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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol
EC Number:
211-477-1
EC Name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol
Cas Number:
647-42-7
Molecular formula:
C8H5F13O
IUPAC Name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol
Details on test material:
- Purity: 99.7%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 52 days old, approximately
- Weight at study assignment: Male Rats – 149-176 grams/Female Rats – 140-163 grams
- Weight at study initiation: Male Rats – 183-222 grams/Female Rats – 147-191 grams
- Fasting period before study: None
- Housing: Stainless steel, wire-bottomed cages, individually, except during cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat’s cage. Beginning no later than gestation day 20, female rats were individually housed in nesting boxes. Each dam and delivered litter was housed in a common nesting box during the postpartum period.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: 8-day pretest period
- Quarantined – not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Targeted at 18-26°C
- Humidity (%): Targeted at 30-70%
- Air changes (per hr): Minimum of 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methylcellulose
Details on exposure:
Dose Administration
Dosages were adjusted based on the most recently recorded body weight and were administered at approximately the same time each day. Formulations were stirred continuously during dosage. The dosage volume was 5 mL/kg, adjusted for body weights recorded before administration.

Male and female rats were administered the test substance and/or the vehicle once daily beginning 70 days before cohabitation, through cohabitation (maximum 14 days), and continuing through the day before sacrifice. F1 generation pups were not directly given the test substance.

Dose Preparation
Suspensions of the test substance were prepared at least once daily at the Testing Facility and were stored at room temperature. The vehicle was prepared at least once weekly at the Testing Facility and was stored refrigerated (2°C to 8°C). Prepared formulations were stirred continuously during dosage administration.

VEHICLE
The vehicle was 0.5% (w/v) aqueous methylcellulose prepared in reverse osmosis processed deionised water. The dosage volume was 5 mL/kg, adjusted for body weights recorded before administration.
Details on mating procedure:
Breeding
Within each dosage group, consecutive order was used to assign P generation rats to cohabitation, one male rat per female rat. The Cohabitation period consisted of a maximum of 14 days. Estrous cycling was evaluated by examination of vaginal cytology beginning 14 days before scheduled cohabitation and continuing through cohabitation until mating was confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period.
Gestation Procedures
Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at day 0 of presumed gestation and assigned to individual housing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose formulations were analysed for test substance by gas chromatography with flame ionisation detector (GC-FID). The method was validated for the analysis of dose formulations at concentrations ranging from 0.46 mg/mL to 50 mg/mL of test substance in 0.5% (w/v) aqueous methylcellulose. Mean measured test substance concentrations for dose formulations at the start, mid, and end of study were within the acceptable limits (±15% of nominal concentration) with the following exception: Group II at start of study (-36.4%). The relative standard deviation (RSD) of the mean of the average concentration values for the top, middle, and bottom of each dose formulation for the start of study was calculated to assess homogeneity. Homogeneity was acceptable (≤5% RSD) for dose formulations at the start of study. Dose formulations at concentrations of 1 mg/mL and 50 mg/mL were stable at 22°C ± 5°C for 24 hours and at 5°C ± 3°C for 15 days. Mean measured concentrations for stored samples were within the acceptable limits (±10% of the initial mean measured concentration).
Duration of treatment / exposure:
Males – up to approximately 84 days
Females - up to approximately 109 days, for females that were not pregnant, and up to approximately 131 days for dams delivering litters.
Male and female rats were dosed for approximately 70 days prior to cohabitation and during the cohabitation period (up to 14 days), and continuing through the day before sacrifice.
Frequency of treatment:
Once daily.
Details on study schedule:
F1 Generation
The pups in each litter were counted and clinical observations were recorded once daily during the postpartum period. Pups were not individually identified during the postpartum period; all parameters were evaluated in terms of the litter. Pup body weights were recorded on lactation days 1, 5, 8, 15, and 22. On lactation day 5, litters were reduced to 8 pups each (4 male and 4 female pups, when possible), using a table of random units or a randomisation program to select pups to be standardised. Pups were not directly given the test substance.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 25, 125, 250 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
20 rats per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the results from three previously-conducted studies with chemically similar test substances, in which similar dose levels were tested. The doses tested ranged from 1 to 250 mg/kg/day. Test substance-related effects varied between the 3 previous studies in specific terms; however, in general terms, the lowest effect levels ranged from 25 to 125 mg/kg/day. Effects seen at these doses included reduction in body weight and food consumption parameters, adverse clinical and necropsy observations, including effects on teeth, the liver, and kidneys (including hepatic peroxisome proliferation and chronic progressive nephropathy). The no-effect levels in the previous studies ranged from 5 to 25 mg/kg/day.
Previous relevant work with the current test material has indicated that the oral LD50 in rats is 1750 mg/kg/day.
In this study, the high dose level of 250 mg/kg/day was expected to produce evidence of toxicity and the high-intermediate dose level of 125 mg/kg/day was expected to be slightly less toxic. Based on these expectations, toxicity was not expected at 25 mg/kg/day and the low dose was set at 5 mg/kg/day to ensure that a no-effect level was established.
- Rationale for animal assignment: Upon arrival, P generation rats were assigned to individual housing on the basis of computer-generated random units. After the acclimation period, rats were selected for study on the basis of physical appearance and body weight. Rats were assigned to 5 dosage groups, 20 rats per sex per dosage group, using a computer-generated (weight-ordered) randomisation procedure.
Positive control:
None.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE/VIABILITY OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: No

ACCLIMATION PERIOD, CLINICAL OBSERVATIONS AND GENERAL APPEARANCE: Yes
- Time schedule: Once weekly, at least

CLINICAL OBSERVATIONS OF EFFECTS OF TEST SUBSTANCE, ABORTIONS, PREMATURE DELIVERIES, AND DEATHS: Yes
- Time schedule: Before dosage and approximately between one and 2 hours after dosage administration.

MATERNAL BEHAVIOR: Yes
- Time schedule: Lactation days 1, 5, 8, 15, and 22.

