Registration Dossier
Registration Dossier
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EC number: 241-527-8 | CAS number: 17527-29-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Link to relevant study records
- Endpoint:
- one-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of 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)
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
- 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
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system 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 - 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, homogenity, 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, homogenity, 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 homogenity, 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 - Dose / conc.:
- 1 mg/kg bw/day (actual dose received)
- Remarks:
- (adjusted for purity)
- Dose / conc.:
- 5 mg/kg bw/day (actual dose received)
- Remarks:
- (adjusted for purity)
- Dose / conc.:
- 25 mg/kg bw/day (actual dose received)
- Remarks:
- (adjusted for purity)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- (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
- 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. - Oestrous 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 (%) - 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:
- effects observed, treatment-related
- Other effects:
- effects observed, treatment-related
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- 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.
- 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
- 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.
- Reproductive effects observed:
- not specified
- 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 P1 generation 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 post-mortem 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 P1 females 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 P1 or 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. F1 pups 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.- Endpoint:
- one-generation reproductive toxicity
- Type of 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: 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.
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
From the toxicokinetic assessment as well as from results of the hydrolysis study it is known that the target chemical 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate becomes hydrolysed in vivo to the alcohol and the acrylic acid moiety. Whereas acrylic acid is intensively investigated for health endpoints and does not show reproductive toxicity, data was required for the corresponding alcohol, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol (the source chemical), thereby being in a position to conclude upon teratogenicity and reproductive toxicity of the target chemical.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both, source chemical as well as target chemical are of high purity (>90%) and it is expected that study results were not compromised by any impurities being present, considering, that typical impurities do only differ by chain length of the fluorinated part of the alcohol moiety.
3. ANALOGUE APPROACH JUSTIFICATION
Based on the TK assessment as well as from results of the hydrolysis study the target chemical 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate breaks down in vivo to the alcohol and the acrylic acid moiety. The resulting alcohol, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol (the source chemical), thereby is being formed in vivo rapidly in the gastrointestinal tract and becomes decisive as metabolite for systemic toxicity as distributed in the organism. Hence, using teratogenicity and reproductive toxicity data from the metabolite 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol for read-across to 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate (target chemical) is justified.
4. DATA MATRIX
Log Pow values are not significantly different (4.54 for source chemical versus 5.07 of target chemical). However, the water solubility of the source chemical is by two orders of magnitude higher (18.8 mg/L) than that of the target chemical (0.185 mg/L) suggesting that distribution in vivo will be quicker reaching reproductive organs, and thus the source chemical may be seen as a worst case surrogate for the target chemical, which requires metabolisation prior to distribution in vivo. In consequence, the data for teratogenicity and reproductive toxicity of the source chemical may be seen as a worst case surrogate for the target chemical 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate, justifying the read-across approach. - Reason / purpose for cross-reference:
- read-across: supporting information
- 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
- 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: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- 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'
- 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
- 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'
- Reproductive effects observed:
- not specified
- 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 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol (the source chemical) 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 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol (the source chemical) 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 live-born 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.
Referenceopen allclose all
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 was associated with correlative test substance-related microscopic findings in the liver and was 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.
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.
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 significant 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 live-born 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 live-born 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 non-surviving rats were non-specific and, therefore, there were no consistent microscopic findings in these rats that could be clearly correlated with a cause of death or moribundity.
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 [pre-culling]/number of live-born pups on postpartum day 1) and the lactation index (number of live pups on postpartum day 21/number of live-born pups on postpartum day 5 [post-culling]). 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 pre-culling, and post-culling, 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.
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). |
||||||
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 100 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- mouse
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Short description of key information: According to the Annex VIII of REACH (8.7.1), screening test for reproductive/developmental toxicity does not need to be conducted, as a pre-natal developmental toxicity study is available. A one generation reproductive study on analogue substance FT-OH (CAS 647-42-7) through oral route (gavage) on mice is available, showing no effects on fertility at a dose of 100 mg/kg/day. NOAEL fertility (oral, gavage, subchronic, mice): >100 mg/kg/day
These findings are supported by an earlier one generation reproductive toxicity study with rats on the same analogue substance FT-OH, showing no effects on fertility or other reproductive indices in the absence of maternal toxicity.
As a result no effects on reproductive indices are seen in surrogate test reports on the corresponding alcohol, the direct metabolite of the reference substance. In conclusion, no further tests to assess reproduction effects of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate are required.
Effects on developmental toxicity
Description of key information
The maternal and developmental NOAEL for 6:2 FTOH is 25 mg/kg/day.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of 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: 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.
