Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 415 on analogue substance (primary breakdown product 6-2 FTOH)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Offspring in the P litters of each treatment level were randomly selected for continued evaluation until approximately PND 40. Addition of developmental landmarks for F1 generation (males and females) and scheduled sacrifice on postnatal day PND 40
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.94%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Male mice: approximately 50 days old / Female mice: approximately 75 days old
- Weight at study initiation: Male mice – 25.0 – 32.0 grams /Female mice – 24.9 – 29.8 grams
- Fasting period before study: None
- Housing: Solid bottom caging with bedding containing Nestlets™ as enrichment. Enrichment was omitted for up to one week before and after expected deliveries. Each cage rack contained only animals of one sex, except during cohabitation when the animals were housed as breeding pairs (female in male’s cage). Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly. During lactation periods, adult females were housed with their litters.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): Targeted at 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methylcellulose; Tween-80 in aqueous methylcellulose
Details on exposure:
Dose Administration
Animals were dosed once daily at approximately the same time (± 2 hours) by intragastric intubation at a dose volume of 5 mL/kg body weight for approximately 70 days (males) or 14 days (females) prior to cohabitation and during the cohabitation period (up to 2 weeks). The amount of test substance each animal received was based on the most recently collected body weight and the formulation concentration. Control animals were dosed with the vehicle (0.5% aqueous methylcellulose, with or without 0.1% Tween-80) at a volume of 5 mL/kg of body weight.

Dose Preparation
Dose formulations of the test substance were prepared within 8 days and stored refrigerated until used. Starting on the seventh week, the 1 mg/kg/day formulation was prepared daily from the 25 mg/kg/day dose level. Dose formulations were prepared with a correction for the sponsor-reported purity.

VEHICLE
The test substance was suspended in 0.5% methylcellulose for the first week of the study. Starting from the second week through the remainder of the study, dose formulations were prepared in 0.1% Tween-80 in 0.5% aqueous methylcellulose. Neither the amount nor nature of any contaminants in the vehicle was expected to affect the integrity or validity of this study.
Details on mating procedure:
Start of Cohabitation: Animals were co-housed for mating after approximately 70 days (males) or 14 days (females) of dosing. The day animals were first co-housed was designated as day 0 of cohabitation.
Duration of Cohabitation Period: Each female was continually housed on a 1:1 basis with a randomly selected, non-sibling male of the same dose level, in the male's cage. Animals were co-housed until evidence of copulation was observed or until 2 weeks elapsed. On the day copulation was confirmed, the female was transferred back to individual cage housing. The cohabitation period ended on the morning of day 14 of cohabitation.
Evidence of Copulation: Twice daily, each female was examined for the presence of an intravaginal copulation plug, which was recorded as evidence of copulation. The day evidence of copulation was observed was designated as day 0 of gestation (GD 0).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test dosing formulation were collected at multiple time points during the study. Minimally, samples were collected near the beginning, approximately monthly, and near the end of the study. Analysis of the first sampling verified mixing uniformity, concentration (average of verification samples), and 15-day refrigerated stability. The subsequent samplings addressed concentration. At the time of analysis, the samples were diluted with methanol and analysed by gas chromatography with flame ionization detection (GC/FID).
The analytical results for stability, homogeneity, and concentration of the formulations were within acceptable limits for the 5, 25, and 100 mg/kg/day groups throughout the dosing period of the study. The analytical results for stability, homogeneity, and concentration of the formulations were within acceptable limits for the 1 mg/kg/day group starting on test day 42 for the males and for the entire dosing period for the females. Prior to test day 42 for the males, the homogeneity, concentration, and/or stability of the formulation for the 1 mg/kg/day group were outside the targeted range due to formulation methods, which were later improved.
Duration of treatment / exposure:
The test substance was administered to P1 generation male and female mice during premating, mating, gestation, and lactation, and to F1 generation male and female adults (post-weaning).
Frequency of treatment:
Once daily
Details on study schedule:
P1 adult mice were administered the test substance daily for 10 weeks (males) and 2 weeks (females) during the premating period, and then up until the day before scheduled sacrifice. Following the premating period, the P1 males and females were co-housed within their respective treatment groups to produce F1 litters. Dams were allowed to deliver and rear their offspring until weaning on postnatal day (PND) 21. At weaning, F1 offspring were randomly selected from the 0, 1, 5, and 25 mg/kg/day groups to continue on study as F1 adults. F1 offspring of P1 animals in the 100 mg/kg/day group were euthanized on PND 21 due to concerns about their viability. The F1 adults from the remaining study groups (0, 1, 5 and 25 mg/kd/day) were administered the test substance from PND 21 until the day before scheduled sacrifice (PND 40-43).