BODY WEIGHT: Yes
- Time schedule for examinations:
Acclimation period - At least weekly
Dosage period
Precohabitation, Cohabitation – Weekly, at least
Gestation – Gestation Days 0, 7, 10, 14, 18, 21, 25
Lactation – Lactation Days 1, 5, 8, 11, 15, 22
Sacrifice

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations:
Dosage period
Precohabitation – Weekly, at least
Gestation – Gestation Days 0, 7, 10, 14, 18, 21, 25
Lactation – Lactation Days 1, 5, 8, 11, 15
Sacrifice
Oestrous cyclicity (parental animals):
Estrous cycling was evaluated by examination of vaginal cytology beginning 14 days before scheduled cohabitation and continuing through cohabitation until mating was confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period.
Sperm parameters (parental animals):
Not examined.
Litter observations:
Lactation Procedures
Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam).
Each litter was evaluated for viability at least twice daily. The pups in each litter were counted daily. Clinical observations were recorded once daily during the postpartum period. Pup body weights were recorded on lactation days 1, 5, 8, 15, and 22.
On lactation day 5, a table of random units or a randomisation program was used to select pups to be standardised, and litters were reduced to 8 pups each (4 male and 4 female pups, when possible).
Postmortem examinations (parental animals):
SACRIFICE AND PATHOLOGY
SACRIFICE SCHEDULE:
Male rats – After completion of the cohabitation period
Female rats – After completion of the 22-day postpartum period
Female rats that did not deliver a litter – Gestation day 25, estimated
Female rats with no surviving pups – After the last pup was found dead of missing, presumed cannibalised.
SACRIFICE: Carbon dioxide asphyxiation
GROSS PATHOLOGY: Yes. See Table 1.

ORGAN WEIGHTS: Yes. (see Table 2).
HISTOPATHOLOGY: Yes. (see Table 2).
Postmortem examinations (offspring):
POSTMORTEM EXAMINATIONS (Offspring surviving to Day 22 scheduled sacrifice)
SACRIFICE AND PATHOLOGY
SACRIFICE SCHEDULE: Day 22 postpartum, or pups that were 15 days of age or older
SACRIFICE: Carbon dioxide asphyxiation.
Pups sacrificed at 14 days of age or younger: Intraperitoneal injection of sodium pentobarbital
GROSS PATHOLOGY for Offspring surviving to Day 22: Yes. Gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
ORGAN WEIGHTS: No.
HISTOPATHOLOGY: No

POSTMORTEM EXAMINATIONS (Pups 14 days of age or younger)
SACRIFICE AND PATHOLOGY
SACRIFICE SCHEDULE: Day 5 postpartum.
SACRIFICE: Intraperitoneal injection of sodium pentobarbital.
Pups that died before initial examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth.
Pups found dead or sacrificed before scheduled termination were examined for gross lesions and for the cause of death or condition when possible. Pups found dead were preserved in appropriate fixative for possible future evaluation.
Statistics:
See Table 3.
Reproductive indices:
Indices of reproductive function that were calculated for the P adults:
Rats that Mated (%)
Fertility Index (%)
Gestation Index (%)
Implantation Sites per Delivered Litter
Offspring viability indices:
Indices of offspring viability calculated for P1 litters:
Pups Liveborn
Viability Index (%)
Lactation Index (%)
Surviving Pups/Litter

