Hexamethylene diacrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- OECD 422; rats; oral; NOAEL maternal toxicity = 250 mg/kg bw/day; NOAEL fertility = 750 mg/kg bw/day (ReachCentrum, 2010).
- oral, rat, 14 days prior to pairing through gestation day 20: NOAEL toxicity to reproduction = 750 mg/kg bw/day (ReachCentrum, 2012).

Read-across to CAS No. 42978-66 -5

- OECD 422; rats; oral; NOAEL (reproduction) >/= 375 mg/kg (ReachCentrum, 2019b)

- OECD 443; rats; oral; NOAEL (reproduction) >/= 100 mg/kg (ReachCentrum, 2019a)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system 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%). However, in a follow-up study (see WIL-738007, included in this dossier) with larger group sizes, no test substance related effects were noted on the mean numbers of corpora lutea and implantation sites for females at a dosage level of 750 mg/kg/day. Therefore, because the results from the current study were not replicated, the higher than expected frequency of gravid females with only 1 implantation site in the 750 mg/kg/day group in the current study were not attributed to test substance administration. 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

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 lower numbers of corpora lutea and implantation sites, but were not considered to be test substance-related because these results were not reproduced at the same dosage level in the follow-up study (see WIL-738007) with larger group sizes. 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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: high dose

Reproductive effects observed:

not specified

Results of Reproductive Performance

	Dosage Level (mg/kg bw/day)				WIL HC^a
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)

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Aug 2018 - 14 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD guidance document supporting OECD test guideline 443 on the extended onegeneration reproductive toxicity test, No. 151

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : 10 weeks

- Basis for dose level selection :
The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats. In this preliminary study, animals were dosed with 0, 40, 125 and 375 mg/kg/day. From dose 125 mg/kg/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet accumulation in the male kidneys at 375 mg/kg/day. Based on the severe findings in the stomach, a parental local NOAEL was established of 40 mg/kg/day in this study. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg/day was selected.

- Inclusion/exclusion of extension of Cohort 1B : Inclusion

- Termination time for F2 : not applicable

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Exclusion

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Exclusion

- Route of administration : The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6-7 week; (F1) 3 wks
- Weight at study initiation: (P) Males: 136 and 187 g; Females: 112 and 153 g; (F1) Males: mean 50 g; Females: 49 g
- Fasting period before study: During motor activity measurements, F0- female animals had no access to food for a maximum of 2 hours. During motor activity measurements, F0-female animals had no access to water for a maximum of 2 hours.
- Housing:
On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III)
During the post-mating phase, males were housed in their home cage (Macrolon type IV) with a maximum of 5 males/cage). Females were individually housed in Macrolon plastic cages (type III).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F0-females were housed individually in a Hi-temp polycarbonate cage
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 45 to 67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly as a solution, formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 2, 6, 20 mg/mL
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (type III).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

see table 1

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

25 (F0) / 20 (F1)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats and in an attempt to produce graded responses to the test item. In this preliminary study, aninmals were dosed with 0, 40, 125 and 375 mg/kg bw/day. From dose 125 mg/kg bw/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet
accumulation in the male kidneys at 375 mg/kg/day. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg bw/day was selected. No (severe) clinical symptoms and effects on body weight and food consumption were noted (i.e. the general health of the animals was good). The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL TESTS – F0-Generation
Functional tests were performed on the selected 5 females during Week 10 of treatment. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

CLINICAL PATHOLOGY - F0-Generation and Cohort 1A animals
Sample collection:
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected from the retro-orbital sinus under anaesthesia using isoflurane in the animal facility.
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.

Oestrous cyclicity (parental animals):

Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.

Sperm parameters (parental animals):

Parameters examined in [F0, Cohort 1A] male parental generations:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:

F1-Generation until Weaning (PND 21):

Mortality/Moribundity Checks
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

Body Weights
Live pups were weighed individually on PND 1, 4, 7, 13 and 21. For animals of Cohort Surplus, a terminal weight was recorded on the day of scheduled necropsy.

Sex
Sex was externally determined for all pups on PND 1 and 4 (sex determination not noted for Day 13)

Anogenital Distance
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Clinical Pathology
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation for determination of thyroid hormone.

F1-Generation from Weaning (PND 21) onwards:

Mortality/Moribundity Checks – Cohorts 1A, 1B and 1C
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations– Cohorts 1A, 1B and 1C
Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and 0-30 minutes after dosing.

Body Weights – Cohorts 1A, 1B and 1C
Animals were weekly weighed individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A and 1B, a terminal weight was recorded on the day of scheduled necropsy.

Food Consumption– Cohorts 1A, 1B and 1C
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

Water Consumption – Cohorts 1A, 1B and 1C
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Vaginal Patency – Cohorts 1A, 1B and 1C
Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.

Balanopreputial Separation – Cohorts 1A, 1B and 1C
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.

Stage of Estrus Determination – Cohorts 1A, 1B and 1C
Estrous stages were determined by examining the cytology of vaginal lavage sample, taken on the day of scheduled necropsy.