Animals / Generation / Sacrifice Schedule
Adult Males / P1 / After siring litters (Test day 107-109)
Pregnant Females / P1 / On day of weaning litters (Day 21 Postpartum)
Nonpregnant Females / P1 / With pregnant females
Culled Pups / F1 / Day 4 Postpartum
Weanlings / F1 / On day of weaning - Day 21 Postpartum [0, 1, 5, and 25 mg/kg/day: Three of 4 animals/sex/litter (when possible); 100 mg/kg/day: All animals (due to animal welfare concerns)]
F1 Adults / F1 / Postnatal day 40-43
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 5, 25, 100 mg/kg/day
Basis:
actual ingested
(adjusted for purity)
No. of animals per sex per dose:
15 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for this study were based on previous studies. A one-generation reproductive toxicity study, a subchronic 90-day study, and a developmental toxicity study were conducted in rats, using dose levels of 5, 25, 125, and 250 mg/kg/day, administered by gavage throughout each study. No test substance-related effects were observed at 5 mg/kg/day. At dose levels of 25 mg/kg/day and above, test substance-related differences were observed in haematology and clinical chemistry parameters, urinalysis parameters (females only), and histomorphologically in the liver. At dose levels of 125 mg/kg/day and above, the following test substance-related effects were observed: mortality, clinical observations (primarily dental effects), reductions in body weight and body weight gain during subchronic and gestational exposure, increases in plasma fluoride levels, decreased food consumption in gestating and lactating females, decreased organ weights (uterus with cervix) in females, decreased pup body weights, increased pup mortality, and foetal skeletal variations (ossification delays in the skull and rib alterations). At a dose level of 250 mg/kg/day, the following additional test substance-related effects were observed: reduced food consumption in females during subchronic and gestational exposure, an effect on the ameloblastic epithelium of the tooth, increased apoptosis in pancreatic acinar cells in males, decreased litter size, differences in pup viability and lactation indices, increased pup abnormalities (dehydrated, not nursing, not nesting, or cold to touch), increased stained or wet fur in females during gestation, and increased incidence of foetal pelvic bone ossification. In addition, in vitro metabolic screening studies were conducted using rat and mouse hepatocytes. Similar metabolic profiles were observed in both species of hepatocytes.
Positive control:
None

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Careful clinical observations were performed each time body weight data were collected and specifically, at least once before study start and approximately weekly throughout the premating, cohabitation, gestation, and lactation phases for the P1 parental animals, and during the postweaning period for the F1 generation until study termination. The careful clinical observation was conducted such that each animal was individually handled and carefully examined for abnormal behaviour and/or appearance.

BODY WEIGHT: Yes
- Time schedule for examinations:
Premating Period and Post-Weaning Period: All animals were weighed approximately once a week. Collection of F1 body weights ended on PND 40. All F1 animals being evaluated for developmental landmarks (vaginal patency, preputial separation) were also weighed on the day of achievement.
Gestation and Lactation Periods: P1 females were weighed on gestation days (GD) 0, 4, 7, 11, 14, and 17 and lactation days (LD) 0, 7, 14, and 21. Females without evidence of copulation, those that copulated and did not deliver a litter, and males continued to be weighed on a weekly schedule.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
During the test period, the amount of food consumed by each animal over the weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight of the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval was determined. From the food consumption and body weight data, the mean daily food efficiency were calculated.