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
From the toxicokinetic assessment as well as from results of the hydrolysis study it is known that the target chemical 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate becomes hydrolysed in vivo to the alcohol and the acrylic acid moiety. Whereas acrylic acid is intensively investigated for health endpoints and does not show reproductive toxicity, data was required for the corresponding alcohol, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol (the source chemical), thereby being in a position to conclude upon teratogenicity of the target chemical.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both, source chemical as well as target chemical are of high purity (>90%) and it is expected that study results were not compromised by any impurities being present, considering, that typical impurities do only differ by chain length of the fluorinated part of the alcohol moiety.
3. ANALOGUE APPROACH JUSTIFICATION
Based on the TK assessment as well as from results of the hydrolysis study the target chemical 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate breaks down in vivo to the alcohol and the acrylic acid moiety. The resulting alcohol, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol (the source chemical), thereby is being formed in vivo rapidly in the gastrointestinal tract and becomes decisive as metabolite for systemic toxicity as distributed in the organism. Hence, using teratogenicity data from the metabolite 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol for read-across to 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate (target chemical) is justified.
4. DATA MATRIX
Log Pow values are not significantly different (4.54 for source chemical versus 5.07 of target chemical). However, the water solubility of the source chemical is by two orders of magnitude higher (18.8 mg/L) than that of the target chemical (0.185 mg/L) suggesting that distribution in vivo will be quicker reaching reproductive organs, and thus the source chemical may be seen as a worst case surrogate for the target chemical, which requires metabolisation prior to distribution in vivo. In consequence, the data for teratogenicity of the source chemical may be seen as a worst case surrogate for the target chemical 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate, justifying the read-across approach. - Reason / purpose for cross-reference:
- read-across source
- Dose descriptor:
- NOAEL
- Effect level:
- 25 mg/kg bw/day (nominal)
- Basis for effect level:
- other: maternal toxicity
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 25 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- skeletal malformations
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The study and the conclusions which are drawn from the study on the source chemical do fulfil the quality criteria (validity, reliability, repeatability).
There was no evidence of either maternal or developmental toxicity at 5 or 25 mg/kg/day. Therefore, under the conditions of the current study on the source chemical, the no-observed-adverse-effect level (NOAEL) for both the dam and the fetus was 25 mg/kg/day. This value may be considered as worst case value for the target substance.
Reference
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 25 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
No developmental toxicity data are available for the test substance. A prenatal developmental toxicity study in rats with 6:2 FTOH was used as a read across to fulfil the data requirement for the test substance. The underlying hypothesis for the read-across between the test substance and 6:2 FTOH is that the source chemical is the primary initial metabolite of the target chemical, and both substances will therefore undergo identical distribution, metabolism and elimination from this point forward. Any toxicity expressed by the source chemical in toxicological studies including, but not necessarily limited to, 90-day sub-chronic oral toxicity in the rat and pre-natal developmental toxicity, will be directly representative of the toxicity of the target chemical (see additional details in the attached document).
Pregnant female rats were administered the 6:2 FTOH via gavage at doses of 0, 5, 25, 125, or 250 mg/kg on gestation days 6 - 20 in a prenatal developmental toxicity study according to OECD Guideline 414. The no-observed-adverse-effect level (NOAEL) for both the dam and the fetus was 25 mg/kg/day. Maternal toxicity, evidenced as changes in weight gain, and signs of developmental toxicity, evidenced as skeletal variations were observed at ≥125 mg/kg. There was no evidence of either maternal or developmental toxicity at 5 or 25 mg/kg/day. Justification for selection of effect on developmental toxicity via oral route: NOAEL is based on read across to a rat developmental toxicity study with 6:2 FTOH (OECD Guideline, GLP study).
Justification for classification or non-classification
Although no data are available for the test substance itself, data are available for the read-across material, 6:2 FTOH. 6:2 FTOH has been evaluated in a developmental toxicity study in rats. Effects on offspring were mostly limited to body weight decrements and/or mortality at maternally toxic doses. Although signs of developmental toxicity were observed at higher doses, the substance was not embryolethal or teratogenic in rats and produced no effects at doses that were not maternally toxic. In addition also in two one generation reproductive toxicity studies using 6:2 FT-OH no effects on reproductive indices were observed in rats and mice, respectively. Therefore, 6:2 FTOH was not uniquely toxic to the offspring or impaired fertility. Based on these considerations, the target substance does not need to be classified for reproductive/developmental toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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