Estrous Cycle Determination – Cohort 1A
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods. During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

Clinical Pathology - F1- animals of Cohort Surplus on PND 22
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group), if possible for determination of Thyroid Hormone. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

Clinical Pathology – Cohort 1A
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy for
- Hematology: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets, Reticulocytes (absolute), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical Chemistry: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT),Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)
- Thyroid hormone: Thyroxine (T4), Thyroid Stimulating Hormone (TSH)
Urinalysis: Specific gravity, White blood cells (WBC-sed.), Clarity Red blood cells (RBC-sed.), Colour Casts, pH, Epithelial cells, Blood Crystals, White blood cells (WBC), Bacteria, Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 μm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:
T-cells, T-helper cells, T-cytotoxic cells, B-cells, NK-cells, Ratio T-helper cells/ T-cytotoxic cells (Th/Tc)
The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

Postmortem examinations (parental animals):

SACRIFICE- F0 Generation
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies:
Males (which sired and failed to sire): After successful mating and a minimum of 10 weeks of treatment.
Females which delivered: LD 23-25.
Females which failed to deliver:
With evidence of mating: Post-coitum Days 25-27 (102,105, 108, 111, 122, 144, 179, 194, 200).
Without evidence of mating: 24 days after the last day of the mating period (Nos. 112, 168)
Females with total litter loss (No. 126): Within 24 hours after the last pup was found dead or missing.

Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.

GROSS NECROPSY- F0 Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no
macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS - F0 Generation
The organs identified in Table 5 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios were calculated. Representative samples of the tissues identified in Table 5 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

Postmortem examinations (offspring):

SACRIFICE -F1 Generation until weaning
Unscheduled Deaths– F1-Generation
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The
stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation.

GROSS NECROPSY
See table 3 for details

Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 89-95. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Cohort 1B
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort 1C
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded. All
animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
Cohort 1A
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.
Cohort 1B
In addition to the procedures described above, the reproductive organs of all Cohort 1B animals were processed to block stage.

Statistics:

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set (at least 3 groups) was compared using an overall Kruskal-Wallis.
Incidence:
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

see table 4

Offspring viability indices:

see table 4

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item related clinical signs were noted up to treatment with 30 mg/kg bw/day. No findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among animals of the 100 mg/kg bw/day dose group during the first 8 weeks of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than being a sign of systemic toxicity. Other clinical signs noted during the treatment period occurred within the range of
background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No test item-related mortality occurred during the study period. One female of the 10 mg/kg bw/day group was euthanized on Lactation Day 1, as she had a total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment up to 100 mg/kg bw/day. A trend towards decreased body weights and body weight gain may appeared in males at 100 mg/kg bw/day up to Week 1 of the mating period. A statistical significant decrease in bodyweight was observed at 100 mg/kg bw/day in Week 8 of treatment. The statistical significant increase in body weight gain at 30 mg/kg bw/day in Week 3 of mating was considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight in animals treated up to 100 mg/kg bw/day was similar to the control level over the treatment period. Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters. Statistically significant lower white blood cell count (WBC) was noted in females at 10 and 100 mg/kg bw/day, which was likely the result of lower lymphocytes levels observed in these animals. In absence of a dose response and as values remained within normal range, these changes were considered unrelated to treatment with the test item. Any other statistically significant changes in haematology parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response. Coagulation parameters of treated rats were considered not to have been affected by treatment up to 100 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes were observed in treated males at 30 and/or 100 mg/kg bw/day:
Increase in urea at 100 mg/kg/day (1.16x, 4.3 mmol/L).
• Increase in sodium at 30 and 100 mg/kg bw/day (1.01x (143.3 mmol/L) and 1.02x (143.8 mmol/L), respectively).
• Increase in potassium at 100 mg/kg bw/day (1.07x, 4.02 mmol/L)
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment with the test item. Serum levels of TSH and T4 in F0-males and -females were not affected by treatment up to 100 mg/kg bw/kg.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis was not affected by treatment with the test item up to 100 mg/kg bw/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. Motor activity (total movements and ambulations) was (non-statistically) slightly increased in high dose animals. This slight increase was considered due to one female, for which a level of total movements and ambulations was recorded.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic alterations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Histopathological examination of reproductive tissues revealed no evidence of a treatment-related effect on reproduction.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were unaffected by treatment with the test item. All females had regular cycles of 4 to 5 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility, concentration and morphology parameters were unaffected by treatment up to 100 mg/kg bw/day. Statistically significant changes noted were considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was considered not to be affected by treatment. With the exception of one control and one 30 mg/kg bw/day female, all females showed evidence of mating. Precoital time was considered not to be affected by treatment. With the exception of one female (mated at Day 14), all females showed evidence of mating within the first 4 days. Number of implantation sites was considered not to be affected by treatment up to 100 mg/kg bw/day. Fertility index was considered not to be affected by treatment. The fertility indices were 79%, 96%, 100% and 88% for the control, 10, 30 and 100 mg/kg bw/day groups, respectively. A total of 5 control females, one female at 10 mg/kg bw/day and 3 females at 100 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Remarks on result:

other: A dose level of 100 mg/kg bw/day was selected as the NOAEL of 40 mg/kg bw/day was established in a dose range finder. Here, adverse findings consisted of ulcers and inflammation of the forestomach and hyperplasia squamous cells from 125 mg/kg bw/day on.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest doses tesed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. For the pup of one Female (10 mg/kg bw/day) which was missing on Day 2, absence of milk in the stomach was noted on Day 1 and for the pup of Female No. 154 which was missing on Day 2, a pale appearance was noted on Day 1. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. No test item-related clinical signs were noted during daily detailed clinical observations in Cohort 1A,1B and 1C or during weekly arena observations.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No mortality occurred during the study period in Cohort 1A, 1B and 1C from weaning onwards that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
Body weights and body weight gains of Cohort 1A, 1B and 1C were considered unaffected by treatment up to 100 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before and after correction for body weight was considered unaffected by treatment up to 100 mg/kg bw/day.
Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment as changes were only minimal and/or no trend was apparent regarding dose and duration of treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A: The following statistically significant changes were noted in male animals at 100 mg/kg bw/day.
• Increased neutrophil levels (1.3x)
• Increased lymphocyte levels (1.16x)
The statistically significant change in white blood cell count (WBC) in males at 10 mg/kg bw/day was considered not to be related to treatment with the test item as it occurred in the absence of a dose-related trend. Coagulation parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female pups, culled at PND 4 and male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment. Serum TSH levels in male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment.
Cohort 1A: Clinical chemistry parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day. Serum levels of thyroid stimulating hormone (TSH) and Total T4 were considered not to be affected by treatment up to 100 mg/kg bw/day.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Cohort 1A: Urinary parameters were unaffected by treatment.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Balanopreputial separation and vaginal patency were considered unaffected by treatment up to 100 mg/kg bw/day. At 30 mg/kg bw/day, an increase in the age of reaching balanopreputial separation was observed (1.03x compared with control). This was likely related to Male No. 355 that had a very low body weight and had reached balanopreputial separation at age PND 54 (versus average age of reaching balanopreputial separation on PND 41.5 for control animals).
At 10 mg/kg/day, an increase in age of reaching first estrus (1.04x compared with controls) was noted in females. In absence of a dose response, this slight increase was considered unrelated to treatment.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 100 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related organ weight changes in the Cohort surplus-animals.

Cohort 1A and 1B:There were no test item-related organ weight changes observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.
Cohort 1A and 1B: There were no test item-related macroscopic alterations in the F1-generation. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in several females in all dose groups in Cohort 1A and 1B, is related to a stage in the estrous cycle and is a normal finding.

Histopathological findings:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no test item-related microscopic alterations in Cohort 1A of the F1-generation.. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Spermatogenesis-staging (Cohort 1A)
Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Ovarian Follicle Counts - Cohort 1A
There were no test item-related effects on the ovarian follicle counts in the F1-females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous Cycle – F1-Generation (Cohort 1A):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 to 5 days. Female No. 636 (30 mg/kg bw/day) had two cycles with a duration of 2 days and one of 5 days and was therefore classified as having an irregular cycle. For Female Nos. 495 (control) and 555 (10 mg/kg bw/day) estrous cycle regularity/length could not be determined. Considering the low incidence and in absence of a dose response, this finding was considered unrelated to treatment with the test item.
Sperm Analysis – F1-Generation (Cohort 1A):
At 100 mg/kg bw/day, an increase in spermcount of the epididymides was observed of 1.23x compared with controls. This was considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no test item-related effects on splenic lymphocyte subpopulations observed up to 100 mg/kg bw/day.
Higher T-cell and T-cytotoxic cell splenic subpopulations and lower B-cell splenic subpopulations observed for females reached statistical significance when compared with controls at 10 mg/kg bw/day. However, these shifts were slight of nature and occurred in the absence of a dose-related response and in absence of any test item-related microscopic findings in the spleen. As such, these shifts were considered to represent biological variability and were considered not to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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 valid data available for the assessment of toxicity to reproduction with 1,6-hexanediol diacrylate an the read-across substance tripropylene glycol diacrylate (Cas No. 42978-66-5).

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. 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 (ReachCentrum, 2010).

The follow-up study (WIL-738007) 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 (ReachCentrum, 2012).

Available data for tripropylene glycol diacrylate (TPGDA, Cas No. 42978-66-5):

The test substance was investigated of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed (OECD 443). Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development untiladulthood, was expected to identify specific target organs in the offspring.