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Clinical pathology evaluation was conducted on all surviving P1 animals on test days 107-109 for males and test days 53, 55-61 for females. Each group of males and females was divided into two approximately equally sized subsets for haematology and clinical chemistry. These animals were not fasted prior to blood collection. Blood samples for haematology or clinical chemistry measurements were collected from the vena cava of each animal while the animal was under isoflurane anaesthesia. Bone marrow smears were prepared at sacrifice from animals undergoing haematology evaluation.
Haematology: The following parameters were determined: red blood cell count, red cell distribution width, haemoglobin, absolute reticulocyte count, hematocrit, platelet count, mean corpuscular (cell) volume, white blood cell count, mean corpuscular (cell) haemoglobin, differential white blood cell count, and mean corpuscular (cell) haemoglobin concentration.
Clinical Chemistry: The following parameters were determined: aspartate aminotransferase, total protein, alanine aminotransferase, albumin, sorbitol dehydrogenase, globulin, alkaline phosphatase, calcium, total bilirubin, inorganic phosphorus, urea nitrogen, sodium, creatinine, potassium, cholesterol, chloride, triglycerides, bile acids, and glucose.
Estrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
The day when delivery was complete was designated as LD 0 for dams and PND 0 for pups. At each examination period (days 0, 4, 7, 14, and 21 postpartum), offspring were individually handled and examined for abnormal behaviour and appearance; any dead or abnormal pups were recorded. Dams that had no live pups remaining during the lactation period were sacrificed. Due to a malfunction with the automated watering equipment, the entire litter of one control female was found dead during lactation.
Day 0 Postpartum: Live and dead pups in each litter were counted by sex as soon as possible after delivery was completed. Live pups in each litter were individually weighed.
Day 4 Postpartum: Pups in each litter were counted by sex and individually weighed. Litters were culled randomly to 8 (4/sex when possible) and the number of pups of each sex recorded. Extra offspring were euthanized (by decapitation) and discarded without anatomic pathology examination. Litters of 8 offspring or fewer were not reduced.
Days 7 and 14 Postpartum: Pups in each litter were counted by sex and individually weighed.
Day 21 Postpartum (Weaning - PND 21): Pups in each litter were counted by sex and individually weighed. For the 0, 1, 5, and 25 mg/kg/day groups, offspring in the F1 litters of each treatment level were randomly selected (one animal/sex/litter when possible) to be evaluated for collection of developmental landmark data. F1 female animals were examined for vaginal patency once daily beginning on PND 21 until achievement. Body weight was recorded on the day of achievement. F1 male animals were examined for preputial separation beginning on PND 21 until achievement. Body weight was recorded on the day of achievement. For the 100 mg/kg/day group, all offspring were sacrificed as weanlings due to concerns about their ability to survive without the dam, based on low pup body weights and high pup mortality.
Postmortem examinations (parental animals):
SACRIFICE AND PATHOLOGY
SACRIFICE SCHEDULE:
Adult males (P1) – After siring litters (Test day 107-109)
Pregnant females (P1) – On day of weaning litters (Day 21 Postpartum)
Nonpregnant females (P1) – With pregnant females

SACRIFICE: Carbon dioxide asphyxiation while under isoflurane anaesthesia

GROSS PATHOLOGY: All P1 adult mice received a complete necropsy that included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities including viscera. For P1 adult females, the number of former implantation sites was recorded.

