Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 78 days old
- Weight at study initiation:
- Fasting period before study: no
- Housing: upon completion of mating single housing
- Diet (ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): Reverse osmosis-purified (on site) drinking water
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 22.7
- Humidity (%): 37.3 - 45.3
- Air changes (per hr): a minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately twice weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 15, 50, 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: YR1134
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Further matings after unsuccessful attempts: no. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Quadruplicate samples for homogeneity and concentration analyses were collected from the middle of the vehicle control formulation and from the top, middle, and bottom stratum of the test substance formulations prepared for the first week of dose administration. In addition, quadruplicate samples for concentration analyses were collected from the middle stratum of the vehicle control and test substance formulations prepared for the remainder of the study. One set of duplicate samples from each collection was subjected to the appropriate analyses. The remaining set of duplicate samples was stored frozen (approximately -70°C) as back-up. All analyses were conducted by the WIL Research Laboratories, LLC Analytical Chemistry Department using a validated high performance liquid chromatography method using ultraviolet absorbance detection.
The analyzed dosing formulations were within protocol-specified range (100 % ± 5 %) and were homogeneous.
Duration of treatment / exposure:
Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses.
Females received 14 daily doses prior to pairing and were dosed through lactation day 4 for a total of 41-49 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating or post cohabitation day 25) for a total of 39-52 doses.
Frequency of treatment:
once daily
Details on study schedule:
no
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
no data
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Each male and female was also observed for signs of toxicity immediately following dosing and at approximately 1 hour following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly beginning approximately 1 week prior to the initiation of dose administration


BODY WEIGHT: Yes
- Time schedule for examinations:
Males: approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until the day of scheduled euthanasia
Females: approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until the day evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20, and on lactation days 0 (when possible), 1, 4, and 5.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for the female with no evidence of mating was measured on a weekly basis until the scheduled euthanasia


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28 for males and lactation day 5 for females)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 6 animals/sex/group
- Parameters were examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count, Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Hemoglobin Distribution Width (HDW),
Differential leukocyte count: (Percent and absolute): Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC),
Platelet estimatea, Red cell morphology (RBC MORPHOLOGY).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28 for males and lactation day 5 for females)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group
- Parameters were examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Bile acids.


URINALYSIS: Yes
- Time schedule for collection of urine: overnight before the scheduled necropsies (study day 28)
- Metabolism cages used for collection of urine: Yes
- How many animals: 6 males/group
- Animals fasted: Yes
- Parameters were examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Color (COL), Clarity (CLA), Protein (PRO), Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult blood (BLD), Leukocytes (LEU), Nitrites (NIT), Microscopy of sediment.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB assessments were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males) and lactation day 4 (females).
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity:
1. Home cage observations: Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure
2. Handling observations: Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone
3. Open field observations: Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing
4. Sensory observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation
5. Neuromuscular observations: Hindlimb extensor strength, Hindlimb foot splay, Grip strength hind and forelimb, Rotarod performance
6. Physiological observations: Catalepsy, Body temperature, Body weight
7. Locomotor activity (measured automatically using a personal computer controlled system): Data were collected in 5 minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
All females were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia.

PARAMETERS EXAMINED
On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.
Daily observations of survival and any abnormalities in (nursing) behaviour; detailed physical examination and determination of body weights on PND 1 and 4.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE: All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 5 or within 24 hours of total litter loss; the numbers of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating).

GROSS PATHOLOGY: Yes
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss. A gross necropsy was conducted on all animals including the female that was found dead during gestation; the numbers of corpora lutea and implantation sites were recorded and recognizable fetuses were examined externally for gross abnormalities. Necropsies included examination of the external surface, all orifices, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

ORGAN WEIGHTS: from F0 animals at the scheduled necropsies, the following organs were weighed: Adrenal glands, Ovaries with oviducts, Brain, Spleen, Epididymides, Testes, Heart, Thymus gland, Kidneys, Thyroids with parathyroids, Liver.