In addition, the study provided and/or confirmed information about the effects of the test substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

Furthermore, the information obtained from the assessments characterized potential effects in those systems. The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test substance in rats. In this preliminary study, animals were dosed with 0, 40, 125 and 375 mg/kg/day. From dose 125 mg/kg/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet accumulation in the male kidneys at 375 mg/kg/day. Based on the severe findings in the stomach, a parental local NOAEL was established of 40 mg/kg/day in this study. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg/day was selected. Further doses selected were 10 mg/kg bw/day and 30 mg/kg bw/day. For the F0-generation, the following parameters and end points were evaluated in this study:

mortality/ moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. In addition, functional observations were evaluated in females in this study.

For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial Separation, day of first estrus, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologicexaminations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).No parental toxicity was observed up to the highest dose level tested (100 mg/kg/day). At 30 and 100 mg/kg/day, a minor increase in sodium was noted in males and additionally, at 100 mg/kg/day, urea and potassium levels were slightly increased. As values were only slightly above the range considered normal (sodium) or remained within normal range (urea and potassium) and no corroborative microscopic findings were found, these changes were considered non-adverse.

No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (females), body weight, food consumption, heamatology, coagulation, thyroid hormone analysis, urinalysis, macroscopic examination, organ weights and microscopic examination). No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg/day). No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the pre-weaning phase.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, duration of gestation, viability and weaning indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), and macroscopic examination). No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the post-weaning phase.

At 100 mg/kg/day, increases in neutrophil and lymphocyte levels were found in males. As values remained within normal range and no corrobative microscopic findings were found, these changes were considered non-adverse.

No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, body weight, food consumption, coagulation, clinical chemistry, thyroid hormone analysis, urinalysis, splenic lymphocyte subpopulation, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, sperm analysis, macroscopic examination, organ weights, ovarian follicle and corpora lutea counts, and microscopic examination, including stagedependent qualitative evaluation of spermatogenesis in the testis).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the no-observed-adverse-effect levels (NOAEL) of the test substance for general toxicity (F0 and F1), reproduction, and development was observed to be at least 100 mg/kg bw/day (ReachCentrum, 2019a).

Additionally Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Estrous cycles examinations, reproductive organ weight assessment and respective histopathology was performed. For the testes of all selected males of the lowest and highest exposure level, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). There were 2/10 control group couples, 1/10 couples treated at 40 mg/kg/day, 3/10 couples treated at 125 mg/kg/day and 1/10 couples treated at 375 mg/kg/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. The length and regularity of the estrous cycle were not considered to have been affected by treatment. Moreover, the mating index, precoital time, number of implantation sites and the fertility index were unaffceted by treatment.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no parental reproductive toxicity was observed up to and including the highest tested dose of 375 mg test item/kg bw/d due to the abscence of respective adverse effects under the conditions of this study (ReachCentrum, 2019b).

Effects on developmental toxicity

Description of key information

- comp. OECD 414; oral, rat, gestation day 6-15: LOAEL maternal toxicity = 750 mg/kg bw/day; NOAEL teratogenicity =750 mg/kg bw/day (Hazleton Labs Inc., 1983).
- OECD 422; oral, rat, gestation day 0 through lactation day 4: NOAEL maternal toxicity = 250 mg/kg bw/day; NOAEL developmental toxicity = 750 mg/kg bw/day (ReachCentrum, 2010).

Read-across to CAS No. 42978-66 -5

- OECD 414; rabbits; oral; 50, 150, 450 mg/kg; NOAEL (development) 450 mg/kg bw/day (ReachCentrum, 2019c)

- equivalent to OECD 414; rat; oral; NOAEL (development) >/= 250 mg/kg bw/day (Hazleton, 1994)

- OECD 422; GLP; rats; oral; 40, 125, 375 mg/kg; NOAEL (development) >/= 375 mg/kg (ReachCentrum, 2019b)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

However, only a single dose was tested which showed maternal toxicity and embryotoxic effects.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

; only a single dose was tested.

Principles of method if other than guideline:

The aim of the study was to evaluate the embryo/fetal toxicity and teratogenic effects of the test substance when administered by gavage to pregnant rats from day 6 to day 15 of gestation. The test material was administered to a group of 22 female rats at a single dose level of 750 mg/kg bw. Another group served as a common control and received only corn oil.

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Kingston, New York
- Age at study initiation: ca. 5 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: individual in elevated wire-mesh cages; during mating two females were housed with one male
- Diet: Purina Rodent Laboratory Chow, ad libitum
- Water: ad libitum
- Acclimation period: ca. 9 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24-25 °C
- Humidity (%): 57 +- 4.8 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The amount of compound required for each group was weighed, filled to volume with the vehicle and stirred on a magnetic stirrer. The prepared dilutions were transferred to amber bottles and labelled. Before use, the prepared dilutions were well shaken and the animals were dosed while the solutions were mixed on a magnetic stirrer. Fresh solutions were prepared weekly.