ORGAN WEIGHTS AND HISTOPATHOLOGY:
The following tissues were weighed from P1 adults sacrificed by design (except those sacrificed due to death of the litter).
Male: testes, epididymides
Female: ovaries (with oviducts), uterus (with cervix)
Both Sexes: brain, liver (with gallbladder), kidneys
See Table 1 for tissues collected from all P1 adult animals sacrificed by design (includes dams that were sacrificed due to death of litter) and preserved in appropriate fixative.
See Table 2 for tissues collected from P1 adults that were found dead, or euthanized prior to scheduled sacrifice (excludes dams that were sacrificed due to death of litter).
Microscopic examination of haematoxylin and eosin (H&E) stained paraffin sections were performed by a veterinary pathologist on all protocol tissues collected from P1 animals from the control and high-dose groups, from all P1 pairs that failed to produce a litter, and from all parental animals dying spontaneously (i.e., found dead) or euthanized in extremis (i.e., unscheduled sacrifice). In addition, the following organs from all P1 adult mice from all dose levels were processed to slides and examined microscopically: liver, nose/teeth, ovaries, uterus, vagina, and mammary gland.
Postmortem examinations (offspring):
SACRIFICE
Pups up to lactation day 4 were euthanized by decapitation. For lactating pups between lactation days 5 and 20, euthanasia was performed by intraperitoneal injection of a suitable commercial euthanasia agent. F1 weanlings and adults were euthanized by isoflurane anaesthesia followed by CO2 inhalation (weanlings) or exsanguination (adults).
GROSS NECROPSY
Pups that either died or were euthanized prior to scheduled sacrifice were examined grossly. All F1 weanlings and adults received a gross pathology evaluation.
HISTOPATHOLOGY / ORGAN WEIGTHS
Gross lesions from weanlings and adults were examined microscopically.
Statistics:
See Table 3.
Reproductive indices:
Indices of reproductive function that were calculated for the P adults:
Mating index (%)
Fertility Index (%)
Gestation Index (%)
Post-Implantation Loss (%)
Offspring viability indices:
Indices of offspring viability calculated for P1 litters:
Pups Born Alive (%)
Viability Index (%)
Lactation Index (%)
Litter Survival (%)

Results and discussion

Results: P0 (first parental animals)

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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS
Adverse, test substance-related clinical signs and mortality were observed at 100 mg/kg/day in P1 males and females. One male and two females at this dose level were found dead or humanely euthanized due to clinical abnormalities. Clinical signs in these animals and three additional animals that survived included clonic and tonic convulsions, ataxia, tremors (head/upper body/forelimbs), increased muscle tone, lethargy, pallor, and/or respiratory impairments. When the clinical observations did not result in death, the clinical signs abated within approximately one week.

BODY WEIGHT AND BODY WEIGHT GAINS (PARENTAL ANIMALS)
P1 males administered 100 mg/kg/day exhibited slight decreases in body weight and body weight gain compared with controls, which were considered test substance-related but not adverse. Among males administered 100 mg/kg/day, mean values for final body weight and overall body weight gain were not statistically significant than control means. There were two transient instances of statistical significance during weekly intervals for each parameter. The differences were considered test substance-related, due to the corroborative differences in nutritional parameters at the same dose level, but non-adverse due to the lack of statistical differences in final body weights or overall weight gain over the duration of the study. Instances of significantly lower body weight and body weight gain observed during individual weekly intervals in males administered 25 mg/kg/day were transient, lacked a dose response relationship in the affected intervals, and were not reflected in statistically or biologically significant differences in final body weight or overall body weight gain; therefore, they were considered spurious.
P1 females administered 100 mg/kg/day exhibited significantly lower body weight gains during the intervals lactation days 0-7 and lactations days 7-14, compared with controls, resulting in significantly lower body weights on lactation day 7 and lactation day 14. During lactation days 14-21, the same females exhibited a significantly lower magnitude of body weight loss, relative to controls. Body weight gains in P1 females administered 1, 5, and 25 mg/kg/day were significantly higher than control values during lactation days 0-7. The differences were transient (they were reversed during the following interval, lactation days 7-14), were in the opposite direction as the difference observed at 100 mg/kg/day, and there was no dose response relationship during any weekly interval or in the overall lactation days 0-21 body weight gain; therefore, the differences were considered spurious.

