Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: Combined Repeated Dose Toxicity with Reproduction/Developmental Toxicity Screening study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted in compliance with GLP regulations
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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
Remarks:
Doses / Concentrations:
75, 250, and 750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent vehicle
Positive control:
no
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.
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
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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.

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.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
-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.

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).

-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.

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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups.
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 (see Overall remarks).
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.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The mean final body weight was 6.8% lower for the 750 mg/kg bw/day group males when compared with the control group; the difference did not achieve statistical significance.
The mean liver weight was higher for the 750 mg/kg bw/day group males and females when compared with the control group. The difference in the absolute liver weight and liver weight relative to brain weight was significant (p<0.05 or p<0.01) for the 750 mg/kg bw/day group females, and the difference in mean liver weight relative to body weight was significant (p<0.01) for both the 750 mg/kg bw/day group males and females.
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.
There were no other test substance-related effects on organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In the 750 mg/kg bw/day group, one female was found dead approximately 11 minutes following dose administration on gestation day 21. At necropsy, this female had 16 dead fetuses with no apparent malformations and 1 early resorption in utero; no remarkable gross findings were noted. All other animals survived to the scheduled necropsies.
At the scheduled necropsy, a pale liver was noted for one male and a thickened nonglandular portion of the stomach was noted for another male in the 750 mg/kg bw/day group. Although these findings were noted in single males, the pale liver finding was associated with swelling and fine vacuolation of periportal hepatocytes and the thickened stomach finding was associated with epithelial hyperplasia and hyperkeratosis. Therefore, these macroscopic findings in the 750 mg/kg bw/day group males were attributed to test substance administration. No other test substance related internal findings were observed at any dosage level in females that failed to deliver, the female with total litter loss, or males and females at the scheduled necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All of the 750 mg/kg bw/day group males and females exhibited mild to moderate epithelial hyperplasia and mild to severe hyperkeratosis in the non-glandular portion of the stomach. This consisted of thickening of the squamous epithelial lining with multiple superficial layers of keratin. This change was also observed in a single 250 mg/kg/day group male and 2 females in the 250 mg/kg bw/day group. It was not observed in any of the 75 mg/kg bw/day group or control group animals.
Nine of 12 males from the 750 mg/kg bw/day group had diffuse vacuolation of periportal hepatocytes. These hepatocytes were swollen with abundant fine well-delineated microvesicular cytoplasmic vacuoles. This change was also observed in 9 of the 250 mg/kg bw/day group males, 4 of the 75 mg/kg bw/day group males, and a single control group male. Although the change was present in 1 control group male, a dose-response relationship was present among the test substance-treated males; therefore, the change was considered to be test substance-related. Eight of 12 of the 750 mg/kg bw/day group females exhibited periportal to midzonal hepatocellular vacuolation. This differed from the periportal vacuolation observed in the 750 mg/kg bw/day group males in that the vacuoles ranged from fine (microvesicular) to large with displacement of the nucleus (macrovesicular), and the hepatocytes did not appear swollen. This change was also observed in 8 of the 250 mg/kg bw/day females and 3 of the 75 mg/kg bw/day females, as well as a single 75 mg/kg bw/day male. Although the change was also observed in a single control group male and a single control group female, a dose response relationship was present among the test substance-treated females; therefore, the change was considered to be test substance-related. There were no other test substance-related histologic changes.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity: body weight; liver weights; clinical chemistry
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Toxicity to reproduction
VIABILITY (OFFSPRING)
In the 750 mg/kg bw/day group, the mean number of pups born (12.8 pups/dam) and live litter size on PND 0 (12.7 pups/dam) were lower than the control group (14.8 and 14.5 pups/dam, respectively); an additional female with only 1 implantation site in the 750 mg/kg bw/day group failed to deliver. These differences were attributed to the test substance-related reductions in the numbers of corpora lutea and implantation sites by the author of the study. These effects were also assessed independently by external experts who did not consent to the conclusions of the study director (see Overall remarks).
Mean postnatal survival on PND 0 (relative to the number born; 88.9% per litter) and consequently from birth to PND 4 (87.4% per litter) was reduced compared to the control group (98.3% and 96.0% per litter, respectively). These differences did not achieve statistical significance and were primarily the result of a single female in the 750 mg/kg bw/day group that delivered only 1 pup and had a total litter loss on PND 0. Therefore, in the absence of an increase in the numbers of pups found dead or missing, the reductions in mean postnatal survival were not attributed to test substance administration. Mean postnatal survival during PND 0-1 and 1-4 and the percentage of males at birth in the 750 mg/kg bw/day group were similar to the control group values.
The mean number of pups born, live litter size, and the percentage of males at birth in the 75 and 250 mg/kg bw/day groups were similar to the control group values. Postnatal survival in the 75 and 250 mg/kg bw/day groups was unaffected by test substance administration.