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
P Males: One rat at 5 mg/kg/day, 4 rats at 125 mg/kg/day and 3 rats at 250 mg/kg/day were humanely euthanised due to adverse clinical signs during the dosage period. All sacrifices, except for the rat at 5 mg/kg/day (male rat 4333) and one of the rats at 125 mg/kg/day (male rat 4367), were considered to be related to the test substance. Observations in male rats 4333 (5 mg/kg/day) and 4367 (125 mg/kg/day) were considered unrelated to the test substance because the findings were not dosage-dependent or the cause of the adverse clinical condition was related to an apparent dosing error, as evident by the postmortem observations. Early euthanasia of the other male rats in the 125 and 250 mg/kg/day that were sacrificed prior to scheduled termination generally occurred after 75 to 86 dosages had been given, and were generally preceded by dental problems (missing/broken/misaligned incisors, whitened teeth, swollen and discoloured gums and/or tongue, perforation in the mouth, ulceration in the mouth or behind the incisors and/or broken palate), swollen snout, swollen upper margins, red substance in the cage pan, signs of respiratory distress (rales or open mouth breathing), red or yellow perioral substance, red substance in the cage, chromodacryorrhea and/or chromorhinorrhea. These clinical signs, particularly the dental problems, are consistent with observations in previous studies evaluating this test substance.
Dental problems (i.e., whitened teeth and missing/broken/misaligned incisors) were observed in male rats dosed with 125 and 250 mg/kg/day test substance and were presumed related to the test substance. The number of affected rats in the 125 and 250 mg/kg/day dosage groups was often significantly increased (p≤0.01) in comparison to the vehicle control group. There was also a significant increase (p≤0.01) in the total number of rats in the 250 mg/kg/day dosage group with slight excess salivation. Whitened teeth were the most frequently occurring of these clinical signs.
At 125 and 250 mg/kg/day, chromorhinorrhea, swollen snout or mouth, perioral substance (orange, red or yellow), red substance in the cage, broken palate, vocalisation to touch occurred at low incidences, but were considered related to the test substance because they occurred in rats that were humanely euthanised, as previously described. In addition, red substance in the cage pan, swollen tongue, red gums, swollen gums, swollen upper margin, ulceration in the mouth, discoloured (purple) tongue and open mouth breathing occurred in one or 2 rats in the 250 mg/kg/day dosage group. Two rats in the 125 mg/kg/day were observed gasping prior to euthanasia. These observations were also attributed to treatment with test substance because the signs occurred in rats that were humanely euthanised, as previously described.
All other clinical observations were considered unrelated to the test substance because: 1) the observations were not dosage-dependent; 2) the observation occurred in only one or 2 rats in any dosage group; and/or 3) the finding is commonly observed in this strain of rat. These observations included moderate excess salivation; a scab on the head, nose, neck, back, left forelimb and left flank; mild or moderate dehydration; soft or liquid faeces; localised alopecia on the neck, limbs or underside; urine-stained abdominal fur; red perinasal substance; sparse hair coat on the limbs and underside; an ulceration on the neck or back; lacrimation; a laceration behind the upper incisors; ptosis; ungroomed coat; pale extremities; enlarged left eye; corneal opacity; swollen left forelimb; limited use of the left forelimb; perforation in the mouth; ataxia; red substance on the penis; paleness in the ears; coldness to the touch; bradypnea; a swollen right ear; a perforation behind the upper incisors; scant faeces; red substance in the mouth; and an abrasion on the neck.
P Females: One rat in the 0 (Vehicle) mg/kg/day dosage group was humanely euthanised due to adverse clinical signs during the precohabitation period. The unscheduled sacrifice was considered unrelated to test substance because it was a single non-dosage-dependent in the vehicle control group.
At 250 mg/kg/day, there was a significant increase (p≤0.01) in the total number of female rats that were either found dead (p≤0.01) or sacrificed due to adverse clinical signs during the study. Each of the deaths/early sacrifices at 250 mg/kg/day were attributed to test substance because they occurred at the highest dosage level tested.
Death/early euthanasia of the female rats in the 250 mg/kg/day that were sacrificed prior to scheduled termination generally occurred after 22 to 114 dosages had been given, and these unscheduled events were generally preceded by urine-stained abdominal fur, excess salivation (slight to extreme), slight to severe dehydration (based on skin turgor), coldness to touch, ungroomed coat, dental problems (missing/broken/misaligned/overgrown incisors and/or whitened teeth), and hunched posture. The dental problems are consistent with observations observed in male rats, as previously described, and in previous studies evaluating this test substance.
Whitened teeth were observed in female rats given with 125 and 250 mg/kg/day test substance and was observed during the precohabitation, gestation and lactation period and presumed related to test substance. The number of affected rats in the 125 and 250 mg/kg/day dosage groups during each of the aforementioned periods was significantly increased (p≤0.01) in comparison to the vehicle control group. In addition, dental problems (missing/misaligned/broken/overgrown incisors), excess salivation, dehydration (based on skin turgor), urine-stained abdominal fur, ungroomed coat, hunched posture and/or coldness to touch were observed during one or more of the observations period in the 125 and/or 250 mg/kg/day in an increased or significantly increased (p≤0.05 or p≤0.01) number of female rats. It is noteworthy to mention that the dental problems (missing/misaligned/broken/overgrown incisors) were more frequently observed in gestating and lactating dams.
Other clinical observations that reach statistically significant increase (p≤0.01) relative to the vehicle control group value during the in-life portion of the study included: chromorhinorrhea, scant faeces, ptosis, decreased motor activity, lethargy, pale extremities and ataxia at 250 mg/kg/day during the gestation period, and pale extremities at 250 mg/kg/day during the lactation period. Although these clinical signs occurred in a small number of animals (N=2 to 4), these findings were presumed related to the test
substance because they occurred at the highest dosage level tested.
All other clinical observations were considered unrelated to the test substance because: 1) the observations were not dosage-dependent; 2) the observation occurred in only one or 2 rats in any dosage group; and/or 3) the finding is commonly observed in this strain of rat. These observations included localised alopecia in the limb(s); sparse hair coat on the underside or limb(s); dyspnea; chromodacryorrhea; swollen snout; yellow fur in the perianal area or lower midline; scant faeces; swollen periorbital (right eye); light brown faeces; soft or liquid faeces; red perioral substance; lacrimation; red perinasal substance; hyperreactivity to touch or sound; prostration; rales; abrasion in the mouth; swollen mouth; red substance in the cage and feed jar; scab or ulceration on the mouth or neck; limited use of the right forelimb; vocalisation upon handling; swollen right forelimb; no use of the right forelimb; hyperactivity without stimuli; exophthalmos; dark red or pale right and/or left eye; tip-toe walk; laboured breathing; tachypnea; dark/brown reddish or red substance on the fur of the nose; red substance in the cage pan; rough coat; corneal opacity; paleness in both eyes; scab on the nose; laceration on the nose and a mass on the right axilla. There was a significant reduction (p≤0.01) in the number of female rats in the 5, 25 and 125 mg/kg/day dosage groups observed with mild dehydration during the gestation period, as compared to the vehicle control group value. This finding was not considered to be test substance-related because the statistical significance reflected an increase in the vehicle control group relative to the absence of this observation in the 5, 25 and 125 mg/kg/day dosage group.

BODY WEIGHT AND WEIGHT GAIN
P Males: Body weight gains were significantly reduced (p≤0.01) in the 250 mg/kg/day dosage group on study days 22 to 29, days 77 to 84 and for the entire dosage period (study days 1 to 101), in comparison to the vehicle control group values. In addition, mean body weights were significantly reduced (p≤0.05 or p≤0.01) at 250 mg/kg/day on study days 57, 64, 70, 84, 91, 98 and 101. The average body weight gain in the 250 mg/kg/day dosage group was 14% lower than the vehicle control group value for the entire dosage period.
Body weight gains and body weights during the study period were unaffected by exposure to the 5 and 25 mg/kg/day dosages of test substance. Body weight gains were significantly reduced (p≤0.01) on study days 8 to 15 at 25 mg/kg/day, and mean body weights were significantly reduced (p≤0.05) at 25 mg/kg/day on study days 57 and 64, and on study day 98 at 125 mg/kg/day. These reductions in body weight and/or body weight gain were not considered test substance related because they were either: 1) single occurrences that did not persist; or 2) the reductions were not dosage-dependent.
P Females: There were no test substance-related effects on body weight or body weight gain observed in the female rats during the precohabitation period (days 1 to 70). At 250 mg/kg/day, there was a significant reduction (p≤0.05) in the body weight gain on study days 50 to 57, as compared to the vehicle control group value. This finding was not considered to be test substance-related because: 1) it was a single occurrence that did not persist; and 2) it did not impact the overall body weight gain for the precohabitation
dosage period (study days 1 to 70).
During the gestation and lactation periods, test substance-related effects on maternal body weight gain were observed in the 250 mg/kg/day dosage group as indicated in the following text. Maternal body weight gains were reduced or significantly reduced (p≤0.01) at 250 mg/kg/day on gestation days 0 to 7, gestation days 18 to 21 and for the entire gestation period (gestation days 0 to 21), as compared to the vehicle control group values. The average body weight for gestating female rats was also reduced or significantly reduced (p≤0.05 or p≤0.01) beginning on gestation day 7 and continuing until gestation day 21, in comparison to the vehicle control group values. In lactating dams, a significant loss (p≤0.05) in body weight occurred in the 250 mg/kg/day dosage group on lactation days 5 to 8, and body weight gains were reduced in this same dosage group at the next tabulated interval (lactation days 8 to 11), as compared to the vehicle control group values. Overall for the lactation period (lactation days 1 to 22), body weight gains were lower in the 250 mg/kg/day dosage group relative to the corresponding vehicle control group value (35% less than the vehicle controls). The average body weights of the lactating dams in the 250 mg/kg/day dosage group were also reduced or significantly reduced (p≤0.05 or p≤0.01) at all intervals throughout the lactation period.
Body weight gains and body weights during the study period were unaffected by dosages of the test substance as high as 125 mg/kg/day.

