Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

1-There is an extended one generation study (OECD 443) performed by Charles River in 2022.


In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following No Observed Adverse Effect Levels (NOAEL) of the test item were established:


- General toxicity (F0 and F1): NOAEL at least 1000 mg/kg bw/day


- Reproduction (F0 and F1): NOAEL at least 1000 mg/kg bw/day


- Developmental (F0, F1 and F2): NOAEL at least 1000 mg/kg bw/day


- Developmental neurotoxicity (F1): NOAEL at least 1000 mg/kg bw/day


 


2-There is a GLP combined repeated dose and reproduction / developmental screening test conducted according to OECD guideline 422, the NOAEL for the reproductive/developmental toxicity was concluded to be higher than 900 mg/kg bw/day in this study.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not available the completion date since the report has to be finalized yet. To be included by Sponsor once the report is finalized.
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:
June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151
Version / remarks:
July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

The design of this study was based on the final decision on a compliance check by ECHA (Decision No. TPE-D-2114501107-65-01/F, date 19 Feb 2020).

- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: the dose levels were selected based on the results of previous toxicity studies with the test item in rats, consisting of a combined reproduction/developmental toxicity screening test (OECD 422, Huntingdon Life Sciences Reference No. RAJ0004), and a prenatal developmental toxicity study in rats (OECD 414, Harlan Reference No. D84032).
In the OECD 422 study, Crl:CD (SD) rats received oral doses (gavage) at 0, 100, 300 or 900 mg/kg bw/day in corn oil. At 900 mg/kg bw/day, a slightly lower mean number of implantation sites (and secondary lower mean litter size), and a slightly longer gestation duration was observed. Since the mean number of corpora lutea was not determined, it could not be confirmed if these changes represented test-item related effects. The slightly longer gestation duration did not show a dose-response, and therefore its relationship to treatment was equivocal. Therefore, although these findings were considered as possible indicators of reproductive toxicity, a NOAEL of 900 mg/kg bw/day was established for parental and reproductive/developmental toxicity.
In the OECD 414 study, Rat, RccHanTM: WIST(SPF) rats received oral doses (gavage) at 0, 100, 300 or 1000 mg/kg bw/day in corn oil. At 1000 mg/kg bw/day, lower fetal body weight (6% lower than control) and maternal body weight gain (corrected body weight gain 8% lower than control) were recorded. In the study report, the maternal NOAEL was established at 1000 mg/kg bw/day and the developmental NOAEL at 300 mg/kg bw/day.
Based on these results, dose levels for this Extended One-Generation Study were selected to be 0, 100, 300 and 1000 mg/kg bw/day. The high dose of 1000 mg/kg bw/day was the limit dose as specified in OECD guideline 443.
- Inclusion/exclusion of extension of Cohort 1B: Cohort 1B has been extended to produce the F2 generation because: 1) the use of the Substance leads to significant exposure of consumers and professionals: the Substance is used by professionals in adhesives/inks/coatings formulations and consumers as coatings, thinners and paint removers; 2) there are indications for endocrine-disrupting modes of action resulting from changes in gestation length, suggesting a minor shift towards a longer gestation length for females: "At 900 mg/kg bw/day group, no females had a 22-day gestation length compared with 3, 5 and 4 females at 0, 700 and 300 mg/kg bw/day, respectively. The incidence of females with a 23.5 day gestation length was marginally outside the Historical control data range." (ECHA decision No. TPE-D-2114501107-65-01/F, date 19 Feb 2020)
- Termination time for F2: the F2 generation was followed to weaning allowing assessment of nursing and lactation of the F1 parents and postnatal development of F2 offspring. Investigations for F2 pups were similar to those requested for F1 pups in OECD TG 443 and described in OECD GD 151. F2 adult animals were sacrificed after they have been found positive for balanopreputial separation or after occurrence of first estrous.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Cohorts 2A and 2B were included since existing information on the Substance derived from the existing 90-day sub-chronic study according to OECD TG 408 (2015) shows evidence of significantly reduced locomotor activity in the mid- and high-dose males. Specifically in the mid-dose group, the reduction is up to 6% at the last time point, in the absence of systemic toxicity (ECHA decision No. TPE-D-2114501107-65-01/F, date 19 Feb 2020).
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Cohort 3 was not included since no particular concern for (developmental) immunotoxicity needs to be further elucidated (ECHA decision No. TPE-D-2114501107-65-01/F, date 19 Feb 2020).
- Route of administration: oral by gavage as also agreed by ECHA (ECHA decision No. TPE-D-2114501107-65-01/F, date 19 Feb 2020).
- Other considerations: rat considered the preferred species (ECHA decision No. TPE-D-2114501107-65-01/F, date 19 Feb 2020).
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 reproduction and developmental toxicity testing and for neurotoxicity and testing by regulatory agencies. The Test Facility has general and reproduction/developmental/neurological 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 and neurological toxicants.
The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The choice of the rat species was also endorsed by ECHA (ECHA decision No. TPE-D-2114501107-65-01/F, date 19 Feb 2020).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (F0 males and females) 11 wks
- Weight at study initiation: (F0) Males: 250-300 g; Females: 200-230 g
- Fasting period before study: not indicated
- Housing: group housed (up to 5 animals of the same sex, same group and same cohort together; for F2 animals in post-weaning period up to 6 animals of the same sex and same dosing group) in polycarbonate cages. During the mating phase males and females were cohabitated on a 1:1 basis in Makrolon plastic cages. During the lactation phase, females were housed in Makrolon plastic cages. Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohort 2B and Surplus, and non-selected F2 animals) or until weaning on PND 21 (Cohorts 1A, 1B, 1C, 2A, and selected F2 animals). During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water for a maximum of 2 hours. The cages, unless otherwise specified, contained appropriate bedding material and were equipped with water bottles. Furthermore, animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. Analysis showed that there were no known contaminants that would interfere with the objectives of the study.
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, F1- Cohort 2A animals did not have access to food for a maximum of 2 hours. The feed analysis confirmed the absence of known contaminants that would interfere with the objectives of the study.
- Water: ad libitum, municipal tap water. During motor activity measurements, F1- Cohort 2A animals did not have access to water for a maximum of 2 hours. Periodic water analyses confirmed the absence of known contaminants that would interfere with the objectives of the study.
- Acclimation period: 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.4 to 22.4°C (actual daily mean)
- Relative humidity: 43 to 71% (actual daily mean)
- Air changes: at least 10 air changes per hour
- Photoperiod: 12 hours of dark / 12 hours of light

IN-LIFE DATES: From: 26 January 2021 To: 10 August 2021
Route of administration:
oral: gavage
Vehicle:
corn oil
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 weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
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: the substance demonstrated to be soluble and stable in the vehicle.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1 (avoiding sibling mating)
- Length of cohabitation: F0 animals, daily, after a minimum of 14 days of treatment; F1 animals (Cohort 1B), daily, after a minimum of 10 weeks of treatment (between PND 90-120) for a maximum of 14 consecutive days.
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as Day 0 post-coitum.
- A maximum of 14 days will be allowed for mating, after which females who have not shown
evidence of mating will be separated from their males without further opportunity of mating.
- Further matings after two unsuccessful attempts: no
- After successful mating, males and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration (all groups) and homogeneity (low- and high-dose groups) samples collections were performed on several occasions (week 1, 3, 6, 9, 12, 15, 18, 21 and 24 of treatment). Analyses were performed using a validated analytical procedure (Test Facility Study No. 20249993).
Samples were stored at target temperature range of 18-22°C. Acceptance criteria for analytical analyses were the following: for concentration, mean sample concentration results within or equal to ± 10% for solutions of theoretical concentration. For homogeneity, relative standard deviation (RSD) of concentrations of =10% for each group.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20249993) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20249993.
Duration of treatment / exposure:
F0 and F1 PARENTS
- F0 Males: treated for a minimum of 10 weeks, including 2 weeks prior to mating and during the mating period, up to and including the day before scheduled necropsy
- F0 Females: treated for a minimum of 8 weeks, including 2 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering.

F1 and F2 OFFSPRING
- Prior to weaning, pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
- From weaning onwards (PND 21), F1 animals of Cohorts 1A, 1B, 1C and 2A and selected F2 animals were dosed up to and including the day before scheduled necropsy. The F1 animals of Cohort 2B, Cohort Surplus and Spares (not assigned to one of the cohorts) and non-selected F2 animals were not dosed.
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until a minimum of 10 weeks treatment have been elapsed after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age (PND 21).
- Age at mating of the mated animals in the study: parents from F1 generation were between PND 90-120.
Dose / conc.:
100 mg/kg bw/day
Remarks:
low-dose (Group 2)
Dose / conc.:
300 mg/kg bw/day
Remarks:
mid-dose (Group 3)
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high-dose (Group 4)
No. of animals per sex per dose:
F0: 25/sex/dose
Cohort 1A: 20/sex/dose
Cohort 1B: 20/sex/dose
Cohort 2A: 10/sex/dose
Cohort 2B: 10/sex/dose
Cohort Surplus: 10/sex/dose
F2: 60/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: please refer to section "Justification for study design".
- Fasting period before blood sampling for clinical biochemistry: yes, for F0 and Cohort 1A animals (maximum of 24 hours, water available). Fasting period not foreseen for F1 culled pups, F1 Cohort Surplus and F2 animals selected for blood sampling.
Parental animals: Observations and examinations:
The following in-life procedures, observations, and measurements were performed for parental animals (F0).

MORTALITY/MORIBUNDITY: Yes
- Time schedule: twice daily, animals were observed for general health/mortality and moribundity within their cages, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily, up to the day prior to necropsy. Conducted prior to dosing and after dosing. The time of onset, grade and duration of any observed signs was recorded and graded for severity.

ARENA OBSERVATIONS: Yes
- Time schedule: once before the first administration of the test item and at weekly intervals during the treatment period. Animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed 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.

FOOD CONSUMPTION: Yes
- Food consumption was 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 over Days 0-4, 4-7, 7-11, 11-14, 14-17 and 17-20 post-coitum and during lactation over PND 1-4, 4-7, 7-14 and 14-21.

WATER CONSUMPTION: Yes
- Time schedule for examinations: recorded on regular basis throughout the study. By visual inspection of the water bottles. If inter group differences are noted, consumption will be assessed by weight.

GENERAL REPRODUCTION DATA: Yes
- Daily from the mating period onwards. Male number paired with, mating date, confirmation of pregnancy, and delivery day will be recorded. Palpation and/or body weight measurement were used to aid in confirmation of pregnancy.
The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 was considered to be the day when the female started to deliver and was defined as Lactation Day (LD) 0 for the dam and PND 0 for the offspring and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females were examined for evidence of premature delivery. Signs of difficult or prolonged parturition were recorded, if applicable. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded, if applicable.
Oestrous cyclicity (parental animals):
ESTROUS CYCLE DETERMINATION: Yes
- For F0 females, daily vaginal lavage was performed beginning 14 days prior to mating and during mating until evidence of copulation is observed. Vaginal lavage 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 to determine the stage of estrous. This was done for all females, except for females that were euthanized in extremis or died spontaneously.
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Sperm parameters (parental animals):
Parameters examined in all surviving males of F0 parental and Cohort 1A 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 hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples. One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at =-15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples. In the case of any abnormalities in the right epididymis, the right side organ(s) was fixed in modified Davidson's solution, and the left side organ was used for evaluation of sperm numbers. If abnormalities were found in both epididymis, both these organs were fixed in modified Davidson's solution and no evaluation of sperm numbers was performed.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and blood collected for thyroid hormone analysis. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was performed.