VEHICLE:
- Duke´s Corn Oil, a yellow liquid was received from the C.F. Sauer Co. Richmond, Virginia and used as vehicle.
- Lot/batch no.: 80235

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Following a health status examination by a staff veterinarian, the males and females (1 male per 2 females) were placed in breeding cages for a maximum of 3 weeks. Females were rotated after the tenth day of mating. Mating was confirmed by the presence of a vaginal plug or by daily examination of vaginal smears for the presence of sperm. The day that mating was confirmed was designated as day 0 of gestation for each female placed on study.

Duration of treatment / exposure:

day 6 to day 15 of gestation

Frequency of treatment:

daily

Duration of test:

until day 20 of gestation

Remarks:

Doses / Concentrations:
750 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

22 mated females

Control animals:

yes, concurrent vehicle

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: mortality, moribundity and clinical signs
- Time schedule: daily

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

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: on day 6-8, 9-11, 12-14, 15-17 and 18-20 of gestation

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20:
On day 20 of gestation, females were sacrificed by carbon dioxide asphyxiation and the fetuses were taken by cesarean section. Following gross examination of each dam, the number of corpora lutea per ovary and the number and placement of implantation sites, early and late resorptions, and live and dead fetuses in each uterine horn were recorded. Fetuses were removed from the placenta, individually identified, examined externally, weighed, sexed, and measured from the frontal-parietal suture to the base of the tail (crown-rump distance). Gravid and nongravid uterine weights (with ovaries attached) were recorded.
- Organ Weights:
Following careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner, the gravid and nongravid uterine weights (with ovaries attached) of each female were taken for organ weighing.
- Tissue Preservation:
The uterus and ovaries of each dam, in addition to unusual lesions, were preserved in 10 % neutral buffered formalin.

Fetal examinations:

- Visceral Examination of Fetuses:
Approximately one-third of the fetuses from each litter were fixed in Bouin's solution, sectioned, and examined by Wilson's freehand razor technique (Wilson and Warkany, 1965), and sealed in plastic. The prepared sections were re-examined against a light box with the aid of magnification.
- Skeletal Examination of Fetuses:
After undergoing external examinations, approximately two-thirds of all fetuses from each litter were opened by longitudinal incision and the viscera examined grossly. The fetuses were then placed in M5 ethyl alcohol and the skeletons were stained in a potassium hydroxide - Alizarin red S solution (modified Staples and Schnell, 1964). Each skeleton was examined with the aid of magnification on a light box for bone alignment, degree of ossification, and anomalies. The number of sternebrae, ribs, caudal vertebrae, and bones of the extremities were noted and recorded. The fetuses examined by Wilson's technique were preserved In Bouin's solution and sealed in plastic after sectioning. The fetuses stained for skeletal examination were preserved in plastic in a glycerin ethanol (1:1) solution with several crystals of thymol to retard bacterial growth.

Statistics:

Survival was statistically analyzed by the National Cancer Institute Package (Thomas, Breslow, & Gart, 1977).
The mean maternal body weight changes (Days 0-6, 6-15, 15-20, and 0-20), mean maternal food and water consumptions (Days 6-9, 9-12, 12-15, 15-18, 18-20, and 6-20), percent males per litter, mean fetal body weights and lengths, fetal viability, percent resorptions, implantation efficiency, gravid and nongravid uterine weights, and the incidence of visceral and skeletal anomalies and variants were analyzed by Box's test for homogeneity of variances (Box, 1949). This test was followed by a one-way classification analysis of variance (ANOVA), if the variances proved to be homogenous. If the variances proved to be heterogenous, a rank transformation of data was performed, which was followed by Box´s test and ANOVA. If ANOVA of untransformed or transformed data was significant, Dunnet´s T-test was used for control vs. treatment group mean comparisons.
Pregnancy rates were analyzed by a test of multiple proportions using one degree of freedom Chi-square test with Yates´continuity correction. All pairwise comparisons were evaluated at the 5.0 % probability (one-tailed) level.

Details on maternal toxic effects:

Details on maternal toxic effects:
Mortality and Clinical Signs:
No mortality occured during treatment. An increased incidence of clincal symptoms was noted (wheezing, dyspnea, urine stains, wasted feed, rough haircoat, hunched pasture, bloody crust on the eyes, nase, snouth, and frontpaws, salivation and alopecia).
- Body weights:
Mean body weight changes lower than the control, but not statistically significant were observed in the treated group during the treatment phase.
- Food and water consumption:
In the treated group a statistically significant increase in water consumption was noted on days 18 and 20 and in total water consumption, compared to control.
- Gross pathology:
The most distinct alterations attributed to treatment were noted in the stomachs of the treated animals. The findings consisted of discoloured material or fluid in the stomach; gas distending the stomach; discoloured, ulcerated or raised areas in the glandular or nonglandular portion of the stomach. glandular mucosa smooth; walls thick or thin/smooth; and nonglandular mucosa thin and pale, or thick and rough. Other gross pathology findings were considered to be incidental in nature and showed no relation to treatment.
- Pregnancy rates, Corpora Lutea and Implantations:
Pregnancy rates, corpora lutea and implantations, and mean implantation efficiency (number of implantations per number of corpora lutea) were generally comparable for treated animals and controls.