CLINICAL SIGNS (OFFSPRING)
The general physical condition of all F1 pups in this study were unaffected by test substance administration. 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 bw/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 (OFFSPRING)
Mean male and female pup body weights and body weight changes in the 75, 250, and 750 mg/kg bw/day groups were unaffected by test substance administration during PND 1-4. No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING)
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 bw/day groups, respectively. Aside from the absence of milk in the stomach, no other internal findings were noted.

Reproductive effects observed:
not specified

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)

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

There are no experimental data for the test substance itself to fully evaluate the toxicity to reproduction (fertility), but there are data available for the structural analogue 1,6 -Hexamethylene Diacrylate, that are adopted via read-across. A detailed justification for this approach is attached in IUCLID chapter 13. As supporting information, Tripropylene Glycol Diacrylate did not have any effects on the reproductive organs in a subchronic dermal repeated dose study in rats.

1,6-Hexamethylene Diacrylate (HDDA) was tested in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and in compliance with GLP regulations by WIL Research Laboratories for ReachCentrum (2010). The test substance, 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 75, 250, and 750 mg/kg bw/day administered at a dosage volume of 5 mL/kg bw. A concurrent control group of 12 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 11 weeks of age at the beginning of test substance administration. 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.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals and locomotor activity data were recorded for 6 males/group following approximately 28 days of dose administration and for 6 females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1clinical observations and body weights were recorded on PND 1 and 4. Pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology, serum chemistry, and urinalysis [males only]) were performed on 6 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period. F0 females were euthanized on lactation day 5 for females that delivered and post-mating or post-cohabitation day 25 for females that failed to deliver. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups; the liver, stomach, and gross lesions from all animals in all dosage groups were also examined microscopically.

No incidences of mortality or moribundity were attributed to systemic toxicity of the test substance. In the 750 mg/kg bw/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 bw/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 bw/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 bw/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 bw/day group males throughout the study and in the 75, 250, and 750 mg/kg bw/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 in the liver at 75, 250, and 750 mg/kg bw/day. This vacuolar change was also present in 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 bw/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 bw/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 bw/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 bw/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 bw/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 bw/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 bw/day. Reproductive parameters in the 75 and 250 mg/kg bw/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.

Based on reduced mean body weights and body weight gains in the 750 mg/kg bw/day group males and adverse changes in serum chemistry parameters associated with increased liver weights in the 750 mg/kg bw/day group males and females, the NOAEL for systemic toxicity was considered to be 250 mg/kg bw/day.

The equivocal effects on implantation processes observed in the 750mg/kg/day group were not confirmed in a follow-up study, thus the NOAEL for reproductive toxicity of HDDA when administered orally by gavage to Crl: CD(SD) rats was set at 750 mg/kg bw/day.

The follow-up study (WIL-738007, ReachCentrum, 2012)was especially designed to evaluate the effects of oral administration of the test substance on specific reproductive parameters, including mating, fertility, numbers of corpora lutea, implantation sites, resorptions, viable fetuses, and reproductive organ weights that are included in the United States EPA (OPPTS 870.3550) and OECD (Test Guideline 421) Reproduction/Developmental Toxicity Screening Test.