F1 AND F2 GENERATION OFFSPRING
The in-life procedures, observations, and measurements listed below were performed for the pups of F1 and F2 generation until weaning (PND 21).

- OBSERVATIONS:
Mortality and moribundity were checked twice daily. Pups were observed for general health/mortality and moribundity. If possible, defects or cause of death were evaluated. Pups were not removed from cage during observation, unless necessary for identification or confirmation of possible findings. Detailed clinical observations were made for all pups at least once a day.

- PARAMETERS EXAMINED:
Body weights were recorded for all live pups individually on PND 1, 4, 7, 13 and 21. From 10 selected litters/group of the non-selected F2-animals of Cohort 1B, terminal body weights were determined for one male and one female pup; sex was determined for all pups on PND 1, 4 and 13; anogenital distance (AGD) was measured for all live pups on PND 1 and normalized to the cube root of body weight; all males were examined for the number of areola/nipples on PND 13. [number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia]


COHORTS 1A, 1B, 1C and 2A
The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort 2B, Cohort Surplus and spare F1-animals as these will be terminated on/before PND 24.

- OBSERVATIONS:
Mortality and moribundity were checked twice daily. Animals were observed for general health/mortality and moribundity. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings. At least twice daily, up to the day prior to necropsy clinical observations were conducted prior to dosing and after dosing. The time of onset, grade and duration of any observed signs was recorded and graded for severity. Arena observations were performed once when the first animals have reached Day 8 of weaning and thereafter at weekly intervals during the treatment period. Animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

- PARAMETERS EXAMINED:
Body weights were measured weekly from weaning onwards. 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. Food consumption was measured weekly from weaning onwards up to the day prior to scheduled necropsy. Water consumption was monitored by visual inspection of the water bottles on a regular basis throughout the study. Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards by visual inspection of the genital area until the separation is present. Estrous cycle was determined on the day of scheduled necropsy by examining the cytology of vaginal lavage samples (only for females of Cohorts 1A and 1B).


COHORTS 1A
The in-life procedures, observations, and measurements listed below were performed for the F1-females of Cohort 1A only, in addition to the procedures mentioned above.

- PARAMETERS EXAMINED:
Estrous cycle was determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all Cohort 1A females starting on the first day after 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. Data relevant for estrous cycle determination were reported from daily vaginal lavage performed from PND 75 to 88.


COHORTS 1B
The in-life procedures, observations, and measurements listed below were performed for the F1-animals of Cohort 1B only, in addition to the procedures mentioned above.

- PARAMETERS EXAMINED:
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. Food consumption was not determined for males and females which are 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. Estrous cycle was determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed starting one day after initiation of the mating period until evidence of copulation is observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

- COHABITATION/MATING PROCEDURE FOR THE PRODUCTION OF F2 GENERATION
Animals were cohabitated as reported under section "Details on mating procedure". The mating period started after a minimum of 10 weeks of treatment (between PND 90-120).

- GENERAL REPRODUCTION DATA
On a daily frequency from the mating period onwards general reproduction data were collected from Cohort 1B animals. Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation and/or body weight measurement were used to aid in confirmation of pregnancy. The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 was considered to be the day when the female started to deliver and is defined as Lactation Day (LD) 0 for the dam and PND 0 for the offspring and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females were examined for evidence of premature delivery. Signs of difficult or prolonged parturition were recorded, if applicable. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded, if applicable.


F2 GENERATION (FROM WEANING ONWARDS)
The in-life procedures, observations, and measurements listed below are applicable for all F2-animals from weaning (PND 21) onwards (i.e. selected F2 animals).

- OBSERVATIONS:
Animals were observed for general health/mortality and moribundity at least twice daily. Clinical observations were performed at least twice daily, up to the day prior to necropsy, and conducted prior to dosing and after dosing. The time of onset, grade and duration of any observed signs was recorded and graded for severity. Vaginal patency and balanopreputial separation were monitored daily for all females and males from PND 25 or PND 35, respectively, onwards until present. Vaginal patency (vaginal opening) was monitored by visual inspection of the vaginal area. Balanopreputial separation (prepuce opening) was monitored by visual inspection of the genital area. Body weight was recorded on the day of acquisition of vaginal patency and balanopreputial separation for females and males, respectively. Estrous stages was determined by examining the cytology of vaginal lavage samples starting on the first day after 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.

- PARAMETERS EXAMINED:
Animals individual weights were recorded weekly. In addition, the body weight was recorded for each female on the day of acquisition of vaginal patency and for each male on the day of acquisition of balanopreputial separation. Food consumption was recorded on a weekly basis. Water consumption was monitored by visual inspection of the water bottles on a regular basis.


COHORT 2A - ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY
The in-life procedures, observations, and measurements listed below were performed for the F1-animals of Cohort 2A only, in addition to the procedures mentioned above.

- Acoustic startle response: once between PND 23-25, acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed in a sound-attenuated room. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses was recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight-second inter-trial interval. Average response amplitude (AveN), average maximum response amplitude (MaxN) and average latency to achieving the maximum response amplitude (Tmax) were analyzed in five blocks of 10 trials each.

- Functional Observation battery (FOB): once between PND 63-75, the FOB tests were conducted in the order of sequence indicated below and were divided between several days. The detailed functional observations and locomotor activity were conducted in separate room(s) specially equipped for these purposes.
1. Detailed functional observations consist of a number of tests conducted in- and out-side the home cage.
2. Rectal temperature was measured immediately after the detailed functional observations.
3. Locomotor activity was tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
4. Hearing ability (Score 0 = normal/present, score 1 = abnormal/absent).
5. Pupillary reflex (both eyes).
6. Fore- and hindlimb grip strength. This was recorded per animal as the mean of three measurements, using a grip strength meter.
7. Landing (hind) foot splay. This was recorded per animal as the mean of three measurements.
Postmortem examinations (parental animals):
F0 GENERATION (refer also to Table 7 and Table 8 under "Any other information on materials and methods incl. tables")

SACRIFICE
- Unscheduled deaths: for animals died on study a necropsy was conducted and specified tissues were saved, but not weighed. If necessary, the animal was refrigerated to minimize autolysis. Animals euthanized for humane reasons were deeply anesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained, but not weighed. The specified tissues which will be retained are mentioned in Table 8.
- Scheduled euthanasia: animals surviving until scheduled euthanasia had the terminal body weight recorded and were deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies are summarized below:
Males which sire: after successful mating and a minimum of 10 weeks of treatment.
Males which fail to sire: at the end of the mating period and after a minimum of 10 weeks of treatment.
Females which deliver: LD 23-25.
Females which fail to deliver: with evidence of mating: post-coitum Days 25-27.
Without evidence of mating: approximately 24-26 days after the last day of the mating period.
Females with total litter loss: dams with no surviving pups will be euthanized within 24 hours after the last pup is found dead or missing.
Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.

GROSS NECROPSY
All animals were subjected to a full postmortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites was recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri was 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
The tissues indicated in Table 8 were prepared for microscopic examination and weighed, respectively. Organs weight was recorded for all scheduled euthanasia animals. Organ weights was not be recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) was calculated. According to Table 8 indications, representative samples of the tissues were collected from all animals and preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). Additional tissue samples were collected to elucidate abnormal findings, if necessary. For females which fail to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) were fixed (if applicable) but were not be examined histopathologically in first instance.
Postmortem examinations (offspring):
F1 AND F2 GENERATION (UNTIL WEANING)

SACRIFICE
- Unscheduled deaths (F1 and F2 generation): pups and fetuses were euthanized for humane reasons as per Test Facility SOPs, if necessary. Recognizable fetuses of females that died spontaneously or were euthanized in extremis were examined externally and sexed (both externally and internally, if possible). Pups that were to be sacrificed in extremis, younger than 7 days, were euthanized by decapitation. Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital.
- Scheduled euthanasia (F2 generation): scheduled necropsy of the non-selected F2-animals of Cohort 1B were conducted on PND 21-23. The animals were be deprived of food overnight. These pups were deeply anesthetized using isoflurane and subsequently exsanguinated.

GROSS NECROPSY
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol if not being subjected to necropsy on the same day. 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 or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all abnormalities was recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
The non-selected F2-animals of Cohort 1B were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. Brain, spleen and thymus were weighed while mammary gland (both sexes) and gross lesions were collected for elucidate eventual findings. All remaining pups were sacrificed by intraperitoneal injection of sodium pentobarbital and were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.


F1 GENERATION (FROM WEANING ONWARDS - Cohort 1A, 1B, 1C, 2A, 2B, Cohort Surplus)

Please refer to Table 9 for a complete overview of the terminal procedures of the animals belonging to Cohort 1A, 1B, 1C, 2A, 2B, Cohort Surplus and spare animals.

SACRIFICE
- Unscheduled deaths: animals euthanized for humane reasons were deeply anesthetized using isoflurane and subsequently exsanguinated.
- Scheduled necropsy of spare F1-animals: animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital.
- Scheduled necropsy of Cohort 1A: scheduled necropsy was conducted on PND 89-95. Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated.
- Scheduled necropsy of Cohort 1B: scheduled necropsy of the F1-Generation of Cohort 1B was conducted on the following days:
F1-Males (which sire and fail to sire): following completion of the mating period.
F1-Females which deliver: LD 21-23
F1-Females which fail to deliver: with evidence of mating: Post-coitum Days 25-27. Without evidence of mating: Approximately 24-26 days after the last day of the mating period.
F1-Females with total litter loss: dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing. The F1-animals of Cohort 1B were not deprived of food overnight before necropsy. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated.
- Scheduled necropsy Cohort of 1C: scheduled necropsy of Cohort 1C were conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated.
- Scheduled necropsy of Cohort 2A and 2B: scheduled necropsy of Cohort 2A was conducted on PND 76-90. Scheduled necropsy of Cohort 2B was conducted on PND 21-22. The animals were not deprived of food overnight before necropsy.
The animals were first anesthetized using isoflurane and were subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution.
- Scheduled necropsy of Cohort Surplus: scheduled necropsy of Cohort Surplus was conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated.