FOOD CONSUMPTION AND FOOD EFFICIENCY (PARENTAL ANIMALS)
Males administered 100 mg/kg/day exhibited slight decreases in food consumption and food efficiency compared with controls, which were considered test substance-related but not adverse. Among males administered 100 mg/kg/day, mean values for overall (test day 0-70) food consumption and food efficiency were not statistically significant than control means, respectively. There were up to two transient instances of statistical significance during weekly intervals for each parameter. The differences were considered test substance related, due to the corroborative differences in body weight parameters at the same dose level, but non-adverse due to the lack of statistical differences in overall food consumption or efficiency over the duration of the study. Up to two instances of significantly higher or lower food consumption or food efficiency were observed during individual weekly intervals in males administered 1, 5, or 25 mg/kg/day. The differences were transient, they lacked a dose response relationship in the affected intervals, and were not reflected in statistically or biologically significant differences in overall food consumption or food efficiency; therefore, they were considered spurious.
P1 females administered 100 mg/kg/day exhibited adverse, test substance related differences in food consumption and food efficiency during the lactation period, compared with controls. P1 females administered 100 mg/kg/day exhibited significantly lower food consumption during the intervals lactation days 0-7, 7-14, and 0-14 compared with controls. Due to lower body weight gain, food efficiency was significantly lower during the same intervals. Due to transiently higher body weight gains in P1 females administered 1, 5, and 25 mg/kg/day during LD 0-7, food efficiency was significantly higher during the same interval. The differences were transient (they were reversed during the following interval, lactation days 7-14), were in the opposite direction as the difference observed at 100 mg/kg/day, and there was no dose response relationship during the affected interval; therefore, the differences were considered spurious.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Liver weight parameters were increased in male and female mice administered 100 mg/kg/day of the test substance (only liver weight relative to body weight was statistically significant). Liver weight increases were greater in females compared to males (liver weight relative to body weight as increased 13 and 24% above control, in males and females, respectively). These liver weight changes correlated with test substance-related microscopic changes in the liver, including hepatocellular hypertrophy.
Kidney weight parameters were increased in the 100 mg/kg/day male group (up to 25% above controls; statistically significant). These increases were not associated with relevant changes in kidney-related clinical pathology parameters or with microscopic changes in the kidney. Therefore, the kidney weight changes in 100 mg/kg/day males were likely test-article related but were considered non-adverse. Kidney weight relative to body weight was minimally higher in the 100 mg/kg/day female group (11% above control; statistically significant). However, there were no statistically significant changes in other kidney weight parameters, and no test substance-related changes in kidney-related clinical pathology parameters or kidney histopathology. Therefore, this change was considered to be unrelated to test substance administration.
There were no other primary test substance-related organ weight changes. Ovarian and uterine weight parameters were moderately to markedly decreased in the 100 mg/kg/day female group. Ovarian weights were decreased by up to 24% compared to controls, and uterine weights were decreased by up to 56% compared to controls (statistically significant for most weight parameters). These organ weight changes were secondary to the marked decrements in body weight and nutritional parameters that occurred during the lactation period at this dose level and correlated with histological changes in the female reproductive tract consistent with anestrus.
Testes weight relative to body weight was statistically increased (15% above control) in the 100 mg/kg/day male group. There were no statistically significant changes in other testes weight parameters (absolute or relative to brain weight) and no test substance-related microscopic changes in the testes. The increase in testes weight relative to body weight was the result of the minimal (though not statistically significant) decrease in terminal body weight in this group. Testes weights are stable during body weight loss so decreases in body weight usually result in an increase in the testes weight relative to body weight.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The liver was grossly recorded as “large” in 1/15 males and 2/15 females administered 100 mg/kg/day. These observations correlated with increased liver weights and microscopic hepatocellular hypertrophy at this dose level and were considered test substance related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Adverse observations of anatomic pathology occurred in the liver and teeth of P1 males and females at 100 mg/kg/day and in the liver of females at 25 mg/kg/day. Liver changes were generally more severe in females and included hepatocellular hypertrophy, oval cell hyperplasia, single cell necrosis of hepatocytes, and cystic degeneration (females only). In males and females at 100 mg/kg/day, microscopic changes in the incisor teeth consistent with fluoride exposure included degeneration and atrophy of ameloblastic epithelium, lamination of dentin, and incomplete decalcification of enamel and/or dentin.
Microscopic findings occurring secondary to the marked decrements in body weight and nutritional parameters in dams administered 100 mg/kg/day included anoestrus with associated atrophic changes in the reproductive tract, and secretory depletion in the mammary gland. The latter change is likely the result of increased duration of nursing in the undernourished pups at the 100 mg/kg/day dose level.
Changes considered test substance-related but non-adverse based on lack of association with organ injury or evidence of decreased function included minimal hepatocellular hypertrophy (5 mg/kg/day males and females, 25 mg/kg/day males), and incomplete decalcification of enamel and dentin (25 mg/kg/day female group).