FOOD CONSUMPTION
P Males: There were no test substance-related effects on the absolute and relative feed consumption values during the dosage period.
There was a significant increase (p≤0.05 or p≤0.01) in relative feed consumption in the 25 mg/kg/day dosage group on test days 43 to 50, and in the 125 and 250 mg/kg/day dosage groups on test days 98 to 101, in comparison to the vehicle control group values. These increases in relative feed consumption were not considered test substance related because: 1) this was a single occurrence that did not persist; 2) it was not dosage dependent; and/or 3) there was no corresponding increase in absolute feed consumption during these intervals. In addition, there was a significantly reduction in (p≤0.01) absolute feed consumption at 250 mg/kg/day on test days 50 to 57, as compared to the vehicle control group value. This reduction was not considered to be test substance-related because it was a single occurrence that did not persist.
P1 Females: There were no test substance-related effects on the maternal absolute and relative feed consumption values during the precohabitation and gestation periods. There was a significant increase (p≤0.05) in the absolute and relative feed consumption values at 125 mg/kg/day on gestation days 18 to 21 and in relative feed consumption values on gestation days 14 to 18 at 250 mg/kg/day. These increases were not considered to be test substance related because: 1) the increase was not dosage-dependent; and/or 2) the increase was a single occurrence that did not persist.
Test substance-related effects on maternal absolute and relative feed consumption values were observed in the 125 and 250 mg/kg/day dosage groups during the lactation period. There were significant reductions (p≤0.05 or p≤0.01) in the absolute and relative feed consumption values on lactation days 5 to 8 at 250 mg/kg/day, and again on lactation days 8 to 11, lactation days 11 to 15 and lactation days 1 to 15 at 125 and 250 mg/kg/day, as compared to the vehicle control group values.
Maternal absolute and relative feed consumption values during the study period were unaffected by dosages of the test substance as high as 25 mg/kg/day. The significant reduction (p≤0.05) in the relative feed consumption on lactation day 11 to 15 that occurred in the 25 mg/kg/day dosage group was not considered to be test substance related because it was a single occurrence that did not persist.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
P Females: The precohabitation estrous cycling observations (mean estrous stages per 14 days, rats
with 6 or more consecutive days in diestrus and rats with 6 or more consecutive days of estrus) were unaffected by dosages of the test substance as high as 250 mg/kg/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
P Males: All mating and fertility parameters (numbers of days in cohabitation, rats that mated, rats with confirmed mating dates during the first week of cohabitation and during the second week of cohabitation and number of pregnancies per number of rats in cohabitation) were unaffected by dosages of the test substance up to and including 250 mg/kg/day.
P Females: All mating and fertility parameters (numbers of days in cohabitation, rats that mated, fertility index [number of pregnancies per number of rats that mated], rats with confirmed mating dates during the first week of cohabitation and number of pregnancies per number of rats in cohabitation) were unaffected by dosages of the test substance as high as 250 mg/kg/day.
Pregnancy occurred in 17, 18, 20, 20 and 14 mated female rats in the 0 (Vehicle), 5, 25, 125 and 250 mg/kg/day dosage groups, respectively, and a total of 17 (100%), 17 (94.4%), 20 (100%), 20 (100%) and 10 (71.4%, statistically different from the vehicle control group value [p≤0.01]) of the respective pregnant dams delivered litters.
Reflecting the overall number of dams that delivered a litter (significantly reduced at p≤0.01), the average number of liveborn pups in the 250 mg/kg/day dosage group was lower than the corresponding vehicle control group value.
No adverse natural delivery or litter observations in the F1 generation pups were attributable to the test substance at dosages as high as 25 mg/kg/day.
Values for the duration of gestation, averages for implantation sites per delivered litter, dams with stillborn pups, the gestation index (number of dams with one or more liveborn pups/number of pregnant), and number of dams with all pups dying postpartum days 5 to 21 were comparable among the 5 groups. The values for mean live litter size at weighing were also comparable among the 5 dosage groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
P Males: There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to administration of test substance because: 1) the incidences were not dosage-dependent; 2) the observations were the result of an intubation error; and/or 3) the observations occurred in only one rat in any dosage group. These necropsy observations included a perforation in the esophagus surrounded by a brown granular material, red gelatinous material on the ventral side of the thoracic area, a pale, spongy appearance to all lobes of the lungs, slight or severe dilation of the pelvis in one or both kidneys, a pitted area in the medulla of the left kidney, dark discoloration in the corticomedullary junction of the kidneys, small ventral side of the prostrate, thickening of the wall of the urinary bladder, numerous red foci on the mucosal surface of the urinary bladder and calculus present in the lumen of the urinary bladder.
P Females: There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to administration of test substance because: 1) the incidences were not dosage-dependent; 2) the observations were the result of an intubation error; and/or 3) the observations occurred in only one or two rats in any dosage group. These necropsy observations included a perforation in the esophagus distal to the thyroid continuing into the right axillary region; discoloration of the lung lobes (mottled red and dark red, red or black areas present); a rough appearance to the kidneys; slight dilation of the pelvis in the kidneys (bilateral); discoloured (black) ovaries and uterus; hydrometra (clear fluid present); numerous red areas on the mucosal surface of the fundic region of the stomach; distention of the stomach with a tan cloudy liquid; and numerous black areas present in the stomach.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P Males: Other than a significant reduction (p≤0.01) in the mean terminal body weight in the 250 mg/kg/day dosage group, there were no other test substance-related effects on terminal body weights, the organ weights or ratio of the organ weights to terminal body weights. The weights of the left epididymis, left testis, right epididymis, right testis, prostate, pituitary and the ratios of these organ weights to terminal body weight were unaffected by dosages of the test substance as high as 250 mg/kg/day.
There was a significant increase (p≤0.05 or p≤0.01) in the absolute and relative weights of the seminal vesicles with the coagulating gland in the 125 mg/kg/day dosage group, as compared to the vehicle control group value. This increase was not considered to be test substance-related because it was not dosage-dependent.
P Females: There was a significant reduction (p≤0.05) in the mean terminal body weight at 250 mg/kg/day, as compared to the vehicle control group value.
In addition, there was a reduction or significant reduction (p≤0.05) in the absolute and relative weights of the uterus with the cervix at 125 and 250 mg/kg/day, in comparison to the vehicle control group values. There were no other test substance-related effects on terminal body weights, absolute organ weights or the ratio of the organ weights to terminal body weights. The absolute weights of the pituitary and ovaries and the ratios of these organ weights to terminal body weight were unaffected by dosages of the test substance as high as 250 mg/kg/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test substance-related microscopic changes were observed in any of the tissues specified for examination from the male and female rats administered test substance at dosages of 250 mg/kg/day.
Many of the microscopic changes observed in the surviving rats were typical of those that occur spontaneously in laboratory rats of this age and strain. The type, incidence, or severity of these changes was not influenced by the test substance.
Several rats on the study died or were sacrificed in moribund condition, including one control female rat and sixteen rats in the 250 mg/kg/day dosage group. There were only a few instances in which a specific cause of death could be determined such as esophageal perforation in the control female rat and gastric mucosal erosions, which may be stress related, in the stomach of three female rats in the 250 mg/kg/day dosage group. There were no significant microscopic changes observed in any of the other rats that died or were sacrificed in moribund condition. The few other changes observed in the nonsurviving rats were nonspecific and, therefore, there were no consistent microscopic findings in these rats that could be clearly correlated with a cause of death or moribundity.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
General Toxicity
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
Fertility
Effect level:
>= 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on fertility were observed at any dose level.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There was a significant increase (p≤0.05 or p≤0.01) in the number of pups found dead, presumed cannibalised or sacrificed due to adverse clinical observations on postnatal day 1 at 125 and 250 mg/kg/day, and on postnatal days 2 to 5, postnatal days 6 to 8 and postnatal days 9 to 15 at 250 mg/kg/day, in comparison to the vehicle control group values. At 250 mg/kg/day, the increased mortality resulted in a significant decrease (p≤0.05 or p≤0.01) in the number of surviving pups per litter on postnatal days 15 and 22, the viability index (number of live pups on postpartum day 5 [preculling]/number of liveborn pups on postpartum day 1) and the lactation index (number of live pups on postpartum day 21/number of liveborn pups on postpartum day 5 [postculling]). One dam in the 250 mg/kg/day dosage group also had all pups die between postpartum days 1 and 4. (See Table 4)
No adverse natural delivery or litter observations in the F1 generation pups were attributable to the test substance at dosages as high as 25 mg/kg/day.