GROSS NECROPSY
- Unscheduled deaths and euthanized in extremis: when an animal died during the study, a necropsy was conducted within 24 hours. If necessary, the animal was refrigerated to minimize autolysis.
- Spare animals: animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally, if possible). Descriptions of all external abnormalities were recorded.
- Cohort 1A: all animals were subjected to a full postmortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Cohort 1B: at necropsy the animals had the terminal body weight recorded and the numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.
- Cohort 1C: terminal body weights were not recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Cohort 2A and 2B: all animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
- Cohort Surplus: all animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- For all animals external abnormalities were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Additional tissue samples were collected to elucidate abnormal findings, if necessary. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) were calculated.
- For Cohort 1A, at necropsy the animals had the terminal body weight recorded. For animals of the control and the high dose groups, one of the ovaries was quantitatively evaluated for follicles (primordial and small growing follicles counted together), as well as corpora lutea initially. In case a treatment-related effect was suspected, this was extended to animals in the intermediate dose groups (Groups 2 and 3).The organs identified for weighing and representative samples of the tissues mentioned in Table 10 were weighed and collected.
- For Cohort 1B, the organs identified for weighing and representative samples of the tissues mentioned in Table 11 were weighed and collected.
- For Cohort 1C, in case of macroscopic abnormalities, only gross lesions were preserved in the most appropriate fixative together with the identification marks.
- For Cohort 2A and 2B, terminal body weight was recorded. After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull. The fixed brains were removed and weighed, and the length and maximum width of the brain were measured for all animals selected for neuropathology. For length of the brain it is intended a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum. For width of the brain it is intended the pituitary region. Measurements were conducted using an digital caliper. Subsequently, the brain were fixed in 10% buffered formalin together with selected PNS tissues. Sections of the brains of all Cohort 2A and 2B animals (all groups) were also be stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet. For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections were obtained. Morphometric (quantitative) analyses of CNS tissues were performed for Cohort 2A and 2B animals of Groups 1 and 4 in first instance. In case a treatment-related effect was suspected, this was extended to animals in the intermediate dose groups (Groups 2 and 3). Representative samples of the tissues mentioned in Table 12 were collected.
- Cohort Surplus: terminal body weight was recorded. The weights of brain, spleen and thymus were recorded and representative samples of brain, gland mammary (both sexes), gross lesions/masses, spleen and thymus were collected for animals reaching scheduled necropsy.

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS - COHORT 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) were 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-cytotoxicity cells, B-cells, NK-cells, ratio T-helper cells/T-cytotoxic cells (Th/Tc). The % lymphoid cells of peripheral blood mononuclear cells (PBMC) was determined using the Forward Scatter and Side Scatter.


F2 GENERATION (FROM WEANING ONWARDS)

SACRIFICE
- Scheduled necropsy of the selected F2-animals of Cohort 1B were conducted after these animals have been found positive for balanopreputial separation or after occurrence of first estrous. The animals were be deprived of food overnight. All pups were sacrificed by intraperitoneal injection of sodium pentobarbital.

GROSS NECROPSY
- These pups were subjected to a limited examination, with special attention being paid to the reproductive organs. The weights of brain, spleen and thymus were recorded and representative samples of brain, gland mammary (both sexes), gross lesions/masses, spleen and thymus were collected for animals reaching scheduled necropsy. Descriptions of all macroscopic abnormalities were recorded. For unscheduled deaths (sacrificed in extremis or found dead), a full list necropsy was performed. See Table 10.
Statistics:
Please refer to chapter "Statistics" under "Any other information on materials and methods incl. tables".
Reproductive indices:
- Mating index males: (Number of males mated / Number of males paired) x 100
- Mating index females: (Number of females mated / Number of females paired) x 100
- Precoital time: number of days between initiation of cohabitation and confirmation of mating
- Fertility index males: (Number of pregnant females / Number of males mated) x 100
- Fertility index females: (Number of pregnant females / Number of females mated) x 100
- Gestation index: (Number of females with living pups on Day 1 / Number of pregnant females) x 100
- Duration of gestation: number of days between confirmation of mating and the beginning of parturition
- Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Offspring viability indices:
- Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
- Weaning index (%): (Number of live offspring on Day 21 after littering / Number live offspring on Day 4 (after culling)) x 100
- Percentage live males at weaning (%): (Number of live male pups on Day 21 after littering / Number of live pups on Day 21 after littering) x 100
- Percentage live females at weaning (%): (Number of live female pups on Day 21 after littering / Number of live pups on Day 21 after littering) x 100

NB: group mean values of precoital time and duration of gestation were calculated from individual values of F0-females and F1- females, the remaining group values were calculated from the total number in each group. Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations and no clinical signs were noted during weekly arena observations.
Salivation seen after dosing among animals of the 100, 300 and 1000 mg/kg bw/day dose groups in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). All other recorded clinical signs were considered to be background findings.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred that was considered to be directly related to treatment with the test item. A total of four females at 1000 mg/kg bw/day were found dead prior to dosing on lactation Days 15, 11, 13 and 16, respectively (pups were cross-fostered to other litters of the same age to the extent possible). There were no relevant clinical signs or body weight changes for these females prior to death. Amongst necropsy findings for these animals, foamy contents in the trachea and lungs that were not collapsed were recorded, which were not recorded for surviving animals. No cause of death could be found histopathologically (it should be noted that histopathological assessment was compromised by advanced tissue autolysis). Although these deaths occurred at the high dose only, none of these animals showed any prior signs of a deteriorated health condition, nor did any of the surviving animals at this dose showed any signs of ill health during dosing administration. Based on the observed foamy contents in the respiratory tract which were noted exclusively for these four animals, it was considered that these deaths were related to a gavage-procedure rather than being directly related to treatment with the test item.
All (other) animals at 100, 300 and 1000 mg/kg bw/day survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 and 1000 mg/kg bw/day, a dose-dependent lower mean body weight gain was recorded for males throughout treatment (statistically significant on most occasions), which was considered to be related to treatment with the test item. Absolute mean body weight for these animals was statistically significantly lower from Week 4 of post-mating and Week 2 of mating onwards, at 300 and 1000 mg/kg bw/day respectively. Mean body weight at the end of treatment was 0.95x and 0.88x of control at 300 and 1000 mg/kg bw/day, respectively. Females at 1000 mg/kg bw/day showed a higher mean body weight gain and absolute mean weight (1.05x of control for absolute mean weight) on Day 21 of lactation only, which was considered to be related to treatment with the test item. Body weight and body weight gain of these females during the preceding period was considered unaffected by treatment with the test item. Given the slight degree of these body weight changes, they were considered non-adverse.
Body weights and body weight gain of males at 100 mg/kg bw/day and of females at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item throughout the treatment period (including post-coitum and lactation).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean absolute and/or relative food consumption was higher from Week 2 of treatment onwards for males, and during most of the post-coitum period and during the last week of the lactation period for females. These changes were considered to be related to treatment with the test item. Mean over mean food intake was 1.20x of control at the end of treatment for males, and 1.11x and 1.10x of control for females during the post-coitum and lactation period, respectively. Given the slight degree of these food intake changes, they were considered non-adverse.
Absolute and relative food consumption of males and females at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant hematological changes at 1000 mg/kg bw/day were considered to be related to treatment with the test item (fold changes in mean values as compared to the control group are indicated between parentheses):
• Lower mean red blood cell counts (RBC) for males and females (0.89x and 0.91x of control, respectively).
• Higher mean reticulocyte counts (RETIC) for males (1.34x of control).
• Lower mean hemoglobin (HGB) concentration for males (0.95x of control).
• Higher mean corpuscular volume (MCV) for males and females (1.07x and 1.05x of control, respectively).
• Higher mean corpuscular hemoglobin (MCH) for males and females (1.06x of control for both sexes).
These changes were indicative of altered red blood cell turn-over. The degree of change in red blood cell counts was however relatively small. These also occurred in the absence of any degenerative morphological findings, and as such they were considered non-adverse.
Two males at 1000 mg/kg bw/day showed higher neutrophil (NEUT) and monocyte (MONO) counts, as a result of which the mean of these parameters were also higher (statistically significant for monocyte counts only). As this concerned two animals only, and since there were no test item-related morphological correlates, these variations were considered not to represent an effect of treatment with the test item.
Hematological parameters at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical chemistry parameters at 1000 mg/kg bw/day were considered to be related to treatment with the test item (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
• Higher mean alanine aminotransferase activity (ALT) for males (1.21x of control; not statistically significant).
• Higher mean aspartate aminotransferase activity (AST) for males (1.18x of control; not statistically significant).
• Higher mean albumin concentration (ALB) for males (1.08x of control).
• Higher mean total bilirubin concentration (TBIL) for males and females (1.65x and 1.31x of control, respectively).
• Higher mean urea concentration for males and females (1.19x and 1.21x of control, respectively).
• Lower mean glucose concentration (GLUC) for males (0.79x of control).
• Lower mean cholesterol concentration (CHOL) for males (0.69x of control; notably low values were recorded for two males).
• Higher mean inorganic phosphate (PHOS) level for males (1.26x of control).
These clinical pathology changes were considered non-adverse as none had morphological correlates.
The statistically significantly higher mean calcium concentrations in females at 300 and 1000 mg/kg bw/day were considered to be unrelated to treatment with the test item as these variations occurred in the absence of a dose-related trend.
Clinical chemistry parameters at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item.
Endocrine findings:
no effects observed
Description (incidence and severity):
Serum levels of TSH and T4 for F0-males and -females were considered not affected by treatment with the test item. Any variations in mean values occurred in the absence of a dose-related trend and/or were within ranges expected for rats of this age and strain.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in urinary parameters were considered to be related to treatment with the test item (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
• Lower mean pH values for males at 300 and 1000 mg/kg bw/day (6.30 and 6.10 vs 7.00 in the control group, respectively).
• Higher mean urinary volume for males at 1000 mg/kg bw/day (2.20x of control; due to high values for two males).
These changes were not associated with morphological correlates and therefore were considered non-adverse.
Urinary parameters at 100 mg/kg bw/day were considered not affected by treatment with the test item.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer also to Table 15 under "Any other information on results incl. tables".

An increased incidence and/or severity of extramedullary hematopoiesis in the spleen was present at 1000 mg/kg bw/day in males up to moderate and females up to slight degree. This microscopic finding likely correlated with the higher spleen weight.
There were no other test item-related histologic changes. The remainder 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 Evaluation of Reproductive Performance – F0-Generation
In the control group there was one female and in the 1000 mg/kg bw/day group there were two females that were not pregnant. In the 100 mg/kg bw/day group, 1/25 couples did not mate. In the 300 and 1000 mg/kg bw/day group, one or two respectively had no offspring.
No abnormalities were seen in the reproductive organs or mammary glands (examined for all control group and 1000 mg/kg bw/day treated males and females), that could explain the absence of pregnancy.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not affected by treatment with the test item.
All females had a regular cycle of 4 days, except for one female at 100 mg/kg bw/day (this female showed evidence of mating and had a normal litter). Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation to treatment with the test item.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm counts, motility and morphology were considered unaffected by treatment with the test item.
The statistically significantly higher progressive sperm count at 1000 mg/kg bw/day was considered not related to treatment with the test item since the control mean was considered to be slightly low compared to historical control data , there was no clear dose-related response and the intra-group variation was essentially similar between the control group and the 1000 mg/kg bw/day group. In addition, this variation comprised an increased motility rather than a decreased motility.
The lower number of cells with detached head at 100 mg/kg bw/day occurred in the absence of a dose-related response and concerned a reduced instead of an increased count. This variation was therefore considered not to be related to treatment with the test item.
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.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTION DATA - F0 GENERATION
Male and female mating index was considered not affected by treatment with the test item. All females, except for one female at 100 mg/kg bw/day, showed evidence of mating.
The incidence of this single case appeared not dose-related and occurred within normal ranges. As such, it was considered unrelated to treatment with the test item.