HISTOPATHOLOGY: Yes
At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin: Adrenal glands (2), Lymph node (Axillary, Mesenteric, Mandibular), Aorta, Bone with marrow (sternebrae), Bone marrow smear ( not placed in formalin), Brain (Cerebrum level 1, Cerebrum level 2, Cerebellum with medulla/pons), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Coagulating glands, Prostate gland, Eyes with optic nerve (2) (in Davidson’s solution), Mandibular salivary glands (2), Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides (2) (fixed in Bouin’s solution), Thymus gland, Thyroids [with parathyroids, if present (2)], Heart, Trachea, Kidneys (2), Urinary bladder, Liver (sections of 2 lobes), Uterus with cervix and vagina (in 10% ammonium sulfide solution), Lungs (including bronchi, fixed by inflation with fixative), All gross lesions.
Microscopic examination was performed on all tissues listed above from all animals in the control and 750 mg/kg/day groups. In addition, the liver, stomach, and all gross lesions from all animals at all dosage levels were examined microscopically.
Postmortem examinations (offspring):
Recognizable fetuses were examined externally for gross abnormalities.
Statistics:
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, clinical pathology values (except gamma glutamyltransferase), pre coital intervals, and continuous FOB data values were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. FOB parameters that yield scalar or descriptive data and histopathological findings in the test substance-treated groups were compared to the control group using Fisher’s Exact test (Steel and Torrie, 1980). Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data, mean litter proportions (percent per litter) of males at birth, and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Reproductive indices:
- Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnancy) / Total No. of Males (Females) Used for Mating x 100
- Male Fertility Index (%) = No. of Males Siring a Litter / Total No. of Males Used for Mating x 100
- Male Copulation Index (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100
- Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating x 100
- Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100


Offspring viability indices:
- Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups on PND 0
- Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) / No. of Litters Per Group x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) / No. of Litters Per Group

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Behaviour-related clinical findings, including wiping mouth on the cage floor and/or walls, excessive pawing of the cage floor and/or walls, and wiping mouth in bedding material following dosing (females only), were noted for the majority of the males and females in the 250 and 750 mg/kg bw/day groups throughout the treatment period. Because these findings were primarily limited to the time of dose administration and generally did not persist to approximately 1 hour following dose administration, they were attributed to the irritative properties of the test substance and were not considered adverse. Other clinical findings attributed to test substance administration included salivation-related findings (salivation prior to or at the time of dose administration and clear material around the mouth) and red material around the mouth for the majority of the animals in the 250 and 750 mg/kg bw/day groups. These findings were noted at the time of and/or approximately 1 hour following dose administration throughout the treatment period; the salivation related findings were also sporadically noted in the 75 mg/kg bw/day group animals and were likely signs of taste aversion to the test substance, which were not considered adverse.
Other clinical findings noted at the daily examinations, at the time of dose administration, or approximately 1 hour following dose administration, including hair loss on various body surfaces, occurred infrequently and/or at similar frequencies in the control group, and were not attributed to test substance administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the 750 mg/kg bw/day group, a single female was found dead prior to evidence of parturition on gestation day 21. This female was noted with clinical findings common to the majority of the other females in this group, including wiping mouth on the cage floor and/or walls, excessive pawing of the cage floor and/or walls, wiping mouth in bedding material following dosing, and wet and clear material around the mouth; these findings were noted at the time of and/or approximately 1 hour following dose administration. In addition, this female was gasping approximately 2 minutes following dose administration on the day of death, and was subsequently found dead approximately 11 minutes after dose administration. Upon macroscopic and microscopic examination, the cause of death of the female was undetermined. Based on these findings, the death of this female was likely attributable to the dose administration procedures and unlikely related to systemic toxicity of the test substance. With the exception of a female in the 750 mg/kg bw/day group that was euthanized on lactation day 0 due to total litter loss, all other males and females at all dosage levels survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
-Males: A test substance-related, significantly (p<0.01) lower mean body weight gain was noted in the 750 mg/kg bw/day group males following the first week of dose administration (study days 0-7) followed by a slightly (not statistically significant) lower mean body weight gain during study days 7-13. Mean body weight changes for these males were comparable to the control group during the remainder of the treatment period (study days 13-28). A mean body weight loss (13 g) was noted for the 750 mg/kg bw/day group males during study days 21-28; however, mean body weight losses were noted across all dosage groups, including the control group, as a result of being food-fasted prior to blood collection on study day 27-28. Due to the initial reductions in mean body weight gains, mean body weight gain in the 750 mg/kg bw/day group males was significantly (p<0.01) lower during the overall pre-mating period (study days 0-13) compared to the control group, and mean body weight was 6.8 % lower (not statistically significant) than the control group value on study day 28.
Mean male body weights and body weight gains in the 75 and 250 mg/kg bw/day groups were similar to the control group throughout the treatment period.