CLINICAL SIGNS (OFFSPRING)
No adverse natural delivery or litter observations in the F1 generation pups were attributable to the test substance at dosages as high as 25 mg/kg/day.
No clinical observations in the F1 generation pups were attributable to maternal dosages of test substance as high as 125 mg/kg/day.
Significantly increased (p≤0.01) number of litters in the 250 mg/kg/day dosage group had pups that appeared dehydrated (based on skin turgor), were not nursing or were not nesting, in comparison to the vehicle control group values. The degrees of dehydration ranged from mild to severe, but the most frequently occurring of these clinical observations was of a mild degree. In addition, an increased number of litters in the same dosage group had pups that were cold to touch (N=4 litters on 12 occasions vs. 1 litter on 13 occasions in the vehicle control group).
All other clinical signs were considered unrelated to exposure of the P generation to test substance. These observations included: no milk band present, a scab present on the tip of the tail or right forepaw, decreased motor activity, whole body or head discoloration (purple), ungroomed coat, sparse hair coat, intermittent to moderate whole body tremors, impaired righting reflex, laboured breathing, pale appearance to the whole body, a lesion or laceration present on the neck, vocalisation to touch, missing skin on the right forelimb and forepaw, circling to the right, head tilt to the right and bent tail.

BODY WEIGHT (OFFSPRING)
Pup body weights per litter were significantly reduced (p≤0.01) in the 125 mg/kg/day dosage group on postnatal days 15 and 22 and in the 250 mg/kg/day dosage group at all tabulated intervals (postnatal days 1, 5 preculling, and postculling, 8, 15, and 22), as compared to the corresponding vehicle control group values. (See Table 4)

GROSS PATHOLOGY (OFFSPRING)
On lactation day 22, all pups were sacrificed and examined for gross lesions. Necropsy on the pups included a single cut at the suture of the frontal and parietal bones of the skull, and the cross-sectioned brain was examined for hydrocephaly. All other tissues were discarded.
At necropsy of the F1 generation pups, significantly fewer (p≤0.01) pups in the 250 mg/kg/day dosage group appeared normal, in comparison to the vehicle control group value. This reduction reflected the significant increase (p≤0.01) in mortality that occurred in this dosage group.
No necropsy observations in the F1 generation pups were attributed to maternal dosages of the test substance as high as 250 mg/kg/day. All necropsy observations were considered unrelated to the test substance because the observations occurred in only one or 2 pups in any litter.
There was a significant increase (p≤0.01) in the incidence (litter or pup) of slight to marked dilation of the pelvis in one or both kidneys, numerous pitted areas in the kidneys, pale kidneys (bilateral) and numerous calculi present in the urinary bladder, in comparison to the vehicle control group values.

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
Reproductive Toxicity and Offspring Viability and Growth
Generation:
F1
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 4

Reproductive Indices – Pups Delivered, Pup Mortality, Viability Index, Lactation Index

Dosage Group

 

I

II

III

IV

V

Dosage (Mg/Kg/Day)

 

0 (Vehicle)

5

25

125

250

 

 

 

 

 

 

 

Delivered Litters with One or More Liveborn Pups

N

17

17

20

20

10

Pups Delivered (Total)

N

225

221

282

269

232

 

Mean ± S.D.

13.2 ± 2.9

13.0 ± 2.2

14.1 ± 2.3

13.4 ± 2.2

12.1 ± 2.0

 

 

 

 

 

 

 

Liveborn

Mean ± S.D.