Precoital time was not affected by treatment with the test item.
Most females showed evidence of mating within 4 days, except for two control females and one female each at 100, 300 and 1000 mg/kg bw/day which showed evidence of mating after 12 to 14 days. The incidence of these longer periods until evidence of mating was detected showed no dose-related trend and was therefore not attributed to treatment with the test item.

Number of implantation sites was considered not affected by treatment with the test item.
One female each at 300 and 1000 mg/kg bw/day had one implantation site (without offspring). The incidence of these findings showed no dose-related trend as such was considered not to be related to treatment with the test item.

Female fertility index (number of pregnant females as percentage of the number of mated females) and male fertility index (number of pregnant females as percentage of the number of mated males) was considered not affected by treatment with the test item. Male and female fertility indices were 96% for the control, 100% for the 100 and 300 mg/kg bw/day groups and 92% for the 1000 mg/kg bw/day group.
One control female and two females at 1000 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no clear dose-related incidence across the dose groups, and recorded incidences can occur for rats of this age and strain in this type of study, this was considered not to be related to treatment with the test item.

DEVELOPMENTAL DATA - F0 GENERATION
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item.
The statistically significantly higher gestation duration at 1000 mg/kg bw/day was considered not to be related to treatment with the test item since the range within which these animals delivered after pairing was the same as for the control group, i.e., after 21 or 22 days of gestation and within the range considered normal for rats of this strain and age. All females at 100 and 300 mg/kg bw/day also delivered after 21 or 22 days of gestation, except for a single female at 100 mg/kg bw/day that delivered after 23 days of gestation. This was considered to be an incidental occurrence, unrelated to treatment with the test item.
Except for the failed pregnancy of one female each at 300 and 1000 mg/kg bw/day (both had one implantation only), all pregnant females had live offspring. These failed pregnancies occurred without related histopathology changes in reproductive organs and were therefore judged to be unrelated to treatment with the test item due to the incidental occurrence and lack of a dose-related trend. The gestation indices were 100% for the control and 100 mg/kg bw/day groups, and 96% for the 300 and 1000 mg/kg bw/day groups.

No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not affected by treatment with the test item. These index values were 95% for the control, 100 and 300 mg/kg/day groups and 92% for the 1000 mg/kg bw/day group.

Litter size was considered not affected by treatment with the test item.
Mean live litter sizes were 12.3, 12.4, 12.0 and 11.5 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Means of all groups remained well within normal ranges.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not affected by treatment with the test item. The live birth indices were 98% for the control group, 100% for the 100 and 300 mg/kg bw/day groups, and 99% for the 1000 mg/kg bw/day group.
A total of six pups over three litters of the control group and two pups over two litters of the 1000 mg/kg bw/day group were found dead at first litter check. The two pups at 1000 mg/kg bw/day and four pups of one litter of the control group were found to have no milk in the stomach at necropsy. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability index (number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) was considered not affected by treatment with the test item. Viability indices were 99% for the control, 100 and 1000 mg/kg bw/day groups and 98% for the 300 mg/kg bw/day group.
A total of three pups over three litters of the control group, two pups of one litter of the
100 mg/kg bw/day group, seven pups over six litters of the 300 mg/kg bw/day group and three pups over three litters of the 1000 mg/kg bw/day group were found dead or missing on PND 2 or 3. Pups missing were most likely cannibalized. Relevant clinical signs recorded for these pups at first litter check were confined to blue appearance and cold to touch for one control pup, and cold to touch and absence of milk in the stomach for one pup at 1000 mg/kg bw/day. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Weaning index (number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling)) was considered not affected by treatment with the test item.
Weaning index was 99% for the control, 100 and 300 mg/kg bw/day groups, and 94% for the 1000 mg/kg bw/day group. As specified under Mortality section, a total of four females at
1000 mg/kg bw/day was found dead during lactation. These deaths were ascribed to the gavage-procedure. Pups of these females were reallocated to other litters of the same group to the extent possible. For two females, only part of the pups could be reallocated and those that could not be reallocated were sacrificed together with the dam. These pups were taken into account when calculating the weaning index for this dose group. As such, the lower calculated weaning index at 1000 mg/kg bw/day is due to the described reallocation of pups and was therefore not directly related to treatment with the test item. One control pup that was found dead on PND 19 was cannibalized to an advanced stage. Relevant clinical signs for the pup at 300 mg/kg bw/day that was found missing on PND 9 included dehydrated and lean appearance between PND 3 and 8 with less milk in the stomach and pale appearance on PND 3. One pup at 1000 mg/kg bw/day was sacrificed for ethical reasons on PND 16 due to a dislocated femur of the left hindleg (confirmed during the in-life phase as abnormal posture of the left hindleg). One pup at 100 mg/kg bw/day was sacrificed for ethical reasons on PND 11 due to severe swelling of the right foreleg.
These intercurrent sacrifices were considered not related to treatment with the test item since these could either be attributed to an injury or occurred incidentally.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1-Generation (Cohort 1B) from Weaning Onwards
No clinical signs were recorded that were considered to be related to treatment with the test item.
Salivation seen after dosing among animals of the 100, 300 and 1000 mg/kg bw/day dose groups in a dose-related manner was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). All other recorded clinical signs were considered to be background findings.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
F1-Generation (Cohort 1B) from Weaning Onwards
No mortality occurred that was considered to be related to treatment with the test item.
One male at 1000 mg/kg bw/day (Cohort 1B) was sacrificed for ethical reasons on PND 25 (Dosing Day 5) prior to dosing as this animal had a lean appearance, and showed lethargy, hunched posture and uncoordinated movements (this animal was replaced by a spare animal). As this death occurred shortly after initiation of dosing in the post-weaning phase, and similar findings were absent for other animals at this dose level, this death was considered unrelated to treatment with the test item.
One female at 1000 mg/kg bw/day (Cohort 1B) was found dead on Lactation Day 16 (dosing Day 113). This female only showed salivation on the days prior to its death and there were no effects on body weight. Main macroscopic findings were beginning autolysis, reddish foci on the thymus, failure of the lungs to collapse and hemorrhagic fluid in the ileum. There were no microscopic findings indicating a cause of morbidity. This death was therefore considered incidental and not related to the test item.
One female at 100 mg/kg bw/day (Cohort 1B) and one female at 300 mg/kg bw/day (Cohort 1B) were sacrificed for ethical reasons (breathing difficulties) on dosing Days 44 and 47, respectively. These females showed hunched posture, laboured respiration, gasping, piloerection and/or lean appearance prior to sacrifice. Necropsy findings for both these animals consisted of esophagus perforation, watery-clear oil like fluid in the thoracic cavity and the thymus having grown together with heart and lungs. For one animal, the macroscopic esophagus perforation was correlated microscopically with slight muscle fiber degeneration/necrosis. For the other animal, the main microscopic finding was slight diffuse fibrosis of the pericardium. Other main macroscopic findings noted for this animal included an accentuated lobular pattern of the liver with yellowish discoloration of the glandular mucosa of the stomach. Based on these macroscopic and microscopic findings the cause of moribundity was considered to be gavage procedure-related.
No further mortality occurred in any dose group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1-Generation (Cohort 1B) from Weaning Onwards
At 1000 mg/kg bw/day, lower mean absolute body weight was recorded for males throughout the dosing period (absolute mean body weights were 0.81x of control at the end of the dosing period). Weight gain of these males was lower than control means throughout the dosing period (not statistically significant on Day 99). For females at this dose level, lower mean absolute body weight was recorded from Day 1 to Day 22 of dosing only (0.93x of control on treatment Day 22). The statistically significant lower mean absolute body weights that were occasionally recorded on subsequent days (prior to the post-coitum phase) for these females were similar to the means encountered at the 100 and 300 mg/kg bw/day groups, and therefore considered not to represent an effect of treatment with the test item and therefore non-adverse. Mean weight gain of these females was lower during the first week of dosing only. Body weight and weight gain during the post-coitum and lactation period were considered not affected by treatment with the test item.
At 100 and 300 mg/kg bw/day, mean body weight gain of males and females was lower from Day 22 onwards (in a dose-related manner for males; statistically significant on most occasions). This was attributed to higher mean absolute body weights on Day 1 of the post weaning phase (not statistically significant for females). Except for slight but statistically significant higher mean absolute weights for males at 300 mg/kg/day on Days 8 and 15, absolute mean body weights of males and females remained similar to control means throughout the dosing period. This was therefore considered not to represent an effect of treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1-Generation (Cohort 1B) from Weaning Onwards
At 1000 mg/kg bw/day, mean relative food intake of males and females was higher than control from Week 2 of dosing onwards (not always statistically significant). Mean absolute food intake was only slightly higher on most occasions. Mean over mean relative food consumption at the end of the dosing period was 1.16x and 1.13x of control for males and females, respectively. Mean over mean relative food consumption at the end of the post-coitum period was 1.17x of control. Absolute and relative food consumption during the lactation phase was considered not affected by treatment with the test item.
The higher mean relative food intake of females at 300 mg/kg bw/day between Days 15 and 22 and between Days 29 and 36 was attributed to the lower mean weights as absolute food consumption remained essentially similar to control means. Between Days 22 and 29, mean relative food intake of these females was lower than the control, due to a slightly lower food consumption in this period.
As these changes were not consistently seen with continuing treatment, these changes in relative food intake were considered not to represent an effect of treatment with the test item.
Absolute and relative food consumption at 100 mg/kg bw/day was considered not affected by treatment with the test item. Any other statistically significant variations in absolute or relative food intake recorded across the dose groups were considered not to represent an effect of treatment with the test item, as these occurred in the absence of a dose-related trend and/or were not consistently seen with continuing treatment.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1B
The statistically significant lower absolute weights of thyroid gland, adrenal gland and epididymides in males at 1000 mg/kg bw/day and the statistically significant higher testes and epididymides weight (relative to body weight) in males at 300 and 1000 mg/kg bw/day were considered to be secondary to a test item-related effect on terminal body weight. For females there were no significant organ weight differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Cohort 1B
There were no test item-related gross observations.
All of the recorded macroscopic findings were considered to be within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Cohort 1B
In the control group there was one female without offspring. In the 100, 300 and the 1000 mg/kg bw/day group there was one female each that was not pregnant. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. There were no test item-related microscopic alterations in the reproductive organs of males and females at 1000 mg/kg bw/day.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Cohort 1B
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.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTIVE DATA - F1 GENERATION

Cohort 1B
Male and female mating index was not affected by treatment with the test item. All females showed evidence of mating.