-Females: Mean female body weights and body weight gains in the 75, 250, and 750 mg/kg bw/day groups were similar to the control group during the pre-mating period (study days 0-13). Mean body weights and body weight gains in the 75, 250, and 750 mg/kg bw/day groups were generally similar to the control group throughout gestation. During lactation, mean body weights and body weight gains in the 75, 250, and 750 mg/kg bw/day group were unaffected by test substance administration.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean male food consumption, evaluated as g/animal/day and g/kg/day, in the 750 mg/kg bw/day group was slightly reduced during the first week of dose administration (study days 0-7). The difference from the control group achieved significance (p<0.05) on a g/kg/day basis only and corresponded to a reduced mean body weight gain during this interval. Mean food consumption in this group was similar to the control group during study days 7-13. Mean male food consumption in the 75 and 250 mg/kg bw/day groups was similar to the control group during the pre-mating period (study days 0-13).

Mean food consumption in the 75, 250, and 750 mg/kg bw/day groups was similar to the control group throughout the pre-mating period (study days 0-13).
Mean food consumption in the 75, 250, and 750 mg/kg bw/day groups was unaffected by test substance administration during gestation. Significantly (p<0.05) higher mean food consumption was noted in the 750 mg/kg bw/day group during gestation days 7-11 (g/animal/day only) and when the overall gestation period (gestation days 0-20; g/kg/day only) was evaluated. However, due to the small magnitude of these differences compared to the control group and the lack of a concurrent similar effect on mean body weight gain, the changes were not attributed to test substance administration.
Mean food consumption in the in the 75, 250, and 750 mg/kg bw/day groups was unaffected by the test substance during lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
see details in the section "repeated toxicity"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details in the section "repeated toxicity"
Urinalysis findings:
no effects observed
Description (incidence and severity):
see details in the section "repeated toxicity"
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see details in the section "repeated toxicity"
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details in the section "repeated toxicity" for the global results.
Regarding the sexual organs: The following organ weight differences were significant (p<0.05 or p<0.01) when compared to the control group but were considered to be a result of the test substance related effect on final body weight: higher left and right testis weight relative to body weight for the 750 mg/kg bw/day group males. The mean and individual absolute testis weights and testis weights relative to body weight for the 750 mg/kg bw/day group males were all within the historical control data range for Crl:CD(SD) rats of a similar age.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See details in the section "repeated toxicity" for the global results.
No effects were observed on sexual organs.
Neuropathological findings:
no effects observed
Description (incidence and severity):
see details in the section "repeated toxicity"
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details in the section "repeated toxicity" for the global results.
No effects were observed on sexual organs.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. None of these differences were statistically significant.
Mean gestation lengths in the 75, 250, and 750 mg/kg bw/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
The mean numbers of corpora lutea and implantation sites in the 750 mg/kg bw/day group females (17.4 corpora lutea and 13.7 implantation sites) were slightly lower compared to the concurrent control group (19.8 corpora lutea and 15.6 implantation sites); the mean number of implantation sites was also lower compared to the historical control mean (15.4 implantation sites; version 2.10). The differences from the concurrent control group were not statistically significant and were primarily the result of a single female that had 11 corpora lutea and 1 implantation site and subsequently delivered 1 pup on gestation day 24; this female had a total litter loss on lactation day 0. However, a female in the 750 mg/kg bw/day group also had only 1 implantation site (1 early resorption) and was excluded from the group mean since it failed to deliver at least 1 pup. The occurrence of high dose group females with only 1 implantation site (2 of 11 gravid females; 18.2%) was much greater than the historical occurrence of 1 implantation site in control group females from developmental and reproductive toxicity studies conducted at WIL Research Laboratories, LLC (4 of 2116 gravid females from 93 data sets over a period of 2005-2010; 0.19%). Therefore, because the frequency of gravid females with only 1 implantation site in the 750 mg/kg bw/day group greatly exceeded the rate observed historically in control group animals, these changes were attributed to test substance administration by the author of the study report. These effects were also assessed independently by external experts who did not consent to the conclusions of the study director.
The mean numbers of corpora lutea and implantation sites in the 75 and 250 mg/kg bw/day groups and the mean numbers of unaccounted-for sites in the 75, 250, and 750 mg/kg bw/day groups were similar to the control group values.