13.0 ± 2.9

12.9 ± 2.1

14.1 ± 2.3

13.0 ± 2.6

11.8 ± 2.4

 

N (%)

222 (98.7)

220 (99.5)

282 (100.0)

261 (97.0)

118 (97.5)

 

 

 

 

 

 

 

Stillborn

Mean ± S.D.

0.2 ± 0.4

0.0 ± 0.2

0.0 ± 0.0

0.2 ± 0.6

0.3 ± 0.7

 

N (%)

3 (1.3)

1 (0.4)

0 (0.0)

5 (1.8)

3 (2.5)

 

 

 

 

 

 

 

Unknown Vital Status

N

0

0

0

3

0

 

 

 

 

 

 

 

Culled

N

84

84

121

92

32

 

 

 

 

 

 

 

Pups found dead, presumed cannibalised, or sacrificed due to adverse clinical observations

Day 1

N/N (%)

1/222 (0.4)

0/220 (0.0)

0/282 (0.0)

5/261 (1.9)**

2/118 (1.7)*

Days 2-5

N/N (%)

3/221 (1.4)

0/220 (0.0)

1/282 (0.4)

6/256 (2.3)

10/98 (10.2)b**

Days 6-8

N/N (%)

0/133 (0.0)a

0/136 (0.0)

0/160 (0.0)

2/158 (1.3)

5/56 (8.9)**

Days 9-15

N/N (%)

0/133 (0.0)

0/136 (0.0)

0/160 (0.0)

3/156 (1.9)

9/51 (17.6)**

Days 16-22

N/N (%)

0/133 (0.0)

0/136 (0.0)

0/160 (0.0)

1/153 (0.6)

1/42 (2.4)

 

 

 

 

 

 

 

Viability Index c

%

98.2

100.0

99.6

95.8

88.0

 

N

218/222

220/220

281/282

250/261

88/100**

 

 

 

 

 

 

 

Lactation Index d

%

100.0

100.0

100.0

96.2

73.2

 

N/N

133/133a

136/136

160/160

152/158

41/56**

Day(s) = Postpartum Day(s)

a. Excludes one pup that had an accidental death.

b. Excludes pups that were sacrificed due to death of dam.

c. Number of live pups on day 5 (preculling) postpartum/number of liveborn pups on day 1 postpartum.

d. Number of live pups on day 21 (weaning) postpartum/number of live pups on day 5 (postculling) postpartum.

* Significantly different from the vehicle control group value (p≤0.05).

** Significantly different from the vehicle control group value (p≤0.01).

Table 4 (Continued)

Reproductive Indices – Pup Survival, Pup Weights

Dosage Group

 

I

II

III

IV

V

Dosage (Mg/Kg/Day)

 

0 (Vehicle)

5

25

125

250

 

 

 

 

 

 

 

Delivered Litters with One or More Liveborn Pups

N

17

17

20

20

10

Surviving Pups/Litter a

 

 

 

 

 

 

 

 

 

 

 

 

 

Day 1 b

Mean ± S.D.

13.0 ± 2.9

12.9 ± 2.1

14.1 ± 2.3

13.0 ± 2.6

11.8 ± 2.4

 

 

 

 

 

 

 

Day 5 Preculling

Mean ± S.D.

12.8 ± 2.8

12.9 ± 2.1

14.0 ± 2.2

12.4 ± 2.5

11.0 ± 4.8

 

 

 

 

 

 

[8]c

Day 5 Postculling

Mean ± S.D.

7.0 ± 0.5

8.0 ± 0.0

8.0 ± 0.0

7.9 ± 0.4

7.0 ± 2.8

 

 

 

 

 

 

[8]c

Day 8

Mean ± S.D.

7.8 ± 0.5

8.0 ± 0.0

8.0 ± 0.0

7.8 ± 0.5

6.4 ± 2.9

 

 

 

 

 

 

[8]c

Day 15

Mean ± S.D.

7.8 ± 0.5

8.0 ± 0.0

8.0 ± 0.0

7.6 ± 0.7

5.2 ± 3.4*

 

 

 

 

 

 

[8]c

Day 22

Mean ± S.D.

7.8 ± 0.5

8.0 ± 0.0

8.0 ± 0.0

7.6 ± 0.8

5.1 ± 3.3*

 

 

 

 

 

 

[8]c

Pup Weight/Litter (Grams)

 

 

 

 

 

 

 

Day 1

Mean ± S.D.

6.8 ± 0.7

6.8 ± 0.4

6.6 ± 0.4

6.6 ± 0.4

6.0 ± 0.3**

 

 

 

 

 

 

 

Day 5 Preculling

Mean ± S.D.

10.2 ± 1.7

10.2 ± 1.3

10.0 ± 0.9

9.9 ± 1.0

7.7 ± 0.6**

 

 

 

 

 

 

[7]a

Day 5 Postculling

Mean ± S.D.

10.3 ± 1.6

10.3 ± 1.3

10.2 ± 0.8

10.0 ± 1.0

7.8 ± 0.7**

 

 

 

 

 

 

[7]a

Day 8

Mean ± S.D.

16.4 ± 2.8

16.6 ± 2.5

16.5 ± 1.6

14.8 ± 2.0

9.0 ± 1.4**

 

 

 

 

 

 

[7]a

Day 15

Mean ± S.D.

34.0 ± 4.7

34.1 ± 4.4

32.4 ± 2.5

26.2 ± 3.5**

16.4 ± 2.8**

 

 

 

 

 

 

[7]a

Day 22

Mean ± S.D.

52.6 ± 6.6

52.2 ± 5.6

50.1 ± 4.4

38.9 ± 6.7**

22.2 ± 3.3**

 

 

 

 

 

 

[7]a

Day = Postpartum Day

[ ] = Number of values averaged

a. Excludes values for dams that were found dead or sacrificed due to no surviving pups.