OTHER FINDINGS (PARENTAL ANIMALS)
Adverse, test substance-related changes in haematology (red and white blood cell) and clinical chemistry (liver-related) parameters were present in P1 males and females administered 100 mg/kg/day. Changes in liver parameters were also present in one female mouse in the 25 mg/kg/day group.
Red blood cell changes in the 100 mg/kg/day groups included minimal to mild decreases in red cell mass parameters (RBC, HGB, and HCT). These changes were slightly higher relative to controls in females compared to males, and in females, were associated with a regenerative response (increased ARET). White blood cell changes in the 100 mg/kg/day male and female groups included minimal to moderate increases in white blood cell parameters (WBC, ALYM, ANEU, and AMON). These changes were likely correlative to the liver effects observed at this dose level.
Changes in liver-related clinical chemistry parameters in the 100 mg/kg/day male and female groups included elevations in AST, ALT, ALP, SDH, and TBA. These changes were greater in females compared to males, with elevations of greater than 5-fold for most parameters in females and less than or equal to 5-fold in males. Increases in these same liver parameters in one 25 mg/kg/day female mouse were associated with correlative test substance-related microscopic findings in the liver and were thus also considered test substance related and adverse. Other clinical chemistry changes observed in the 100 mg/kg/day male and female groups included decreases in BUN, CREA, and CHOL (males only). These changes were greater in males than in females and were considered to be secondary to the liver toxicity observed at this dose level. Minimal elevations in serum potassium were also observed in the 100 mg/kg/day male and female groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Differences in clinical chemistry and histopathology at 25 mg/kg/day, both of which pertained to the liver and were consistent with observations at 100 mg/kg/day.
Dose descriptor:
NOAEL
Remarks:
(reproductive toxicity)
Effect level:
> 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproductive outcome were observed at any dosage.

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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
no effects observed

Details on results (F1)

CLINICAL SIGNS AND VIABILITY (OFFSPRING)
Litters of dams administered 100 mg/kg/day exhibited test substance-related reductions in pup survival (45% reduction in lactation index) but no differences in the live born or viability indices. There were no differences in pup viability, survival, or clinical observations at any other dose level.
At the dose level of 100 mg/kg/day, pup survival was significantly lower than control on lactation days 14 and 21 and the interval lactation days 4-21 (lactation index). The gross observations at necropsy for the dead pups were typical for pups that fail to thrive; however the high incidence of mortality was sufficient to warrant attribution to test substance exposure. Pups in four litters at this dose level were observed with closed eyes at lactation day 21, due to delayed maturation, which was also considered to be test substance-related. F1 pups at 100 mg/kg/day were euthanized on PND 21 due to concerns about their viability.
Across dose levels, two pups were observed with wounds and one pup was accidentally killed; these observations were not related to test substance exposure. Any remaining clinical observations in pups occurred with low incidences, without a dose response relationship, and were not considered test substance-related.

BODY WEIGHT (OFFSPRING)
At 100 mg/kg/day, mean pup body weights per litter were significantly lower than control on lactation days 4, 7, 14, and 21 (64.9% lower on lactation day 21), which was attributed to the test substance. F1 pups at 100 mg/kg/day were euthanized on PND 21 due to concerns about their viability.

SEXUAL MATURATION (OFFSPRING)
There were no test substance-related effects or statistically significant differences in the timing of preputial separation or vaginal patency achievement or corresponding body weight, in F1 males or females administered up to 25 mg/kg/day. No data were collected at 100 mg/kg/day, due to early euthanasia (at weaning) of this entire dose level.

GROSS PATHOLOGY (OFFSPRING)
All gross observations in F1 pups were non-specific findings commonly seen in found dead rodent pups at this age. Increased incidences of some of these findings at 100 mg/kg/day were the result of increased numbers of found dead pups submitted for necropsy at this dose level.
The incidences of pups grossly diagnosed as small was increased in the 100 mg/kg/day male and female F1 weanlings. This finding was consistent with in-life findings of markedly decreased body weight at weaning and was considered test substance related.
The incidence of gross discoloration of the liver was increased in the 100 mg/kg/day male and female weanlings (6/19 and 7/31, respectively; not observed in other groups). However, there was no microscopic correlate to this finding (except in one 100 mg/kg/day female, which had diffuse vacuolation) and, therefore, it was not considered to be a specific test substance-related finding.
All other gross findings occurred in low incidences (1 or 2 weanlings) and were typical of nonspecific lesions common to rodents of this age.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
(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: Clinical observations of delayed development in pups, and reductions in pup survival and pup body weight during lactation at 100 mg/kg/day.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
NOAEL (reproductive): >100 mg/kg/day
NOAEL (viability and growth of offspring): 25 mg/kg/day
NOAEL (systemic toxicity; males): 25 mg/kg/day
NOAEL (systemic toxicity; females): 5 mg/kg/day
Executive summary:

The test substance was administered via gavage to Pgeneration male and female mice during premating, mating, gestation, and lactation, and to F1 generation male and female adults (post-weaning). P1 adult CD-1 mice (15/sex/group) were administered the test substance daily in a vehicle of 0.1% Tween-80 in 0.5% aqueous methylcellulose at dosages of 0, 1, 5, 25, or 100 mg/kg body weight/day (mg/kg/day) for 10 weeks (males) and 2 weeks (females) during the premating period, and then up until the day before scheduled sacrifice. Following the premating period, the P1 males and females were co-housed within their respective treatment groups to produce F1 litters. Litters were culled to 4 pups/sex/litter (litter size permitting) on postnatal day 4; all remaining pups were discarded without further evaluation. Dams were allowed to deliver and rear their offspring until weaning on postnatal day (PND) 21. At weaning, selected F1 offspring (one pup/sex/litter when possible) were randomly selected from the 0, 1, 5, and 25 mg/kg/day groups to continue on study as F1 adults. F1 offspring of P1 animals in the 100 mg/kg/day group were euthanized on PND 21 due to concerns about their viability. The F1 adults from the remaining study groups (0, 1, 5 and 25 mg/kg/day) were administered the test substance from PND 21 until the day before scheduled sacrifice (PND 40-43), and were evaluated for developmental landmarks. Clinical observations, body weight, and food consumption were determined weekly throughout the study. Litter examinations (live or dead pups, individual pup weights, clinical observations) were determined at birth, on PND 4, and weekly during the lactation period. The age and body weight at either vaginal opening or preputial separation were recorded for the F1 generation. P1 animals surviving to scheduled sacrifice were divided approximately equally into subsets for haematology or clinical chemistry evaluation. Gross postmortem examinations were performed on P1 and F1 adults and selected organs from P1 animals were weighed and/or examined microscopically.

Adverse, test substance-related clinical signs and mortality were observed at 100 mg/kg/day in P1 males and females. One male and two females at this dose level were found dead or humanely euthanized due to clinical abnormalities. Clinical signs in these animals and three additional animals that survived included clonic and tonic convulsions, ataxia, tremors (head/upper body/forelimbs), increased muscle tone, lethargy, pallor, and/or respiratory impairments. When the clinical observations did not result in death, the clinical signs abated within approximately one week. In addition, test substance-related reductions in body weight, body weight gain, food consumption, and food efficiency were observed at this dose level in P1 males over the course of the study and in P1females during the lactation period. Marked reductions in food consumption/efficiency among P1 females at 100 mg/kg only during the lactation period resulted in mean body weights on day 14 that were 20.4% lower than controls; by comparison, mean final body weights among males in this group were 4.8% lower than controls. There were no effects on any in-life parameter in P1or F1 males or females, at dose levels of 25 mg/kg/day and lower. F1 litters of P1 adults administered 100 mg/kg/day exhibited clinical observations of delayed maturation (closed eyes at lactation day [LD] 21), a 45% reduction in lactation index (pup survival), and reductions in mean pup body weight per litter (64.9% lower on LD 21), compared with controls. The failure to thrive and delayed maturation of the pups were considered to be adverse, test substance-related effects, which were likely a result of the overt systemic toxicity observed in the dams. F1pups at 100 mg/kg/day were euthanized on PND 21 due to concerns about their viability. There were no effects on reproductive performance, litter size, sex ratio, live born index, or viability index at any dose level. There were no effects on litter parameters and no effects on developmental landmark achievement in F1 males or females at dose levels of 25 mg/kg/day and lower.