Precoital time was considered not to be affected by treatment with the test item.
Most females showed evidence of mating within four days. A total of two out of twenty females at 1000 mg/kg bw/day showed evidence of mating at thirteen days after commencement of pairing. The mating date of one animal was estimated at 21 days prior to delivery date as mating was overlooked initially. The incidence of females having a longer precoital time that four days occurred within the range expected for rats of this age and strain, and was therefore considered not to represent an effect of treatment with the test item.

Number of implantation sites was considered not to be affected by treatment with the test item.

Female fertility index (number of pregnant females as percentage of the number of mated females) and male fertility index (number of pregnant females as percentage of the number of mated males) was considered not to be affected by treatment with the test item. The male and female fertility indices were 100% for the control and 95% for the 100, 300 and 1000 mg/kg bw/day groups.
One female each in the 100, 300 and 1000 mg/kg bw/day groups were non-pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment with the test item.

DEVELOPMENTAL DATA - F1 GENERATION

Cohort 1B
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item. Gestation index was 95% for the control group (one female had implantations only), and 100% for the 100, 300 and 1000 mg/kg bw/day groups. All pregnant females had live offspring.

No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment with the test item. Post-implantation survival indices were 92, 96, 90 and 92% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Litter size was considered not affected by treatment with the test item.
Live litter sizes were 10.2, 10.4, 9.8 and 9.7 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test item. Live birth indices were 100% for the control and 100 mg/kg bw/day groups and 99% for the 300 and 1000 mg/kg bw/day groups.
One pup at 300 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found dead at first litter check. These dead pups were considered not related to treatment as the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability indices were 99% for the control 300 and 1000 mg/kg bw/day groups, and 100% for the 100 mg/kg bw/day group.
One pup of the control group and one pup at 300 mg/kg bw/day were sacrificed in extremis on PND 4 based on a wound on the left foreleg (related to the identification procedure). One pup at 300 mg/kg bw/day and two pups at 1000 mg/kg bw/day were found missing on PND 2 or 3. These pups were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose related trend and remained within the range considered normal for pups of this age.

Weaning index (number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling)) was considered not to be affected by treatment with the test item. The weaning indices were 100% for the control group, 99% for the 100 and 300 mg/kg bw/day groups and 94% for the 1000 mg/kg bw/day group.
One pup at 100 mg/kg bw/day was found dead on PND 19. This pup showed pallor and lethargy on PND 18. One pup at 300 mg/kg bw/day was found missing on PND 9 and was most likely cannibalized. All pups from one litter at 1000 mg/kg bw/day were sacrificed on PND 16 after the dam was found dead on Lactation Day 16. This also resulted in a higher breeding loss at 1000 mg/kg bw/day. As also described under section "Mortality", the death of this female was considered as incidental and not related to treatment with the test item. One additional pup at 1000 mg/kgbw/day was sacrificed in extremis on PND 16 due to edema of the head. No toxicological relevance was attributed to these dead/missing/sacrificed pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups - Lactation phase
No clinical signs occurred among surviving pups that were considered to be related to treatment with the test item.
Any clinical signs that were recorded occurred within the range of background findings to be expected for pups of this age and strain which are housed under the conditions in this study. These clinical signs did not show any apparent dose-related trend, and at the incidence observed, these were considered to be unrelated to treatment with the test item.

Cohort 1A, 1C and Cohort 2A from weaning onwards
Please refer to clinical signs findings reported under "Results: P1 (second parental generation)" which also report Cohort 1B results. No clinical signs were recorded that were considered to be related to treatment with the test item.
One female at 1000 mg/kg bw/day (Cohort 1A) showed abnormal posture of the left hindleg and abnormal gait , and one female at 1000 mg/kg bw/day (Cohort 2A) showed abdominal swelling during the dosing period. Since these clinical signs were not recorded for any other animal of the same dose group, these were considered unrelated to treatment with the test item.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Cohort 1A, 1C and Cohort 2A from weaning onwards
Please refer to mortality findings reported under "Results: P1 (second parental generation)" which also report Cohort 1B results. No mortality occurred that was considered to be related to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 pups - Lactation phase
At 1000 mg/kg bw/day, a statistically significantly higher mean pup weight was recorded for both sexes on PND 1 (combined sexes: 1.08x of control), which was considered to be related to treatment with the test item.
Body weights of pups at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item.

Cohort 1A, 1C and Cohort 2A from weaning onwards
Please refer to body weight findings reported under "Results: P1 (second parental generation)" which also report Cohort 1B results. Overall, effects on body weight were observed in both sexes across all dose groups without any relationship to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A, 1C and Cohort 2A from weaning onwards
Please refer to food consumption findings reported under "Results: P1 (second parental generation)" which also report Cohort 1B results.
Overall, absolute and relative food consumption fluctuations were observed across all dose groups, particularly for the 300 and 1000 mg/kg bw/day animals. These observations were not considered treatment-related.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
The following changes in hematology parameters were considered to be related to treatment with the test item:
• Higher mean monocyte counts (MONO) for males and females at 1000 mg/kg bw/day (1.35x and 1.78x of control, respectively; not statistically significant), and at 300 mg/kg bw/day for females only (1.57x of control; not statistically significant).
• Lower mean red blood cell counts (RBC) for males and females at 1000 mg/kg bw/day (0.90x and 0.89x of control, respectively).
• Higher mean reticulocyte count (RETIC) for males and females at 1000 mg/kg bw/day (1.27x and 1.24x of control, respectively).
• Higher mean value for mean corpuscular volume (MCV) for males and females at 1000 mg/kg bw/day (1.07x and 1.08x of control, respectively).
• Higher mean value for mean corpuscular hemoglobin (MCH) for males and females at 1000 mg/kg bw/day (1.07x and 1.06x of control, respectively; not statistically significant for females). These changes were indicative of altered red blood cell turn-over. The degree was considered relatively small, as were the recorded severities of extramedullary hematopoiesis. These also occurred in the absence of any degenerative morphological findings, and as such they were considered non-adverse.
Hematology parameters at 100 mg/kg bw/day were considered not affected by treatment with the test item.

At 1000 mg/kg bw/day, mean prothrombin time (PT) was longer for males and females (1.06x of control for both sexes). The effect was relative small and not considered adverse.
Coagulation parameters at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 pups (pre-weaning)
Serum T4 levels in male and female pups, culled at PND 4 were considered not affected by treatment with the test item. A very slight apparent dose-related trend towards increased T4 values was recorded over the dose groups. As these represented only small variations, and means remained within ranges considered normal for rats of this age and strain, these were considered not to be related to treatment with the test item.

F1 Cohort Surplus
Serum T4 and TSH levels in male and female pups of Cohort Surplus at PND 22 were considered not affected by treatment with the test item.

Cohort 1A
The following changes in clinical chemistry parameters were considered to be related to treatment with the test item:
• Higher mean alanine aminotransferase activity (ALT) for males at 1000 mg/kg bw/day (1.21x of control).
• Higher mean aspartate aminotransferase activity (AST) for males at 1000 mg/kg bw/day (1.31x of control; not statistically significant).
• Higher mean total bilirubin concentration (TBIL) for males at 1000 mg/kg bw/day (1.21x of control; not statistically significant).
• Higher mean urea concentration for males at 300 and 1000 mg/kg bw/day (1.15x and 1.36x of control, respectively; not statistically significant at 300 mg/kg bw/day) and for females at 100, 300 and 1000 mg/kg bw/day (1.19x, 1.16x and 1.39x of control, respectively; not statistically significant at 300 mg/kg bw/day).
• Lower mean glucose concentration (GLUC) for males at 300 mg/kg bw/day (0.88x of control; not statistically significant), and for males and females at 1000 mg/kg bw/day (0.71x and 0.90x of control, respectively; not statistically significant for females).
• Lower mean cholesterol concentration (CHOL) for males and females at 100, 300 and 1000 mg/kg bw/day (males: 0.88x of control at 100 and 300 mg/kg bw/day, and 0.66x at 1000 mg/kg bw/day; not statistically significant at 100 and 300 mg/kg bw/day. Females: 0.94x, 0.91x and 0.79x of control at 100, 300 and 1000 mg/kg bw/day, respectively; not statistically significant).
• Higher mean potassium concentration (K) for females at 1000 mg/kg bw/day (1.08x of control).
• Higher mean inorganic phosphate concentration (PHOS) for males and females at 1000 mg/kg bw/day (1.16x and 1.38x of control, respectively).
None of these clinical pathology changes had morphological correlates and were therefore considered non-adverse.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
The following changes in urinary parameters were considered to be related to treatment with the test item:
• Lower mean urinary pH of males at 1000 mg/kg bw/day (6.10 vs. 6.60 in the control group) and of females at 300 and 1000 mg/kg bw/day (6.20 and 6.15, respectively, vs. 6.80 in the control group).
• Higher mean specific gravity for females at 1000 mg/kg bw/day (1.01x of control).
Urinalysis parameters at 100 mg/kg bw/day were considered not affected by treatment with the test item.
None of these clinical pathology changes had morphological correlates and were therefore considered non-adverse.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups (pre-weaning)
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not affected by treatment with the test item.
The statistically significantly higher mean normalized anogenital distance of male pups at 300 and 1000 mg/kg bw/day occurred without a clear dose-related trend. These variations were therefore considered not to be related to treatment with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 pups (pre-weaning)
Treatment up to 1000 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:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
Please refer to organ weight findings reported under "Results: P1 (second parental generation)" for Cohort 1B results. Table 16 also reports selected organ weights data for Cohort 1A.

There was a statistically significant higher liver weight in males (relative to body weight) and females (absolute and relative to body weight) at 1000 mg/kg bw/day. For males there was no microscopic correlate to the higher liver weight. These findings were considered non-adverse at the severities recorded at histopathology and in the absence of any degenerative findings.
There was a statistically significant higher kidney weight in males at 300 and 1000 mg/kg bw/day (absolute and relative to body weight) and in females at 1000 (absolute and relative to body weight). The significant higher kidney weight (relative to body weight) in females at 100 and 300 mg/kg bw/day were considered incidental and not test item-related. There was no microscopic correlate for the higher kidney weight and therefore this was considered non-adverse.
There was a statistically significant higher spleen weight in males (relative to body weight) and females (absolute and relative to body weight) at 1000 mg/kg bw/day.
The statistically significant lower absolute weights and/or higher relative weights of brain, heart, pituitary gland, thyroid gland, thymus, adrenal gland, testes, epididymides and seminal vesicles weight in males at 1000 mg/kg bw/day were considered to be secondary to a test item-related effect on terminal body weight. The statistically significant higher weight of axillary lymph node (absolute and relative to body weight) in females at 100 mg/kg bw/day lacked a dose response and was considered incidental and not test item-related.

Cohort 2A and 2B
Fixed brain weights: fixed brain weights of Cohort 2A and 2B animals were considered not affected by treatment with the test item.
Statistically significant higher fixed brain weights (relative to body weights) noted in the 300 and 1000 mg/kg bw/day Cohort 2A males were attributed to the lower terminal body weight and considered not a direct effect of the test item.