Details on results (P0)

Global assessment on reproductive performance :
Mean reproductive indices were generally comparable to control group values at all dosage levels. However, 2 of 11 gravid females in the 750 mg/kg bw/day group were noted with only 1 implantation site; 1 female delivered 1 pup and subsequently had a total litter loss shortly thereafter on PND 0 whereas the other female had 1 early resorption and failed to deliver. As a result of the single female in the 750 mg/kg bw/day group that delivered 1 pup, a lower mean number of implantation sites, number of pups born, and liver litter size PND 0 were noted in this group. Although these differences were not statistically significant compared to the control group, the presence of only 1 implantation site is uncommon in laboratory rats. The occurrence of high dose group females with only 1 implantation site (2 of 11 gravid females; 18.2%) was much greater than the historical occurrence of 1 implantation site in control group females from developmental and reproductive toxicity studies conducted at WIL Research Laboratories, LLC (4 of 2116 gravid females from 93 data sets over a period of 2005-2010; 0.19%). Therefore, because the frequency of gravid females with only 1 implantation site in the 750 mg/kg bw/day group greatly exceeded the rate observed historically in control group animals, a test substance-related effect on the process of implantation was suspected in these 2 females in the 750 mg/kg bw/day group by the author of the study. In addition, the mean number of corpora lutea in the 750 mg/kg bw/day group was lower compared to the control group.
Therefore, under the conditions of this screening study, a test substance-related effect on the process of implantation was presumed in the 750 mg/kg bw/day group females by the study director. Based on these results, a dosage level of 250 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of HDDA when administered orally by gavage to Crl:CD(SD) rats.

However, the results described in the present study were also assessed and discussed by an external expert which was also taken into consideration for final assessment of the study results.

The average number of CL was 17.4 at 750 mg/kg bw vs. 19.8 in the control. The historical average of the test facility is 16.6, and the min/max range from 223 control groups is 13.8-18.8, indicating that the 750 mg/kg bw-value is higher than the historical average and well in the historical range while the concurrent control value is distinctly above the historical range. The reason for these higher numbers is likely to be difficulties in getting an accurate count of CL graviditatis after birth, because they are difficult to distinguish from cyclic CL of the postpartum estrous. Thus, this parameter should be assessed with some cautiousness.

The average number of implants was 13.7 at 750 mg/kg bw vs. 15.6 in the control. The historical average of the test facility is 15.4, and the min/max range from 223 control groups is 12.6-17.1, indicating that the 750 mg/kg bw-value is still in the historical range of the test facility, though on the lower end.

One female in the high-dose group delivered only one pup which died shortly after parturition. It had 11 CL in the right ovary and at least one implant in the right uterine horn, and neither CL in the left ovary nor implants in the left uterine horn. It took 9 days until copulation was detected for this female which is distinctly longer than for the rest of the animals in this study. The test facility brings forward the argument, that not the lack of corpora lutea but the low number of implants for this female is of toxicological concern. However, a potential HDDA-related effect on any of the processes between ovulation and implantation would best be indicated by changes in pre-implantation loss, i.e. a changed difference between numbers of ovulated egg cells (i.e. = count of CL graviditatis) and implantations.
It is obvious that the individual pre-implantation loss data are highly variable, ranging between 0 – 12 in all treated groups and control. The respective female was within that range although it had a single-horn pregnancy.
Altogether, this female took unusually long to become pregnant, had no CL on the left ovary (in the absence of other findings), had a single-horn pregnancy, and had 10 pre-implantation losses, which were, however, in the range of all groups including control. An association of this variety of findings to the treatment should at least be assumed as equivocal.