** Significantly different from the vehicle control group value (p≤0.01).

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
On the basis of these data, the male and female no-observable-effect level (NOEL) for general toxicity of test substance is 25 mg/kg/day. At 125 and 250 mg/kg/day, mortality was increased in the male rats only, adverse clinical signs (primarily dental problems) were increased in both sexes, feed consumption values were significantly reduced in the lactating female rats, and organ weights (uterus with the cervix) were significantly reduced in the female rats. At 250mg/kg/day, mortality was increased in the female rats, and both sexes had significantly reduced body weights and body weight changes during the study.
The reproductive NOEL and the NOEL for viability and growth in the offspring were also 25 mg/kg/day. At 125 and 250 mg/kg/day, there was a significant reduction in pup body weights and a significant increase in pup mortality. At 250 mg/kg/day, the increased pup mortality resulted in a significant decrease in the litter size, as well as the indices for viability and lactation. There was also an increase in the number of pups that appeared dehydrated, were not nursing, were not nesting, or were cold to touch at 250 mg/kg/day.
Executive summary:

One hundred male and 100 female Crl:CD(SD) rats were randomly assigned to 5 dosage groups, 20 rats per sex per dosage group. The test substance or vehicle, 0.5% (w/v) aqueous methylcellulose prepared in R.O. deionised water, was administered orally via gavage to the male and female rats once daily beginning 70 days before cohabitation, through cohabitation (maximum 14 days) and continuing through the day before sacrifice. The dosages were 0, 5, 25, 125, and 250 mg/kg/day. The dosage volume was 5 mL/kg, adjusted for body weights recorded before administration. All P generation rats were observed for viability at least twice each day of the study. Observations for clinical signs were made before dosage and approximately between one and 2 hours after dosage administration. Body weights were recorded at least weekly during the acclimation period, at least weekly during the dosage period, including gestation days 0, 7, 10, 14, 18, 21, and 25 (female rats) and lactation days 1, 5, 8, 11, 15, and 22 (female rats), and at sacrifice. Feed consumption values were recorded weekly during the dosage and precohabitation periods and on gestation days 0, 7, 10, 14, 18, 21, and 25 (female rats) and lactation days 1, 5, 8, 11 and 15 (female rats) and at sacrifice. Maternal behaviour was observed on lactation days 1, 5, 8, 15, and 22. The pups in each litter were counted and clinical observations were recorded once daily during the postpartum period. Pup body weights were recorded on lactation days 1, 5, 8, 15, and 22.

After completion of the cohabitation period, all male rats (with the exception of early deaths) were sacrificed and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. To assess the potential toxicity of the test article on the male reproductive system, reproductive organs were weighed and retained for possible histopathological evaluation.

Female rats (with the exception of early deaths) were sacrificed after completion of the 22-day postpartum period and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. The number and distribution of implantation sites was recorded.

The following organs were retained from all P generation male rats for possible histological evaluation: pituitary gland, gross lesions, testes, epididymides, seminal vesicles with coagulating gland, and prostate. The following organs were retained from all P generation female rats for possible histological evaluation: pituitary gland, gross lesions, ovaries, mammary gland, vagina, and uterus with cervix.

On lactation day 5, litters were reduced to 8 pups each (4 male and 4 female pups, when possible). Pups with gross lesions were preserved in Bouin's solution. All other tissues were discarded.

On lactation day 22, all pups were sacrificed and examined for gross lesions. Necropsy on the pups included a single cut at the suture of the frontal and parietal bones of the skull, and the cross-sectioned brain was examined for hydrocephaly. All other tissues were discarded.

Results

P Generation Male Rats

One male rat at 5 mg/kg/day, 4 rats at 125 mg/kg/day and 3 rats at 250 mg/kg/day were humanely euthanised due to adverse clinical signs during the dosage period. All sacrifices, except for the rat at 5 mg/kg/day and one of the rats at 125 mg/kg/day, were considered to be related to the test substance. The findings in the rats at 5 and 125 mg/kg/day were considered unrelated to the test substance because they were either not dosage-dependent or the cause of the adverse clinical condition was related to an apparent dosing error, as evident by the postmortem observations. All other male rats survived until scheduled sacrifice.

Dental problems (i.e., whitened teeth and missing/broken/misaligned incisors) were observed in male rats dosed with 125 and 250 mg/kg/day and were presumed related to the test substance. The number of affected rats in the 125 and 250 mg/kg/day dosage groups was often significantly increased. There was also a significant increase in the total number of rats in the 250 mg/kg/day dosage group with excess salivation. Whitened teeth were the most frequently occurring of the clinical signs observed in male rats.

At 125 and 250 mg/kg/day, chromorhinorrhea, swollen snout or mouth, perioral substance (orange, red or yellow), red substance in the cage, broken palate, vocalisation to touch occurred at low incidences, but were considered related to the test substance. Red substance in the cage pan, swollen tongue, red gums, swollen gums, swollen upper margin, ulceration in the mouth, discoloured (purple) tongue and open mouth breathing occurred in one or 2 rats in the 250 mg/kg/day dosage group. Two rats in the 125 mg/kg/day were observed gasping prior to euthanasia. These observations were also attributed to treatment with test substance.

Body weight gains were significantly reduced in the 250 mg/kg/day dosage group on study days 22 to 29, study days 77 to 84 and for the entire dosage period (study days 1 to 101) in comparison to the control group values. Mean body weights were significantly reduced at 250 mg/kg/day on study days 57, 64, 70, 84, 91, 98 and 101. The average body weight gain in the 250 mg/kg/day dosage group was 14% lower than the vehicle control group value for the entire dosage period.

There were no test substance-related effects on the absolute and relative feed consumption values during the dosage period.

There were no test substance-related necropsy observations.

Other than a significant reduction in the mean terminal body weight in the 250 mg/kg/day dosage group, there were no other test substance-related effects on terminal body weights, the organ weights or ratio of the organ weights to terminal body weights.

P Generation Female Rats

One rat in the 0 (Vehicle) mg/kg/day dosage group was humanely euthanised due to adverse clinical signs during the precohabitation period. The unscheduled sacrifice was considered unrelated to the test substance. At 250 mg/kg/day, there was a significant increase in the total number of female rats that were either found dead or sacrificed due to adverse clinical signs during the study. Each of the deaths/early sacrifices at 250 mg/kg/day were attributed to test substance because they occurred at the highest dosage level tested.

Death/early euthanasia of the female rats in the 250 mg/kg/day that were sacrificed prior to scheduled termination generally occurred after 22 to 114 dosages had been given, and these unscheduled events were generally preceded by urine-stained abdominal fur, excess salivation (slight to extreme), slight to severe dehydration (based on skin turgor), coldness to touch, ungroomed coat, dental problems (missing/broken/misaligned/overgrown incisors and/or whitened teeth), and hunched posture. The dental problems are consistent with observations observed in male rats, and in previous studies evaluating this test substance.

