number of alterations observed in foetuses from dams administered test material either occurred at low frequencies and/or were not dose related.
There were 6, 1, 0 and 2 malformed foetuses in the 0, 100, 300 and 1000 mg/kg/day groups, respectively. Malformations observed among the control group included three foetuses from the same litter, as follows: one foetus with fused cervical centra, extra thoracic rib and vertebrae, one foetus with missing caudal vertebrae, fused cervical centra and extra lumbar vertebrae, and one foetus with cervical and thoracic hemivertebrae, fused cervical centra, extra thoracic rib and vertebrae, and fused thoracic vertebrae. A control group litter had a single malformed foetus with missing lumbar vertebrae. In the low-dose group, one foetus was observed with a missing thoracic vertebrae and rib. In the high-dose group, one foetus exhibited haemorrhage in the pericardial fluid and another was found with a cervical rib.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related adverse effects at the highest dose tested

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

Conclusions:

Oral gavage administration of the test material up to and including the limit dose of 1000 mg/kg/day resulted in no maternal toxicity and no indications of embryonal/foetal toxicity or teratogenicity. Therefore, the NOEL for maternal toxicity and for developmental toxicity was 1000 mg/kg/day, the highest dose level tested.

Executive summary:

During the study groups of 25 time-mated female rats were administered suspensions of test material, by gavage, at targeted dose levels of 0, 100, 300 or 1000 mg/kg/day on days 6 through 20 of gestation in order to evaluate the maternal and developmental toxicity potential of this compound. In-life maternal study parameters included clinical observations, body weight, body weight gain, and feed consumption. On day 21 of gestation, all 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. Approximately one half of the foetuses were examined for visceral and craniofacial alterations, while skeletal examinations were conducted on the remaining foetuses.

Under the conditions of the study oral gavage administration of the test material up to and including the limit dose of 1000 mg/kg/day resulted in no maternal toxicity and no indications of embryonal/foetal toxicity or teratogenicity. Therefore, the NOEL for maternal toxicity and for developmental toxicity was 1000 mg/kg/day, the highest dose level tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 500 mg/kg bw/day
Study duration:: subacute
Species:: rabbit
Quality of whole database:: Two probe studies were conducted to make a preliminary evaluation of the maternal toxicity and embryo/foetal lethality potential of the test material in rats and rabbits. Both probe studies were conducted under GLP conditions and followed sound scientific principles; both studies were therefore assigned a reliability score of 2 in line with the criteria of Klimisch et al. (1997). Two developmental toxicity studies were then conducted in one in rats and one in rabbits. Both studies were conducted under GLP conditions and in accordance with standardised guidelines; both studies were subsequently assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The overall quality of the data set is 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

Two probe studies are available, one in rats and one in rabbits. The purpose of the studies was to make a preliminary evaluation of the maternal toxicity and embryo/foetal lethality potential of the test material in rats and rabbits.

The study with rats was conducted under GLP conditions. During the study groups of eight time-mated female rats were administered test material, by gavage, at targeted dose levels of 0, 250, 500, 750, or 1000 mg/kg/day on days 6-20 of gestation. In-life parameters evaluated included: clinical observations, body weights, body weight gains, and feed consumption. On gestation day 21, all surviving rats were euthanised and examined for gross pathologic alterations. Liver and kidney weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead foetuses.

Under the conditions of the study, oral 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 adverse effects on embryo/foetal survival. Therefore, the no-observed-effect level (NOEL) for maternal toxicity and embryo/foetal effects was 1000 mg/kg/day, the highest dose level tested.

The study with rabbits was also conducted under GLP conditions. During the study groups of seven time-mated New Zealand White rabbits were administered test material via gavage at targeted dose levels of 0, 250, 500, 750, and 1000 mg/kg/day on gestation days 7-27. In-life parameters evaluated included: clinical observations, body weights, body weight gains, and feed consumption. On gestation day 28, all surviving rabbits were euthanised and examined for gross pathologic alterations. Liver and kidney weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead foetuses.

Oral gavage administration of 750 or 1000 mg test material/kg/day to time-mated New Zealand White rabbits resulted in changes in the consistency and/or amount of faeces, and dose-related decreases in body weight gains and feed consumption early in the dosing period. The magnitude of the change in body weight gains would preclude the use of the 750 and 1000 mg/kg/day dose levels in the subsequent developmental toxicity study. Therefore, these groups were terminated on gestation days 17 and 10, respectively. Treatment-related gross pathologic observations occurring in these rabbits included erosions/ulcers in the stomach and watery stomach contents, which were likely secondary to decreased feed consumption and stress. At 250 and 500 mg/kg/day, treatment-related decreases in body weight gains and feed consumption occurred throughout the dosing period. Relative to controls, the mean body weight gains of the 250 and 500 mg/kg/day groups over the dosing period (gestation days 7-28) were decreased 53 % and 33 %, respectively. At 500 mg/kg/day, statistically significant decreases in absolute (16 % decrease) and relative (14 % decrease) liver weights were identified, while at 250 mg/kg/day slight, non-statistically significant, decreases in absolute (12 % decrease) and relative (7 % decrease) liver weights were noted. Kidney weights were not affected at 250 or 500 mg/kg/day, nor were there any treatment-related gross pathologic observations or evidence of embryo/foetal lethality attributable to treatment in the 250 or 500 mg/kg/day groups.

Therefore, under the conditions of the study, the no-observed-effect-level (NOEL) for maternal effects was less than the lowest dose tested (250 mg/kg/day), while the NOEL for embryo/foetal lethality was 500 mg/kg/day.

Following the probe studies, two developmental toxicity studies were conducted in rats and rabbits.

In the study with rats, the developmental toxicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414, EPA OPPTS 870.3700, EEC 0378 -6978, and JMAFF 4200.

In the study with rabbits, the developmental toxicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414, EPA OPPTS 870.3700, EEC 0378-6978 and JMAFF 2-1-18.

Based upon these results, the no-observed- effect-levels (NOEL) for maternal and developmental toxicity were 250 mg/kg/day and 500 mg/kg/day, respectively.

Justification for selection of Effect on developmental toxicity: via oral route:
The rabbit study was selected as the rabbit can be considered to be the more sensitive species.

Toxicity to reproduction: other studies

Additional information

The test material was evaluated for preliminary teratogenicity screening in the ex vivo whole embryo culture test. Seven non-pregnant female rats ("serum donors") received 1000 mg/kg/day of test material via gavage for three consecutive days. Four hours after the last dose the rats were exsanguinated and their blood was centrifuged to obtain serum. Seven control rats were similarly gavaged with 0.5 % methylcellulose vehicle and bled. Rat conceptuses from a separate group of untreated, timed-pregnant rats were explanted on the afternoon of gestation day 9 and were cultured in the sera (100 %) from the control or test material serum donors. After approximately 42 hours in culture, embryos were evaluated for viability, growth and morphology.

All serum donors appeared normal throughout the test period. Body weight gains in the test material treated rats were not significantly different from those of the control rats. There were no apparent differences between embryos cultured in test material serum vs. control serum for any parameter of embryo viability, growth or morphology.

Based on these findings, the test material was considered negative for teratogenic potential in this screening test.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to either reproductive or developmental toxicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to either reproductive or developmental toxicity.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

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Diss Factsheets

2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Toxicity to reproduction: other studies

Additional information

Justification for classification or non-classification

Additional information

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