The test substance, 1,6-Hexamethylene Diacrylate (HDDA), in the vehicle (corn oil) was administered orally by gavage once daily to one group of Crl: CD(SD) rats, consisting of 25 males and 25 females. The dosage level was 750 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 25 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test substance administration. 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 gestation day 19 for a total of 34-46 doses; the female with no evidence of mating was dosed through the day prior to euthanasia (7 days following the end of the mating period) for a total of 34 doses. All animals were observed twice daily for mortality and moribundity. Detailed physical examinations, body weights, and food consumption were recorded at appropriate intervals. Complete necropsies were conducted on all animals. For females, the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and selected organs were weighed from all males and females. Selected tissues were collected and preserved for possible future histopathological examination from all animals at the time of necropsy.

All animals survived to the scheduled necropsies. Test substance-related clinical findings were noted in the 750 mg/kg/day group males and females and included salivation or evidence thereof (clear material on various body surfaces) and yellow and red material primarily around the mouth and/or forelimbs. None of the aforementioned clinical findings were considered adverse because they were primarily noted at the time of dose administration and/or approximately 1 hour following dose administration, and were attributed to the irritative properties of the test substance rather than systemic toxicity.

Test substance-related lower mean body weight gains were noted for males in the 750 mg/kg/day group when the overall pre-mating (study days 0-13) and treatment (study days 0-28) periods were evaluated. As a result, mean body weight in this group was 11.3% lower than the control group on study day 28. Corresponding lower mean food consumption was noted for males in the 750 mg/kg/day group following the first week of dosing (study days 0-7), but was similar to the control group thereafter. For females, mean body weights and body weight gains were unaffected by test substance administration during the pre-mating period. During gestation, test substance-related lower mean body weight gains were noted in the 750 mg/kg/day group during the latter portion of gestation (gestation days 14-20), resulting in a lower mean body weight gain when the entire gestation period (gestation days 0-20) was evaluated. However, mean body weights in this group were similar to the control group throughout gestation, and therefore the lower mean body weight gains were not considered to be adverse. Mean food consumption in the 750 mg/kg/day group females was unaffected by test substance administration during the pre-mating and gestation periods and mean gravid uterine weight in the 750 mg/kg/day group was similar to the control group value.

Male and female mating and fertility, male copulation and female conception indices, and the mean number of days between pairing and coitus were unaffected by test substance administration at 750 mg/kg/day.

Test substance-related macroscopic findings at the scheduled necropsies of males and females in the 750 mg/kg/day group consisted of a thickened portion of the nonglandular stomach. This finding was likely a manifestation of local irritation rather than a systemic effect; therefore, it was not considered to be systemically adverse. There were no test substance-related effects on organ weights at 750 mg/kg/day.

Intrauterine survival in the 750 mg/kg/day group was unaffected by test substance administration.

Due to the absence of test substance-related effects on reproductive endpoints, including male and female mating and fertility, male copulation and female conception indices, the mean number of days between pairing and coitus, corpora lutea, implantation sites, resorptions, viable fetuses, and reproductive organ weights in the current study, a dosage level of 750 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.


Short description of key information:
Read-across to CAS 13048-33-4
- oral, rat, 14 days prior to pairing through lactation day 4: NOAEL maternal toxicity = 250 mg/kg bw/day; NOAEL fertility = 750 mg/kg bw/day (OECD TG 422; ReachCentrum SPRL 2010);
- oral, rat, 14 days prior to pairing through gestation day 20: NOAEL toxicity to reproduction = 750 mg/kg bw/day (ReachCentrum SPRL 2012).

Effects on developmental toxicity

Description of key information
- oral, rat, gestation day 6-15: NOAEL maternal toxicity = 250 mg/kg bw/day; NOAEL teratogenicity / embryotoxicity = 250 mg/kg bw/day (Hazleton Labs Inc., 1983, Val. 2).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report. However, only a single dose was tested which showed maternal toxicity and embryotoxic effects.
Principles of method if other than guideline:
The test substance was administered by gavage to 22 pregnant rats from day 6 to day 15 of gestation to evaluate the embryo/fetal toxicity and
terratogenic effects.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 5 weeks upon receipt, ca. 14 weeks at start of mating period
- Weight at study initiation: females ca. 233 g on day 0
- Housing: individually, 1 male with 2 feamles during mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24.5 °C )76°F (1.0 S.D.)
- Humidity (%): 57% (4.8 S.D.)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solution was prepared fresh weekly by mixing a specified amount of the test material with the
vehicle.