Cohort Surplus
Brain, thymus and spleen weights were considered not affected by treatment with the test item.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups (until weaning)
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test item.
All recorded macroscopic findings were considered to occur within the range of lesions to be expected for pups of this age and strain.

Cohort 1A and 1C
Please refer to gross pathological findings reported under "Results: P1 (second parental generation)" which also report Cohort 1B results.
There were no test item-related gross observations.
All of the recorded macroscopic findings were considered to be within the range of background gross observations encountered in rats of this age and strain.

Cohort 2A and 2B
Brain dimensions: please refer to Table 17 under "Any other information on results incl. tables" reporting Cohort 2A brain dimensions data.
For Cohort 2A, at PND 76-87 there was a statistically significant lower brain width in males treated at 1000 mg/kg bw/day and in females at 100 and 1000 mg/kg bw/day. For males, the small width difference (with a statistically non-significant brain length difference) was considered secondary to the lower terminal body weight and the resulting lower brain weight (relative to body weight). For females the significant differences were considered incidental in the absence of a dose response and considering the most pronounced decrease was at the low dose. There were no other statistically significant changes in brain width or length.
For Cohort 2B, brain dimensions (length and width of brain) at PND 21-22 were considered not affected by treatment with the test item.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
Please refer to Table 18 under "Any other information on results incl. tables" reporting selected histopathological findings.
Spleen: an increased incidence and severity of extramedullary hematopoiesis was present at 1000 mg/kg bw/day in males and females up to moderate degree. This microscopic finding likely correlated with the higher spleen weight. Similarly to the F0 generation, these findings were not considered adverse based on the low incidence and severity, and in absence of any degenerative morphological findings.
Liver: hepatocellular vacuolation was present in females at 1000 mg/kg bw/day up to slight degree and hepatocellular hypertrophy was present in females at 1000 mg/kg bw/day at minimal degree. For females, these microscopic findings likely correlated with the higher liver weight. These findings were considered non-adverse at the current severities and in the absence of any degenerative findings.
There were no other test item-related histologic changes. The remainder 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.

Cohort 2A and 2B
Morphometric analysis of the brain on PND 21-23 (Cohort 2B) and PND 76-87 (Cohort 2A) revealed no test item-related differences in animals administered the test item at 1000 mg/kg bw/day.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups (pre-weaning)
Sex ratio was considered not affected by treatment with the test item.

Cohort 1A
Estrous cycle: estrous cycle length and regularity were considered not affected by treatment with the test item. For most females, regular cycles of 4 to 5 days were recorded.
One control female had an irregular estrous cycle, one female at 100 mg/kg bw/day had an extended estrous (this animal had one full cycle of 3 days in length), and for two females at 100 mg/kg bw/day, the estrous stage/length of the estrous cycle could not be determined as only one full cycle was completed. Given the incidental nature and the absence of a dose-related incidence, these single occurrences were considered not to indicate a relation to treatment with the test item.

Sperm analysis: sperm motility, concentration and morphology were considered not affected by treatment with the test item.
For one male at 1000 mg/kg bw/day, a high number of sperm cells with detached head was recorded as well as a low sperm count. As this was confined to a single case at this dose level this was considered not to represent an effect of the test item.

Splenic Lymphocyte Subpopulation: splenic lymphocyte subpopulations were considered not affected by treatment with the test item.
Low T-cell and T-helper cell and high NK-cell splenic subpopulations were recorded for individual control animals (one male and one female), as a result of which control means of these parameters were slightly low.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Cohort 2A

Acoustic Startle Response
At 1000 mg/kg bw/day, mean over mean maximum response amplitude of the startle response were slightly lower (0.90x and 0.81x of control for males and females, respectively). Females at 1000 mg/kg bw/day also showed a marginally lower mean over mean average response amplitude. Means did not achieve a level of statistical significance and means remained within the historical control range. Latency to maximum response amplitude was considered not affected by treatment with the test item.
Acoustic startle response parameters at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item. Any statistically significant variations in these parameters at 100 and 300 mg/kg bw/day were considered not to represent an effect of treatment with the test item, as these occurred in the absence of a dose-related trend and/or were not consistently recorded across all test blocks.

Detailed Clinical Observations
Detailed clinical observations revealed no symptoms that were considered to be related to treatment with the test item.
The clinical symptoms that were observed were considered to be within the normal range of behavioral findings for this type of study and were generally also observed in control animals. These findings were therefore considered not to be related to treatment with the test item.
Increased activity was recorded for 1/10 females at 1000 mg/kg bw/day which was not recorded among control animals. However, this finding was considered not to be related to treatment with the test item given the absence of this finding among any other animal of this dose group, and subsequent absence of a clear dose-related response.

Rectal Temperature
Rectal temperature was considered not affected by treatment with the test item.

Motor Activity Test
At 300 and 1000 mg/kg bw/day, lower mean motor activity was recorded for females for both total movements and ambulations (0.74x and 0.60x of control for total movements, respectively, and 0.73x and 0.59x of control for ambulations, respectively; means remained within the historical control range. It should be noted that the mean and standard deviation of the control group was to some extent affected by a high value of these parameters for one control female. Without this value, the mean number of total movements and ambulations for the control group was 5562 and 1894 counts, respectively. Subsequently, means for total movements and ambulations at 300 and 1000 mg/kg bw/day would then be 0.80x and 0.65x of control for total movements, respectively, and 0.77x and 0.62x of control for ambulations, respectively. These findings occurred in the absence of any morphological changes in examined central and peripheral neuronal tissues or supportive clinical signs. Also, means of these parameters occurred within the range considered normal for rats of this age and strain. As such, the recorded changes in these parameters were considered not to represent an adverse effect of the test item.
Motor activity at 100 mg/kg bw/day was considered not affected by treatment with the test item. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Functional Observations
At 1000 mg/kg bw/day, a lower mean grip strength of the hind leg was recorded for males (0.87x of control). Also, a lower mean foot splay value was recorded for males and females at 1000 mg/kg bw/day (0.83x or 0.84x of control, respectively; not statistically significant), and also appeared marginally lower at 300 mg/kg bw/day (0.87x and 0.85x of control for males and females, respectively; not statistically significant). Means for these parameters remained within the historical control range. These findings occurred in the absence of any morphological changes in examined central and peripheral neuronal tissues or supportive clinical signs. Also, means of these parameters occurred within the range considered normal for rats of this age and strain. As such, the recorded changes in these parameters were considered not to represent an adverse effect of the test item.
Mean grip strength of the forelegs of males at 1000 mg/kg bw/day, and fore- and hindlimb grip strength of females at 1000 mg/kg bw/day and of animals at 100 and 300 mg/kg bw/day, as well as mean foot splay at 100 mg/kg bw/day were considered not affected by treatment with the test item.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
developmental neurotoxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were recorded that were considered to be related to treatment with the test item.
Relevant clinical signs that were recorded for the female at 300 mg/kg bw/day that was sacrificed in extremis on Day 22 is detailed under "Mortality".
Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups in a dose related manner was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
All other recorded clinical signs were considered to be background findings or were considered not related to treatment with the test item as they occurred in the absence of a dose-related incidence. These included hunched posture, piloerection, shaking ,and a lean appearance for one male at 100 mg/kg bw/day after dosing on Day 9 and 10 post-weaning (ascribed to misaligned teeth).
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No mortality occurred that was considered to be related to treatment with the test item.
One female each at 100 and 300 mg/kg bw/day was found dead or sacrificed in extremis, respectively, (likely) as the result of the gavage procedure as detailed below.
One female at 100 mg/kg bw/day was found dead on Day 18 post-weaning. No clinical signs or changes in body weight were observed for this animal prior to its death. At necropsy, beginning autolysis was noted. In addition, the lungs failed to collapse and the thoracic cavity contained greenish material and watery-clear contents. Based on the necropsy findings, it was conceivable that its death was related to the gavage procedure.
One female at 300 mg/kg bw/day was sacrificed in extremis on Day 22 post-weaning. Clinical signs recorded after dosing for this animal on the day of sacrifice included calm behavior, hunched posture, labored respiration and gasping, piloerection, ptosis and hypothermia. This female showed normal weight gain. Necropsy findings confirmed that this death was related to the gavage procedure and consisted of a perforated esophagus, lungs that failed to collapse, and oil-like, watery-clear, gelatinous content in the thoracic cavity.
No further mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean weight gain of both sexes and absolute mean weight of females was lower between Days 1 and 8 post-weaning, after which weight gain and absolute body weights became essentially comparable to control means. Given that these changes were relatively slight and temporary, they were considered not to be adverse.
Higher mean weight gain and/or absolute mean body weight was occasionally recorded at 100 and 300 mg/kg bw/day. As these variations occurred in the absence of a dose-related trend they were considered not to be related to treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, absolute and relative food consumption was slightly lower than control means for both males and females over Days 1 to 8 post-weaning (absolute food consumption: 0.79x and 0.86x of control for males and females, respectively; relative food consumption: 0.81x and 0.90x of control for males and females, respectively). Means did not achieve a level of statistical significance for absolute food consumption of males). Given that these changes were relatively slight and temporary, they were considered not to be adverse.
The higher absolute and/or relative food intake for both sexes at 1000 mg/kg bw/day over Days 15 to 22 post-weaning may have been a compensatory response to the lower food intake during the first week of treatment, but was considered not relevant in toxicological terms.
Absolute and relative food consumption at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item. Any other statistically significant variations in absolute and relative food consumption across the dose groups were considered not to represent an effect of treatment with the test item as these occurred in absence of a dose related trend.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, a lower mean thyroid stimulation hormone concentration (TSH) was recorded for males (0.68x of control; not statistically significant and mean remained within the historical control range of Cohort Surplus animals). As such, it was considered that TSH concentration in the F2 generation was not affected by treatment with the test item.
TSH levels at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item. Also, no relevant differences were noted for total T4 levels between the dose groups. Mean T4 values of males at 1000 mg/kg bw/day appeared slightly lower than the control mean (not statistically significant). However, the variation in mean T4 values over the dose groups was similar between both sexes and remained well within historical data of Cohort Surplus animals. As such, this variation was considered not to represent an effect of treatment with the test item.
Sexual maturation:
not specified
Description (incidence and severity):
At 1000 mg/kg bw/day, a delayed onset of vaginal opening was recorded (PND 33.6 vs. PND 32.4 in the control group; means remained within the historical control data range). Mean body weight of females at 1000 mg/kg bw/day on the day at which vaginal opening was confirmed was higher than the control mean, which indicated that this change could not be ascribed to growth retardation. Also, a similar change occurred in the F1-generation. It should be noted however that the mean at 1000 mg/kg bw/day was similar to the mean at 100 mg/kg bw/day, and therefore a relationship to treatment with the test item was equivocal. Therefore, although a relationship to treatment with the test item could not be excluded, this was considered not to represent an adverse effect of the test item.
Balanopreputial separation, time to first estrous (the interval between achieving vaginal patency and occurrence of first estrous) and age of first estrous were considered not affected by treatment with the test item. The apparent earlier mean onset of balanopreputial separation and vaginal opening at 300 mg/kg bw/day occurred in the absence of a dose-related trend and were therefore considered unrelated to treatment with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2 (cohort 1B)
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose Formulation Analyses


Accuracy


The concentrations analyzed in the formulations of low-, mid- and high-dose groups were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90-110% of target concentration).