Another female in the high-dose group failed to deliver but had one early resorption, detected by visual inspection. The proof that there were really no other early resorptions by staining the uterus with ammonium sulfide was not conducted. The ammonium sulfide stain might have had identified more implants, and after rinsing the uterus with water it would still have been available for histopathology. Thus, it cannot be decided finally, whether this animal suffered from an increased pre-implantation or post-implantation loss.

In conclusion, although both females apparently suffered from the same finding („one-implantation pregnancy“) there is no evidence that the cause for this finding is the same in both animals when one looks at all surrounding circumstances. There is not enough information to decide finally whether these findings were treatment-related or not. From the available study data, there is no evidence at all that the implantation process was influenced adversely. The comparison to historical data for the incidence of „one implantation site“ as it was done in the report may be misleading because the study-related incidence of 18.2% was based on 12 animals and the historical incidence was based on several thousand animals. The historical rate does not tell anything about the distribution of such findings among studies. So it may well be that 1-2 control animals with „one implantation site“ have been seen in any of the historical studies, although it cannot be proven.

Taken all these arguments into account, the NOAEL for reproductive toxicity of HDDA after gavage administration to Crl:CD(SD) rats was considered to be 750 mg/kg bw/day.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
(reproductive performance)
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition of all F1 pups in this study were unaffected by test substance administration.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pups (litters) that were found dead numbered 4(3), 5(5), 3(2), and 2(2) in the control, 75, 250, and 750 mg/kg/day groups, respectively. Three (3), 3(1), 1(1), and 1(1) pups (litters) in these same respective groups were missing and presumed to have been cannibalized.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean male and female pup body weights and body weight changes in the 75, 250, and 750 mg/kg/day groups were unaffected by test substance administration during PND 1-4.
No statistically significant differences from the control group were noted.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy of dead pups only:
The numbers of pups (litters) found dead during PND 0-4 numbered 4(3), 5(5), 3(2), and 2(2) in the control, 75, 250, and 750 mg/kg/day groups, respectively.
Aside from the absence of milk in the stomach, no other internal findings were noted.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Results of Reproductive Performance

 

Dosage Level (mg/kg bw/day)

WIL HCa

Parameter

0

75

250

750

Mean (Range)

Male Mating Index (%)

100.0

100.0

91.7

100.0

96.7 (84.0-100.0)

Female Mating Index (%)

100.0

100.0

91.7

100.0

98.2 (86.7-100.0)

Male Fertility Index (%)

100.0

83.3

91.7

91.7

91.0 (60.0-100.0)

Female Fertility Index (%)

100.0

83.3

91.7

91.7

93.2 (60.0-100.0)

Male Copulation Index (%)

100.0

83.3

100.0

91.7

94.4 (71.4-100.0)

Female Conception Index (%)

100.0

83.3

100.0

91.7

94.9 (65.2-100.0)

Pre-Coital Interval (days)

2.8

2.9

2.3

3.4

3.0 (1.8-5.5)

a= WIL historical control data (version 2.10)

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, a test substance-related effect on the process of implantation was noted in the 750 mg/kg/day group females. Based on these results, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of HDDA when administered orally by gavage to Crl:CD(SD) rats. Based on reduced mean body weights and body weight gains in the 750 mg/kg/day group males and adverse changes in serum chemistry parameters associated with increased liver weights in the 750 mg/kg/day group males and females, the NOAEL for systemic toxicity was considered to be 250 mg/kg/day. In the absence of any effects on the general physical condition of the F1 pups, the NOAEL for neonatal toxicity was 750 mg/kg/day.
Executive summary:

The test substance, 1,6-Hexamethylene Diacrylate (HDDA), in the vehicle, corn oil, was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 12 males and 12 females. Dosage levels were 0, 75, 250, and 750 mg/kg/day

administered at a dosage volume of 5 mL/kg. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 4 for a total of 41-49 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39-52 doses.