VEHICLE
- Justification for use and choice of vehicle (if other than water): none given
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: maximum of 3 weeks
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no data]
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
days 6 through 15 of gestation
Frequency of treatment:
once daily
Duration of test:
animals were sacrificed on day 20 of gestation
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
22 female animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: mortality at 500mg/kg, for details see supporting study
- Rationale for animal assignment: using a table of random permutations
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily


BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 9, 12, 15 and 20 of gestation


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus and ovaries of each dam
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter ]
- Skeletal examinations: Yes: [2/3 per litter ]
- Head examinations: No data
Statistics:
various statistical methods used: National Cancer Institue Package, Box's test for homogeneity of variances, one-way classification analysis of variance
(ANOVA), Dunnett's T-test, one degree of freedom Chi-square test with Yates continuity correction, Fisher's Exact test. All pairwise comparisons were
evaluated at the 5.0% probability (one-tailed) level.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
1 out of 22 animals died, but without apparent relation to treatment. Clinical signs such as rough haircoat (3), hunched posture (2), bloody crusts on nose, mouth, eyes, and/or front paws (1) occured only in single animals - exact number given in brackets - and were likely caused by the irritant properties of the test substance. These signs were not considered indicative of systemic toxicity. No significant differences in body weight and food and water consumption were noted compared to controls.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No statistically significant effects compared to controls were observed. A single pub was diagnosed with cleft palate, and situs inversus was observed in one control pub. Due to the low incidence, these finding are considered incidental. There were no visceral abnormalities. The number of visceral variations was comparable between control and treatment groups (62 of 172 pubs and 73 of 191 pubs, respectively). Sex distribution, fetal viability, fetal body weights and uterine weights were also unaltered by treatment.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Species:
rat
Additional information

In a teratology screening study comparable to OECD 414, the test substance was administered by gavage to 22 pregnant rats from day 6 to day 15 of gestation at a dose level of 250 mg/kg bw/d to evaluate the embryo/fetal toxicity and teratogenic effects (Hazleton Labs Inc., 1983, Val. 2). As determined in a previous maternal tolerance study, 500mg/kg led to mortality in one of six animals. The dose level for the main study was thus set at one half of the lethal dose.

1 out of 22 animals died, but without apparent relation to treatment. Clinical signs such as rough haircoat (3), hunched posture (2), bloody crusts on nose, mouth, eyes, and/or front paws (1) occured only in single animals - exact number given in brackets - and were likely caused by the irritant properties of the test substance. These signs were not considered indicative of systemic toxicity. No significant differences in body weight and food and water consumption were noted compared to controls.

There were no differences in the number of corpora lutea, implantation sites, and pre- and post-implantation loss. Sex distribution, fetal viability, fetal body weights and uterine weights were also unaltered by treatment. There was no treatment related increase in gross malformation and visceral abnormalities or variations.

As a consequence, the NOAEL for maternal toxicity is 250 mg/kg bw/day and the NOAEL for embryotoxicity/teratogenicity is also 250 mg/kg bw/day.

The structural analogue 1,6-Hexamethylene Diacrylate (HDDA) was tested in the same study described above. The substance caused reduced ossification in the offspring, which is considered secondary to the observed maternal toxicity (reduced body weight gain, local irritation / ulceration in the stomach, salivation, increased water consumption, and bad general state). The NOAEL for developmental toxicity was just set at the highest dose of 750mg/kg.

In addition, no effects on the pups were observed in the reproductive screening assay performed with HDDA (WIL, 2010).

Justification for classification or non-classification

Based on the results of the available studies,Tripropylene Glycol Diacrylate does not need to be classified for developmental toxicity or fertilty according to CLP/EU-GHS requirements.