No test item was detected in the control group formulations.


Homogeneity


The formulations of low- and high-dose groups were homogeneous (i.e. coefficient of variation ≤ 10%).


 


Table 13 - Summary of the accuracy and homogeneity tests














































































































































































































































Group (mg/kg bw/day)



Week of analysis



Target concentration (mg/mL)



Mean recovery (%)



Coefficient of variation (%)



0 (Control)



1



0



n.a.



n.a.



100



25



97



1.0



300



75



98



n.a.



1000



250



99



2.3



0 (Control)



3



0



n.a.



n.a.



100



25



103



3.9



300



75



102



n.a.



1000



250



100



3.9



0 (Control)



6



0



n.a.



n.a.



100



25



102



1.0



300



75



103



n.a.



1000



250



103



1.6



0 (Control)



9



0



n.a.



n.a.



100



25



107



4.7



300



75



106



n.a.



1000



250



103



2.0



0 (Control)



12



0



n.a.



n.a.



100



25



107



3.8



300



75



105



n.a.



1000



250



102



2.2



0 (Control)



15



0



n.a.



n.a.



100



25



99



2.1



300



75



99



n.a.



1000



250



99



1.7



0 (Control)



18



0



n.a.



n.a.



100



25



100



4.9



300



75



107



n.a.



1000



250



103



5.6



0 (Control)



21



0



n.a.



n.a.



100



25



97



1.9



300



75



100



n.a.



1000



250



98



2.7



0 (Control)



24



0



n.a.



n.a.



100



25



99



4.9



300



75



98



n.a.



1000



250



101



4.5



n.a., not applicable (in the control group no test item was detected; in the mid-dose group no homogeneity analysis was performed)


 


F0-Generation


Table 14 - Mean Percent Organ Weight Differences from Control Groups - F0-Generation


















































































































































 



Males



Females



Dose level (mg/kg bw/day):



100



300



1000



100



300



1000



 



 



 



 



 



 



 



BODY WEIGHT



-4



-6**



-15**



-1



0



-1



 



 



 



 



 



 



 



LIVER



 



 



 



 



 



 



               Absolute



-1



0



5



2



5



7



               Relative to body weight



2



6*



23**



4



6



8*



 



 



 



 



 



 



 



KIDNEY



 



 



 



 



 



 



               Absolute



-1



4



16**



6



3



11**



               Relative to body weight



3



11**



36**



6**



4



11**



 



 



 



 



 



 



 



SPLEEN



 



 



 



 



 



 



               Absolute



-4



0



4



1



3



8



               Relative to body weight



0



6



22**



2



5



10*



*: P<0.05, **: P<0.01.


 


Table 15 - Summary Test Item-Related Microscopic Findings – F0-Generation
























































































 



Males



Females



Dose level (mg/kg bw/day):



0



100



300



1000



0



100



300



1000



 



 



 



 



 



 



 



 



 



SPLEEN a



25



24



25



25



25



25



24



25



    Hematopoiesis extramedullary



 



 



 



 



 



 



 



 



       Minimal



17



16



15



9



10



12



10



14



       Slight



1



-



1



10



2



1



-



4



       Moderate



-



-



-



5



-



-



-



-



a  =  Number of tissues examined from each group.


 


Table 16 - Mean Percent Organ Weight Differences from Control Groups – Cohort 1A































































































































































 



Males



 



 



Females



 



 



Dose level (mg/kg bw/day):



100



300



1000



100



300



1000



 



 



 



 



 



 



 



BODY WEIGHT



1



-3



-17**



-1



-2



-4



 



 



 



 



 



 



 



LIVER



 



 



 



 



 



 



               Absolute



3



0



-3



3



8



15**



               Relative to body weight



3



3



17**



4



9



19**



 



 



 



 



 



 



 



KIDNEY



 



 



 



 



 



 



               Absolute



4



8*



14**



4



4



19**



               Relative to body weight



4



12**



37**



5*



5*



22**



 



 



 



 



 



 



 



SPLEEN



 



 



 



 



 



 



               Absolute



2



2



-5



-1



1



14*



               Relative to body weight



1



4



14**



1



3



18**



 



 



 



 



 



 



 



*: P<0.05, **: P<0.01.


 


Table 17 - Summary Brain Dimensions - F1-Generation Males and Females Cohort 2A



















































































 



Males



 



 



 



Females



 



 



 



Dose level (mg/kg bw/day):



0



100



300



1000



0



100



300



1000



 



 



 



 



 



 



 



 



 



BRAIN DIMENSION a



10



10



10



10



10



10



10



10



      



 



 



 



 



 



 



 



 



Brain Length (mm)



24.26



23.55



24.71



23.40



23.65



23.06



23.76



23.18



Brain Width (mm)



15.80



15.35



15.86



15.09*



15.77



14.78**



15.45



15.10*



a  =  Number of tissues examined from each group.


* Dunnett-test based on pooled variance significant at 5% (*) level.


** Dunnett-test based on pooled variance significant at 1% (**) level


 


Table 18 - Summary Test Item-Related Microscopic Findings – F1-Generation Cohort 1A
































































































































































 



Males



 



 



 



Females



 



 



 



Dose level (mg/kg bw/day):



0



100



300



1000



0



100



300



1000



 



 



 



 



 



 



 



 



 



SPLEEN a



20



20



20



19



20



20



20



20



    Hematopoiesis extramedullary



 



 



 



 



 



 



 



 



       Minimal



9



12



11



13



8



11



12



12



       Slight



1



2



1



3



1



-



-



4



       Moderate



-



-



-



2



-



-



-



3



LIVER a



20



2



-



19



20



20



20



20



    Vacuolation hepatocellular



 



 



 



 



 



 



 



 



       Minimal



-



-



-



-



-



-



-



7



       Slight



-



-



-



-



-



-



-



1



    Hypertrophy hepatocellular



 



 



 



 



 



 



 



 



       Minimal



-



-



-



-



-



-



-



8



a  =  Number of tissues examined from each group.

Conclusions:
In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following No Observed (Adverse) Effect Levels (NO(A)EL) of the test item were established:

- General toxicity (F0 and F1): NOAEL at least 1000 mg/kg bw/day
- Reproduction (F0 and F1): NOAEL at least 1000 mg/kg bw/day
- Developmental (F0, F1 and F2): NOAEL at least 1000 mg/kg bw/day
- Developmental neurotoxicity (F1): NOAEL at least 1000 mg/kg bw/day
Executive summary:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of the test item on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, 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 item 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 behavior, conception, pregnancy, parturition, and lactation.


Furthermore, the information obtained from the developmental neurotoxicity and assessments characterized potential effects in those systems.


The dose levels in this GLP-compliant OECD TG 443 study were selected to be 0, 100, 300 and 1000 mg/kg bw/day, based on results of previous toxicity studies with the test item in rats. The experimental study design was defined on the basis of the ECHA Decision No. TPE-D-2114501107-65-01/F (19 Feb 2020). The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week. F0-males (25 per group) were treated for a minimum of 10 weeks, including 2 weeks prior to mating and during the mating period, up to and including the day before scheduled necropsy. F0-females (25 per group) were treated for a minimum of 8 weeks, including 2 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Animals were allowed to rear their litters until the weaning day (PND 21). From weaning onwards, the F1 animals were splitted in Cohorts of different size. Animals of Cohorts 1A, 1B (20 animals/sex/group) and 1C (10 animals/sex/group) were used to investigate reproductive toxicity endpoints. Animals of Cohort 2A and 2B (10 animals/sex/group) were used to study neurotoxicity endpoints. A Cohort Surplus including 10 animals per sex per dose was dedicated to the investigations of thyroid hormones. Cohort 1B animals were also mated to produce an F2 generation. F1-animals of Cohorts 1A, 1B, 1C and 2A and selected F2 animals were dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort 2B, Cohort Surplus and Spares (not assigned to one of the cohorts) and non-selected F2 animals were not dosed.


Chemical analyses of formulations were conducted at regular intervals during the study to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.


For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations.


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, functional observations including acoustic startle response, clinical pathology including measurement of thyroid hormones, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations, neurohistopathological examinations and morphometric analysis.


For the F2-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, organ weights and gross necropsy findings.


In addition, the following reproduction/developmental parameters were determined for the F1- and F2-generation animals: estrous cycle, 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.


 


F0-Generation


No adverse findings were observed up to the highest dose level tested (1000 mg/kg bw/day).


Four females at 1000 mg/kg bw/day were found dead prior to dosing between lactation Days 11 and 16 which were ascribed to the gavage-procedure. There were no relevant clinical signs or body weight changes for these females prior to death. Foamy contents in the trachea and lungs that failed to collapse were recorded at necropsy, which were not recorded for surviving animals. A cause of death could not be established histopathologically, also because of advanced tissue autolysis. Although these deaths occurred at the high dose only, none of these animals showed any prior signs of a deteriorated health condition, nor did any of the surviving animals at this dose showed any signs of ill health during dosing administration. Based on the observed foamy contents in the respiratory tract which were noted exclusively for these four animals, it was considered that these deaths were related to a gavage-procedure rather than being directly related to treatment with the test item.


At 100 mg/kg bw/day, no test item-related changes were recorded.


At 300 mg/kg bw/day, males showed a non-adverse marginally lower mean weight gain throughout treatment without test item-related changes in food intake. Males also showed a non-adverse higher relative liver and absolute kidney weight and non-adverse lower urinary pH.


At 1000 mg/kg bw/day, the following non-adverse findings were recorded. Slightly lower mean weight gain was recorded for males throughout treatment along with a higher food consumption from Week 2 of treatment onwards. Females at this dose level showed a slightly higher body weight (gain) and food intake at the end of lactation only, as well as higher food consumption during most of the post-coitum period. An increased incidence and/or severity of extramedullary hematopoiesis was present in the spleen in both sexes (up to moderate or slight degree), which likely correlated with the higher spleen weight and lower red blood cells counts and increased formation of new (immature) red blood cells (reticulocytes). Associated changes in red blood cell parameters for males and/or females consisted of higher mean corpuscular volume and mean corpuscular hemoglobin and lower hemoglobin concentration. Additionally, a higher relative liver weight and absolute kidney weight was recorded for both sexes. Clinical pathology changes consisted of higher alanine and aspartate aminotransferase activity, inorganic phosphate and albumin in males, lower glucose and cholesterol concentration in males, higher total bilirubin and urea levels in males and females and lower urinary pH values and higher urinary volume in males.


No test item-related changes were noted in any of the other F0-Generation parameters investigated in this study (i.e., mortality, clinical appearance, clotting parameters, serum levels of TSH and T4 for F0-males and -females and macroscopic examination)


 


Reproduction results – F0-generation


No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/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 parameters (F0), and histopathological examination of reproductive organs).


 


Developmental results – F0 generation / F1 generation (pre-weaning)


No adverse developmental findings were observed up to the highest dose level tested (1000 mg/kg bw/day).