Dose descriptor:

LOAEL

Effect level:

750 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Gross Pathology:
Skeletal examinations included incidences of cleft palate in 1 pup. A high incidence of dilated ureters was noted in both treated and control groups.
- Visceral and Skeletal Examinations:
A higher than control mean incidence of visceral variants was noted (but not statistically significant). Statistically significant increased mean incidence of skeletal variants was observed. The majority of the findings were due to delayed ossification of the various bone structures examined. The authors suggested that this response was resulting from the maternal toxicity noted.

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Only a single dose of 750 mg/kg bw was tested in this study. Taken together, maternal toxic effects as well as embryotoxic effects were observed. According to the authors, the embryotoxic effects resulted from maternal toxicity. Therefore, the results obtained for the offspring at a dose causing maternal toxicity (750 mg/kg bw/d), were not considered relevant for assessing the teratogenic potential of the test material.

Reference 2

Endpoint:

developmental toxicity

Remarks:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2009 - 02 March 2010 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system 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

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.

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

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

Duration of test:

41-49 days

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

Maternal 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).

Ovaries and uterine content:

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.

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.

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

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No test substance-related mortality or moribundity was noted at any dosage level. Dose related incidences of clinical findings indicative of aversion to test substance administration were noted prior to, at the time of, or approximately 1 hour following dose administration throughout the treatment period at dosage levels of 75, 250, and 750 mg/kg bw/day group. However, these findings were attributed to the irritative properties of the test substance and were not considered adverse. Statistically significant reductions in mean body weight gain and food consumption were noted in the 750 mg/kg bw/day group males during the pre-mating period, resulting in a mean body weight that was 6.8% lower (not statistically significant) than the control group on study day 28. No other differences in mean body weights, body weight gains, or food consumption at any dosage level when compared to the control group were considered indicative of parental toxicity.
Following microscopic examination, test substance-related findings included micro- to macrovesicular vacuolar change within the liver at 75, 250, and 750 mg/kg bw/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. In addition, 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. At 750 mg/kg bw/day, higher liver weights and statistically significant changes in serum chemistry parameters were noted for males and females; these changes were attributed to test substance administration were considered adverse. 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.
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.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The general physical condition of all F1 pups in this study were unaffected by test substance administration. 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. 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. Upon visual examination of the pups, no malformations were detected. In the absence of any effects on the general physical condition of the F1 pups, the NOAEL for neonatal toxicity was 750 mg/kg bw/day.

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: No effects on the general physical condition observed.

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jun 2018 - 25 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 15-21 weeks old
- Weight at study initiation: between 3043 g and 4268 g
- Housing: housed individually in cages with perforated floors (Ebeco, Germany)
- Diet: ad libitum, Pelleted diet for rabbits (Global Diet 2030 from Harlan)
- Water: ad libitum, Municipal tap water
- Acclimation period: least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 38 to 97
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension; protected from light. If not used on the same day of preparation, the aliquots were stored in the refrigerator set to maintain 4°C for a maximum of 8 days. On the day of use, they were removed from the refrigerator and allowed to warm to room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during dosing.m Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: 1% Aqueous carboxymethyl cellulose with 0.5% Tween 80

Analytical verification of doses or concentrations:

yes

Remarks:

Acquity UPLC system

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

from Day 6 to Day 28 post-coitum

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the tolerability study and dose range finder and in an attempt to produce graded responses to the test item. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity including mortality was observed at 750 mg/kg. Therefore, the dose levels selected in the current main teratology study were 0, 50, 150 and 450 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
All animals (including female no. 75 euthanized before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. In addition, the gastro-intestinal tract was examined for signs of irritation/corrosion; the oesophagus was examined internally. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed. Each ovary and uterine horn of all animals was dissected and examined as quickly as possible.

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: [all per litter]

- Skeletal examinations: Yes: [all per litter]
Subsequently, the skeletal examination was done on all fetuses from all groups from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

- Head examinations: Yes: [half per litter]

Statistics:

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

see table 1

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicological relevant findings up to 450 mg/kg.
Reduced faeces production (up to a moderate degree) was observed in 14, 18, 19 and 18 females in the control, 50, 150 and 450 mg/kg groups, respectively, on several days during treatment. As all groups were affected (including the vehicle control group) and in the absence of a dose-related trend, no toxicological significance was attached to this observation. Incidental findings of note were red fluid on the manure tray in one high dose female, and lean appearance and/or piloerection in another high dose female. These findings were considered not to be toxicological relevant as they were observed in the vehicle control group at the same incidence (red fluid on the manure tray, lean appearance), were transient (lean appearance and piloerection) and all gravid females in this study had a normal pregnancy.
One female in Group 3 was noted with missing toes (two missing toes on the right foreleg and 1 missing toe on the left foreleg; taken from the study daybook). This finding was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain, and/or were observed in control females only. They were therefore of no toxicologically relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatmentwith the test item.
One female from the high dose group had to be euthanized after 4 days of treatment. Immediately after dosing on post-coitum Day 9, she was observed with respiratory problems (gasping) and moribund. At necropsy, red foamy content of the trachea, several reddish foci in left caudal lobe of the lungs and grey-white discolouration of the left caudal lobe of the lungs were noted. Taken together, these findings were indicative of complications during the oral gavage procedure. Therefore, the preterm sacrifice of this female was considered unrelated to the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight, body weight gain before and after correction for (gravid) uterus weight of treated Group 2, 3 and 4 females were unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 450 mg/kg. In the high and mid dose group each, a trend towards slightly lower absolute and relative food consumption compared to the concurrent control group was noted from Days 6-15 and Days 12-15 post-coitum, respectively. As changes were only slight (reaching no statistical significance) and transient (complete recovery was seen from Days 15-18 post-coitum onwards), it was not considered as adverse.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Moreover, the examination of the gastro-intestinal tract and the internal examination of the oesophagus did not reveal any signs of irritation and/or corrosion. The observation of missing toes in one Group 3 female was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Remaining incidental findings among control and treated animals included watery-clear cysts of the oviducts, alopecia, scabs or an isolated wound. These findings are occasionally seen among rabbits used in these types of study, and in the absence of a dose relationship they were considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Overall, the number of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and treatment groups were considered to be unaffected by treatment. Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Remarks on result:

other: Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 450 mg/kg.
Mean combined (male and female) fetal body weights were 37.8, 38.2, 39.1 and 38.0 gram for the control, 50, 150 and 450 mg/kg groups, respectively.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 450 mg/kg.
Mean sex ratios (males:females) were 46:54, 50:50, 44:56 and 56:44 for the control, 50, 150 and 450 mg/kg groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.4, 9.8, 10.1 and 9.3 fetuses/litter for the control, 50, 150 and 450 mg/kg groups, respectively. As all values remained within the available historical control range the slightly lower litter size in the high dose group was considered unrelated to treatment.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 450 mg/kg/day.
Two external malformations were observed in this study. Two fetus of Groups 2 and 4, respectively, both had an open eye with relating findings of the eye/lens noted at soft tissue cephalic examination (i.e. eyelids not joined, lens attached to cornea and/or lens misshapen). Due to the single occurrence of this malformation in a low and high dose fetus, it was considered to be chance finding. A flexure of the carpal and/or tarsal was the other malformation and this occurred in the control group in one fetus (both tarsals, without apparent skeletal origin) and in late resorptions in two fetuses (one or both carpals, not skeletally examined). Because carpal and/or tarsal flexures were observed at the control level only, this was considered spontaneous in origin. External variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment at 450 mg/kg/day.
The only fetus with a malformation was one fetus of the high dose, which had a vertebral anomaly with associated rib anomaly. A vertebral anomaly is the most common skeletal malformation among historical control fetuses and at the single occurrence noted in this study, it is considered spontaneous in origin. A statistical significant increase in the litter incidence of fetuses with caudal shift of pelvic girdle was observed in the high dose group. This variation occurred at an incidence of 21.5%
versus 8.7% per litter in the concurrent control group. Because the litter incidence of caudal shift of pelvic girdle was well within the historical control data range (mean 16.5% per litter; P5 – P95: 2.8 - 34.5% per litter), the higher incidence of this finding in Group 4 was considered to have occurred by chance and not to be toxicologically relevant. All other variations noted were not considered treatment related as they occurred infrequently, at frequencies that were within the range of available historical control data, or were observed in control fetuses only.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 450 mg/kg/day. Visceral malformations occurred in 0 (0), 1 (1), 4 (3) and 3 (2) fetuses (litters) in the control, 50, 150 and 450 mg/kg groups, respectively.
In Group 4, the three malformed foetuses (out of two litters) had a malpositioned testis. This malformation was also noted in Group 3 fetus, but not in fetuses at the control and low dose levels. Nevertheless, because a malpositioned testis is one of the most common visceral malformations in historical control fetuses, this was considered not to be treatment related. The other malformed Group 3 fetuses all had a multiple cardiovascular malformation. As no cases occurred in Group 4 and none of
the individual malformations was uncommon among historical control fetuses these were considered not toxicologically relevant. The affected fetus in Group 2 had a diverticulum of the intestine, which at the single occurrence at the low dose was considered spontaneous in origin.
All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Species:

rat

Additional information

There are valid data available for the assessment of developmental toxicity with 1,6-hexanedioldiacrylate an the read-across substance triporpylene glycol diacrylate (Cas No. 42978-66-5).

In a developmental toxicity study comparable to OECD 414, 22 pregnant female rats were exposed via gavage to 750 mg/kg bw/day test substance daily during gestation days 6-15. This dose level was determined in a previous maternal tolerance study. The LOAEL for maternal toxicity was 750 mg/kg bw/day and the NOAEL for teratogenicity was 750 mg/kg bw/day.