No incidences of mortality or moribundity were attributed to systemic toxicity of the test substance. In the 750 mg/kg/day group, a single female was found dead following dose administration on gestation day 21. However, due to the lack of evidence of test

substance-related toxicity in this female, as well as the time of mortality relative to dose administration (11 minutes following dosing), this single mortality was not attributed to systemic toxicity of the test substance. All other animals in all dosage groups survived to

the scheduled necropsies. Test substance-related clinical findings were noted in the 250 and 750 mg/kg/day group males and females and included wiping mouth on cage floor and/or walls, excessive pawing of cage floor and/or walls, wiping mouth in bedding

material following dosing (females only), salivation-related findings, and red material around the mouth. The salivation-related findings were also occasionally noted in the 75 mg/kg/day group animals. Because the aforementioned clinical findings were noted at the time of dosing and/or approximately 1 hour following dose administration, they were attributed to the irritative properties of the test substance and not considered adverse. In the 750 mg/kg/day group males, test substance-related lower mean body weight gain and food consumption were noted during the pre-mating period, resulting in mean male body weight that was 6.8% lower than the control group on study day 28. Mean body weights, body weight changes, and food consumption were unaffected by test substance administration in the 75 and 250 mg/kg/day group males throughout the study and in the 75, 250, and 750 mg/kg/day group females during the pre-mating, gestation, and lactation periods. No test substance-related effects were noted during the FOB or locomotor activity evaluations at any dosage level.

Test substance administration was associated with micro- to macrovesicular vacuolar change within the liver at 75, 250, and 750 mg/kg/day. This vacuolar change was also present within the liver of 3 control group animals (2 males and 1 female). The change in

the control group animals and all test substance-treated animals was minimal to mild and there was no evidence of cellular or tissue damage; therefore, the change was not considered to be an adverse effect. At 750 mg/kg/day, higher liver weights, higher serum

bile acid values, and higher urea nitrogen values were noted in both males and females, a higher total bilirubin value was noted in males, and higher ALT, cholesterol, triglycerides, calcium, and phosphorous values were noted in females. At 250 mg/kg/day, a higher ALT value was noted for females; however, this difference was not statistically significant and was not considered to be an adverse effect. Test substance administration at dosage levels of 250 and 750 mg/kg/day to males and females was associated with squamous epithelial hyperplasia and hyperkeratosis in the non-glandular stomach. This was a manifestation of local irritation rather than a systemic effect; therefore, it was not considered to be systemically adverse.

Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance administration at all dosage levels. Non-statistically significantly lower postnatal survival on PND 0 (relative to the number born) and from birth to PND 4 and a lower mean number of implantation sites, number of pups born, and live litter size on PND 0 were noted in the 750 mg/kg/day group due to a single female that delivered 1 pup and had a total litter loss on PND 0. In addition to this female, another female in the 750 mg/kg/day group was noted with only 1 implantation site (1 early resorption) and failed to deliver. The mean number of corpora lutea was also lower (not statistically significant) in the 750 mg/kg/day group compared to the control group. The mean number of unaccounted-for sites and the percentage of males per litter were unaffected by dose administration at 750 mg/kg/day. Reproductive parameters in the 75 and 250 mg/kg/day groups and mean pup body weights and body weight gains at all dosage levels were unaffected by dose administration. No test substance-related clinical findings were noted for the F1 pups and there were no remarkable macroscopic findings in the F1 pups at the scheduled necropsy at any dosage level.

Under the conditions of this screening study, a test substance-related effect on the process of implantation was noted in the 750 mg/kg/day group females. Based on these results, a dosage level of 250 mg/kg/day was considered to be the NOAEL for reproductive toxicity of HDDA when administered orally by gavage to Crl:CD(SD) rats. Based on reduced mean body weights and body weight gains in the 750 mg/kg/day group males and adverse changes in serum chemistry parameters associated with increased liver weights in the 750 mg/kg/day group males and females, the NOAEL for systemic toxicity was considered to be 250 mg/kg/day. In the absence of any effects on the general physical condition of the F1 pups, the NOAEL for neonatal toxicity was 750 mg/kg/day.