Whitened teeth were observed in female rats given with 125 and 250 mg/kg/day test substance and was observed during the precohabitation, gestation and lactation period and presumed related to test substance. The number of affected rats in the 125 and 250 mg/kg/day dosage groups during each of the aforementioned periods was significantly increased. Dental problems (missing/misaligned/broken/overgrown incisors), excess salivation, dehydration (based on skin turgor), urine-stained abdominal fur, ungroomed coat, hunched posture and/or coldness to touch were observed during one or more of the observations period in the 125 and/or 250 mg/kg/day in an increased or significantly increased number of female rats. It is noteworthy to mention that the dental problems (missing/misaligned/broken/overgrown incisors) were more frequently observed in gestating and lactating dams.

Other clinical observations that reach statistically significant increase relative to the vehicle control group value during the in-life portion of the study included: chromorhinorrhea, scant faeces, ptosis, decreased motor activity, lethargy, pale extremities and ataxia at 250 mg/kg/day during the gestation period, and pale extremities at 250 mg/kg/day during the lactation period. Although these clinical signs occurred in a small number of animals (N=2 to 4), these findings were presumed related to the test substance because they occurred at the highest dosage level tested.

There were no test substance-related effects on body weight or body weight gain observed in the female rats during the precohabitation period. During the gestation and lactation periods, test substance-related effects on maternal body weight gain were observed in the 250 mg/kg/day dosage group. Maternal body weight gains were reduced or significantly reduced at 250 mg/kg/day on gestation days 0 to 7, gestation days 18 to 21, and for the entire gestation period. The average body weight for gestating female rats was also reduced or significantly reduced beginning on gestation day 7 and continuing until gestation day 21. In lactating dams, a significant loss in body weight occurred in the 250 mg/kg/day dosage group on lactation days 5 to 8, and body weight gains were reduced in this same dosage group at the next tabulated interval (lactation days 8 to 11). Overall for the lactation period (lactation days 1 to 22), body weight gains were lower in the 250 mg/kg/day dosage group relative to the corresponding vehicle control group value (35% less than the vehicle controls). The average body weights of the lactating dams in the 250 mg/kg/day dosage group were also reduced or significantly reduced at all intervals throughout the lactation period.

There were no test substance-related effects on the maternal absolute and relative feed consumption values during the precohabitation and gestation periods. Test substance-related effects on maternal absolute and relative feed consumption values were observed in the 125 and 250 mg/kg/day dosage groups during the lactation period. There were significant reductions in the absolute and relative feed consumption values on lactation days 5 to 8 at 250 mg/kg/day, and again on lactation days 8 to 11, lactation days 11 to 15 and lactation days 1 to 15 at 125 and 250 mg/kg/day.

The precohabitation estrous cycling observations (mean estrous stages per 14 days, rats with 6 or more consecutive days in diestrus and rats with 6 or more consecutive days of estrus) were unaffected by dosages of the test substance as high as 250 mg/kg/day. All mating and fertility parameters were unaffected by dosages of the test substance as high as 250 mg/kg/day.

There were no test substance-related necropsy observations in the female rats.

There was a significant reduction in the mean terminal body weight at 250 mg/kg/day. In addition, there was a reduction or significant reduction in the absolute and relative weights of the uterus with the cervix at 125 and 250 mg/kg/day.

Pregnancy occurred in 17, 18, 20, 20, and 14 mated female rats in the 0 (Vehicle), 5, 25, 125, and 250 mg/kg/day dosage groups, respectively, and a total of 17 (100%), 17 (94.4%), 20 (100%), 20 (100%), and 10 (71.4%) of the respective pregnant dams delivered litters. Reflecting the overall number of dams that delivered a litter, the average number of liveborn pups in the 250 mg/kg/day dosage group was lower than the corresponding vehicle control group value.

F1 Generation Pups

There was a significant increase in the number of pups found dead, presumed cannibalised or sacrificed due to adverse clinical observations on postnatal day 1 at 125 and 250 mg/kg/day, and on postnatal days 2 to 5, 6 to 8, and 9 to 15 at 250 mg/kg/day. At 250 mg/kg/day, the increased mortality resulted in a significant decrease in the number of surviving pups per litter on postnatal days 15 and 22, the viability index and the lactation index. One dam in the 250 mg/kg/day dosage group also had all pups die between postpartum days 1 and 4. Pup body weights per litter were significantly reduced in the 125 mg/kg/day dosage group on days 15 and 22 and in the 250 mg/kg/day dosage group at all tabulated intervals.

Significantly increased number of litters in the 250 mg/kg/day dosage group had pups that appeared dehydrated, were not nursing or were not nesting. The degrees of dehydration ranged from mild to severe, but the most frequently occurring of these clinical observations was of a mild degree. In addition, an increased number of litters in the same dosage group had pups that were cold to touch.

At necropsy of the F1 generation pups, significantly fewer pups in the 250 mg/kg/day dosage group that appeared normal. This reduction reflected the significant increase in mortality that occurred in this dosage group. No necropsy observations in the F1 generation pups were attributed to maternal dosages of the test substance as high as 250 mg/kg/day.

On the basis of these data, the male and female no-observable-effect level (NOEL) for general toxicity of test substance is 25 mg/kg/day. At 125 and 250 mg/kg/day, mortality was increased in the male rats only, adverse clinical signs (primarily dental problems) were increased in both sexes, feed consumption values were significantly reduced in the lactating female rats, and organ weights (uterus with the cervix) were significantly reduced in the female rats. At 250mg/kg/day, mortality was increased in the female rats, and both sexes had significantly reduced body weights and body weight changes during the study.

The reproductive NOEL and the NOEL for viability and growth in the offspring were also 25 mg/kg/day. At 125 and 250 mg/kg/day, there was a significant reduction in pup body weights and a significant increase in pup mortality. At 250 mg/kg/day, the increased pup mortality resulted in a significant decrease in the litter size, as well as the indices for viability and lactation. There was also an increase in the number of pups that appeared dehydrated, were not nursing, were not nesting, or were cold to touch at 250 mg/kg/day.

 

 

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