Adverse, test substance-related changes in haematology (red and white blood cell) and clinical chemistry (liver-related) parameters were present in P1males and females administered 100 mg/kg/day. Changes in liver parameters were also present in one female mouse in the 25 mg/kg/day group. Red blood cell changes in the 100 mg/kg/day groups included minimal to mild decreases in red cell mass parameters (RBC, HGB, and HCT). These changes were slightly higher relative to controls in females compared to males, and in females, were associated with a regenerative response (increased ARET). White blood cell changes in the 100 mg/kg/day male and female groups included minimal to moderate increases in white blood cell parameters (WBC, ALYM, ANEU, and AMON). These changes were likely correlative to the liver effects observed at this dose level. Changes in liver-related clinical chemistry parameters in the 100 mg/kg/day male and female groups included elevations in AST, ALT, ALP, SDH, and TBA. These changes were greater in females compared to males, with elevations of greater than 5-fold for most parameters in females and less than or equal to 5-fold in males. Increases in these same liver parameters in one 25 mg/kg/day female mouse were associated with correlative test substance-related microscopic findings in the liver and were thus also considered test substance related and adverse. Other clinical chemistry changes observed in the 100 mg/kg/day male and female groups included decreases in BUN, CREA, and CHOL (males only). These changes were greater in males than in females and were considered to be secondary to the liver toxicity observed at this dose level. Minimal elevations in serum potassium were also observed in the 100 mg/kg/day male and female groups. There were no adverse test substance-related changes in clinical pathology parameters in male mice administered 25 mg/kg/day or less, or in female mice administered 5 mg/kg/day or less.

Adverse observations of anatomic pathology occurred in the liver and teeth of P1 males and females at 100 mg/kg/day and in the liver of females at 25 mg/kg/day. Liver changes were generally more severe in females and included hepatocellular hypertrophy, oval cell hyperplasia, single cell necrosis of hepatocytes, and cystic degeneration (females only). Increased liver weight was also observed at 100 mg/kg/day in males and females. In males and females at 100 mg/kg/day, microscopic changes in the incisor teeth consistent with fluoride exposure included degeneration and atrophy of ameloblastic epithelium, lamination of dentin, and incomplete decalcification of enamel and/or dentin. Microscopic findings occurring secondary to the marked decrements in body weight and nutritional parameters in dams administered 100 mg/kg/day included anoestrus with associated atrophic changes in the reproductive tract, and secretory depletion in the mammary gland. The latter change is likely the result of increased duration of nursing in the undernourished pups at the 100 mg/kg/day dose level. Changes considered test substance-related but non-adverse based on lack of association with organ injury or evidence of decreased function included increased kidney weights (100 mg/kg/day males), minimal hepatocellular hypertrophy (5 mg/kg/day males and females, 25 mg/kg/day males), and incomplete decalcification of enamel and dentin (25 mg/kg/day female group). Test substance related gross findings in F1 generation mice were limited to increased incidences of weanlings grossly diagnosed as small. This finding was consistent with the in-life finding of markedly decreased body weight at weaning. There were no adverse pathology findings in P1 or F1 male mice administered 25 mg/kg/day or less, or in female mice administered 5 mg/kg/day or less.

Under the conditions of the study, the test substance was not a selective reproductive toxicant. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was 25 mg/kg/day (males) and 5 mg/kg/day (females). Observations of systemic toxicity in P1 males and females at 100 mg/kg/day included mortality, clinical abnormalities, and differences in body weight, nutritional parameters, haematology (red and white blood cell), clinical chemistry (liver-related), liver weights, and histopathology (liver, teeth, reproductive tract, and mammary gland). Observations of systemic toxicity in P1 females at 25 mg/kg/day included differences in clinical chemistry and histopathology, both of which pertained to the liver and were consistent with observations at 100 mg/kg/day. The NOAEL for reproductive toxicity was > 100 mg/kg/day; no effects on reproductive outcome were observed at any dosage. The NOAEL for viability and growth of the offspring was 25 mg/kg/day, based on clinical observations of delayed development in pups, and reductions in pup survival and pup body weight during lactation at 100 mg/kg/day.