No mortality occured during treatment. An increased incidence of clincal symptoms was noted (wheezing, dyspnea, urine stains, wasted feed, rough haircoat, hunched pasture, bloody crust on the eyes, nase, snouth, and frontpaws, salivation and alopecia). Mean body weight changes were lower than the control, but not statistically significant. In the treated group a statistically significant increase in water consumption was noted on days 18 and 20 and in total water consumption, compared to control. Alterations attributed to treatment were noted in the stomachs of the treated animals. The findings consisted of discoloured material or fluid in the stomach, gas distending the stomach, discoloured, ulcerated or raised areas in the glandular or nonglandular portion of the stomach, glandular mucosa smooth, walls thick or thin/smooth and nonglandular mucosa thin and pale, or thick and rough. Pregnancy rates, corpora lutea and implantations, and mean implantation were generally comparable for treated animals and controls. Skeletal examinations included incidences of cleft palate in 1 pup. A high incidence of dilated ureters was noted in both treated and control groups. A higher than control mean incidence of visceral variants was noted, but not statistically significant. Statistically significant increased mean incidence of skeletal variants was observed. The majority of the findings were due to delayed ossification of the various bone structures examined. The authors suggested that these responses were resulting from the maternal toxicity noted.

Taken together, maternal toxic effects as well as embryotoxic effects were observed. According to the authors, the embryotoxic effects resulted from maternal toxicity. Therefore, the results obtained for the offspring at a dose causing maternal toxicity (750 mg/kg bw/d), were not considered relevant for assessing the teratogenic potential of the test material (Hazleton Labs Inc., 1983).

  

In addition, 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 was conducted. 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. F1 clinical observations and body weights were recorded on PND 1 and 4. Pups were euthanized and discarded on PND 4. Clinical pathology evaluations (haematology, 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 test substance-related mortality or moribundity was noted at any dosage level. Dose related incidences of clinical findings indicative of aversion to test substance administration were noted prior to, at the time of, or approximately 1 hour following dose administration throughout the treatment period at dosage levels of 75, 250, and 750 mg/kg bw/day group. However, these findings were attributed to the irritative properties of the test substance and were not considered adverse. Statistically significant reductions in mean body weight gain and food consumption were noted in the 750 mg/kg bw/day group males during the pre-mating period, resulting in a mean body weight that was 6.8% lower (not statistically significant) than the control group on study day 28. No other differences in mean body weights, body weight gains, or food consumption at any dosage level when compared to the control group were considered indicative of parental toxicity.

Following microscopic examination, test substance-related findings included micro- to macrovesicular vacuolar change within the liver at 75, 250, and 750 mg/kg bw/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. In addition, 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. At 750 mg/kg bw/day, higher liver weights and statistically significant changes in serum chemistry parameters were noted for males and females; these changes were attributed to test substance administration were considered adverse. 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. 

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 general physical condition of all F1 pups in this study were unaffected by test substance administration. 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. 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. Upon visual examination of the pups, no malformations were detected. Aside from the absence of milk in the stomach, no other internal findings were noted. In the absence of any effects on the general physical condition of the F1 pups, the NOAEL for neonatal toxicity was 750 mg/kg bw/day (ReachCentrum, 2010).

Available data for tripropylene glycol diacrylate (TPGDA, Cas No. 42978-66-5):

The test substance was investigated in a teratogenicity study according to OECD 414 and in compliance with GLP. The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg/day, based on the results of the dose range finderand tolerability study. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity was observed at 750 mg/kg. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsyfindings, number of corpora lutea, (gravid) uterine weight and uterine contents, and maternal pregnancy data. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance of at least 450 mg/kg was established. Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificedin extremison Days 4 and 7 of treatment at 750 mg/kg (ReachCentrum, 2019c).

In addition no indications of a possible developmental toxic effect were observed in a recent OECD 422 study in Wistar Han rats treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days. Estrous cycles examinations, reproductive organ weight assessment and respective histopathology was performed. For the testes of all selected males of the lowest and highest exposure level, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). There were 2/10 control group couples, 1/10 couples treated at 40 mg/kg/day, 3/10 couples treated at 125 mg/kg/day and 1/10 couples treated at 375 mg/kg/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. The length and regularity of the estrous cycle were not considered to have been affected by treatment. Moreover, the mating index, precoital time, number of implantation sites and the fertility index were unaffceted by treatment. Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no parental reproductive toxicity was observed up to and including the highest tested dose of 375 mg test item/kg bw/d due to the abscence of respective adverse effects under the conditions of this study (ReachCentrum, 2019b).

In a supporting 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. As determined in a previous maternal tolerance study, at 500 mg/ kg pathlogical findings in stomach, liver and lung are described in additon. 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. Oneout 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) occurred 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 at least 250 mg/kg bw/day and the NOAEL for embryotoxicity/teratogenicity is also at least 250 mg/kg bw/day (Hazleton Labs Inc., 1194).

Justification for classification or non-classification

Based on the available data for developmental toxicity or toxicity to reproduction ,the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).