No test item-related mortality occurred among pups during the lactation period. Recorded mortality incidences showed no dose-related trend and remained within the range considered normal for pups of this age. Mortality cases (dead, missing or sacrificed pups) consisted of a total of ten pups in the control group, and two, seven and six pups at 100, 300 and 1000 mg/kg bw/day, respectively.


At 1000 mg/kg w/day, a non-adverse slightly higher mean pup body weight was recorded for both sexes on PND 1 only.


No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e., litter size, gestation, live birth, viability, post-implantation and weaning indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).


 


Developmental results – F1 generation (post-weaning)


No adverse developmental findings were observed up to the highest dose level tested (1000 mg/kg bw/day).


Two animals at 1000 mg/kg bw/day were sacrificed or found dead on dosing Days 5 or 113. These deaths were considered incidental and not related to treatment with the test item. One female each at 100 and 300 mg/kg bw/day were sacrificed for ethical reasons due to gavage procedure-related ill health.


At 100 mg/kg bw/day, non-adverse higher urea (females) and lower cholesterol (males and females) were recorded.


At 300 mg/kg bw/day, non-adverse changes consisted of a lower motor activity (females) and lower foot splay value (both sexes), higher urea, monocyte counts and lower cholesterol (males and females), lower glucose (males), lower urinary pH (females) and higher kidney weight (males).


At 1000 mg/kg bw/day, the age at which vaginal opening was achieved was higher, similar to the change recorded in the F2-generation. It should be noted that the mean value at 1000 mg/kg bw/day in the F2-generation was similar to the mean recorded at 100 mg/kg bw/day. Additionally, this change occurred in the absence of any other test item-related changes in reproduction parameters up to the highest dose level tested (1000 mg/kg bw/day) including estrous cycle length and regularity and mating performance. Also, means remained within the historical control data range. Therefore, although a relationship to treatment with the test item could not be excluded, this was considered not to represent an adverse effect of the test item.


At 1000 mg/kg bw/day, a non-adverse lower startle response and foot splay value (both sexes), lower motor activity (females), and a lower grip strength of the hind legs (males) was recorded. A non-adverse lower mean absolute body weight and/or weight gain was recorded for males throughout the dosing period, and for females during the initial weeks of dosing only. These body weight changes were accompanied by slightly higher mean relative food intake from Week 2 of dosing onwards. Similar to the hematological and associated morphological effects seen in the F0-generation, a non-adverse increased incidence and severity of extramedullary hematopoiesis in the spleen was present at in both sexes (up to moderate degree), along with higher spleen weight and with hematological changes. Other non-adverse changes in clinical chemistry parameters consisted of higher alanine and aspartate aminotransferase activity and total bilirubin (males), higher inorganic phosphate, urea and monocyte counts, lower glucose and cholesterol and longer prothrombin time (both sexes), higher potassium, lower urinary pH (both sexes), and higher specific gravity of the urine (females). Also, higher liver weights were recorded for both sexes, correlating to a non-adverse degree of hepatocellular vacuolation and hepatocellular hypertrophy for females (up to slight or minimal degree, respectively). Additionally, a non-adverse higher kidney weight was recorded for both sexes.


No test item-related changes were noted in any of the other F1-developmental post-weaning parameters investigated in this study (i.e., mortality, clinical observations, estrous cycle, sperm analysis, coagulation, splenic lymphocyte subpopulation analysis, macroscopy, neurohistopathology and brain morphometry).


 


Reproduction results – F1-generation


No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/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 and histopathological examination of reproductive organs).


 


Developmental results – F1-generation / F2-generation (pre-weaning)


No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).


No mortality occurred that was considered to be related to treatment with the test item. One control pup, one pup at 100 mg/kg bw/day, and four pups each at 300 and 1000 mg/kg bw/day were found dead or missing or were sacrificed for humane reasons. In addition, all pups from one litter at 1000 mg/kg bw/day were sacrificed after the dam was found dead. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.


No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e., litter size, gestation, live birth, viability, post-implantation and weaning indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, organ weights and macroscopic examination).


 


Developmental results – F2-generation (post-weaning)


No adverse developmental findings were observed in the post-weaning phase up to the highest dose level tested (1000 mg/kg bw/day).


One female each at 100 and 300 mg/kg bw/day were found dead or sacrificed in extremis during the post-weaning phase (likely) as the result of the gavage procedure.  


At 1000 mg/kg bw/day, a non-adverse temporarily lower weight gain and food consumption was recorded during the first week of dosing for both sexes, which became at least comparable to control levels as dosing progressed. Also, a non-adverse delayed onset of vaginal opening was recorded at 1000 mg/kg bw/day, similar to the change which occurred in the F1-generation. Mean body weight on the day at which vaginal opening was confirmed was higher than the control mean, which indicated that this change could not be ascribed to growth retardation. Means however remained within the historical control data range and the mean at 1000 mg/kg bw/day was similar to the mean at 100 mg/kg bw/day. Therefore, although a relationship to treatment with the test item could not be excluded, this was considered not to represent an adverse effect of the test item.


No test item-related changes were noted in any of the other F1-developmental post-weaning parameters investigated in this study (i.e., mortality, clinical observations and macroscopy).


 


In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following No Observed Adverse Effect Levels (NOAEL) of the test item were established:


- General toxicity (F0 and F1): NOAEL at least 1000 mg/kg bw/day


- Reproduction (F0 and F1): NOAEL at least 1000 mg/kg bw/day


- Developmental (F0, F1 and F2): NOAEL at least 1000 mg/kg bw/day


- Developmental neurotoxicity (F1): NOAEL at least 1000 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Stannard's study is considered to be reliable, and was performed according to the OECD TG 443
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

1-Extended one generation study with F2 and cohort 1 and 2 A and B (CRL, 2022):


The objective of this study was to provide an evaluation of the pre- and postnatal effects of the test item on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring.


The dose levels in this GLP-compliant OECD TG 443 study were selected to be 0, 100, 300 and 1000 mg/kg bw/day, based on results of previous toxicity studies with the test item in rats. The experimental study design was defined on the basis of the ECHA Decision No. TPE-D-2114501107-65-01/F (19 Feb 2020).


F0-Generation


No adverse findings were observed up to the highest dose level tested (1000 mg/kg bw/day).


Four females at 1000 mg/kg bw/day were found dead prior to dosing between lactation Days 11 and 16 which were ascribed to the gavage-procedure. There were no relevant clinical signs or body weight changes for these females prior to death. Foamy contents in the trachea and lungs that failed to collapse were recorded at necropsy, which were not recorded for surviving animals. A cause of death could not be established histopathologically, also because of advanced tissue autolysis. Although these deaths occurred at the high dose only, none of these animals showed any prior signs of a deteriorated health condition, nor did any of the surviving animals at this dose showed any signs of ill health during dosing administration. Based on the observed foamy contents in the respiratory tract which were noted exclusively for these four animals, it was considered that these deaths were related to a gavage-procedure rather than being directly related to treatment with the test item.


At 100 mg/kg bw/day, no test item-related changes were recorded.


At 300 mg/kg bw/day, males showed a non-adverse marginally lower mean weight gain throughout treatment without test item-related changes in food intake. Males also showed a non-adverse higher relative liver and absolute kidney weight and non-adverse lower urinary pH.


At 1000 mg/kg bw/day, the following non-adverse findings were recorded. Slightly lower mean weight gain was recorded for males throughout treatment along with a higher food consumption from Week 2 of treatment onwards. Females at this dose level showed a slightly higher body weight (gain) and food intake at the end of lactation only, as well as higher food consumption during most of the post-coitum period. An increased incidence and/or severity of extramedullary hematopoiesis was present in the spleen in both sexes (up to moderate or slight degree), which likely correlated with the higher spleen weight and lower red blood cells counts and increased formation of new (immature) red blood cells (reticulocytes). Associated changes in red blood cell parameters for males and/or females consisted of higher mean corpuscular volume and mean corpuscular hemoglobin and lower hemoglobin concentration. Additionally, a higher relative liver weight and absolute kidney weight was recorded for both sexes. Clinical pathology changes consisted of higher alanine and aspartate aminotransferase activity, inorganic phosphate and albumin in males, lower glucose and cholesterol concentration in males, higher total bilirubin and urea levels in males and females and lower urinary pH values and higher urinary volume in males.


No test item-related changes were noted in any of the other F0-Generation parameters investigated in this study (i.e., mortality, clinical appearance, clotting parameters, serum levels of TSH and T4 for F0-males and -females and macroscopic examination)


 


Reproduction results – F0-generation


No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/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 parameters (F0), and histopathological examination of reproductive organs).


 


Developmental results – F0 generation / F1 generation (pre-weaning)


No adverse developmental findings were observed up to the highest dose level tested (1000 mg/kg bw/day).


No test item-related mortality occurred among pups during the lactation period. Recorded mortality incidences showed no dose-related trend and remained within the range considered normal for pups of this age. Mortality cases (dead, missing or sacrificed pups) consisted of a total of ten pups in the control group, and two, seven and six pups at 100, 300 and 1000 mg/kg bw/day, respectively.


At 1000 mg/kg w/day, a non-adverse slightly higher mean pup body weight was recorded for both sexes on PND 1 only.


No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e., litter size, gestation, live birth, viability, post-implantation and weaning indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).


 


Developmental results – F1 generation (post-weaning)


No adverse developmental findings were observed up to the highest dose level tested (1000 mg/kg bw/day).


Two animals at 1000 mg/kg bw/day were sacrificed or found dead on dosing Days 5 or 113. These deaths were considered incidental and not related to treatment with the test item. One female each at 100 and 300 mg/kg bw/day were sacrificed for ethical reasons due to gavage procedure-related ill health.


At 100 mg/kg bw/day, non-adverse higher urea (females) and lower cholesterol (males and females) were recorded.


At 300 mg/kg bw/day, non-adverse changes consisted of a lower motor activity (females) and lower foot splay value (both sexes), higher urea, monocyte counts and lower cholesterol (males and females), lower glucose (males), lower urinary pH (females) and higher kidney weight (males).


At 1000 mg/kg bw/day, the age at which vaginal opening was achieved was higher, similar to the change recorded in the F2-generation. It should be noted that the mean value at 1000 mg/kg bw/day in the F2-generation was similar to the mean recorded at 100 mg/kg bw/day. Additionally, this change occurred in the absence of any other test item-related changes in reproduction parameters up to the highest dose level tested (1000 mg/kg bw/day) including estrous cycle length and regularity and mating performance. Also, means remained within the historical control data range. Therefore, although a relationship to treatment with the test item could not be excluded, this was considered not to represent an adverse effect of the test item.


At 1000 mg/kg bw/day, a non-adverse lower startle response and foot splay value (both sexes), lower motor activity (females), and a lower grip strength of the hind legs (males) was recorded. A non-adverse lower mean absolute body weight and/or weight gain was recorded for males throughout the dosing period, and for females during the initial weeks of dosing only. These body weight changes were accompanied by slightly higher mean relative food intake from Week 2 of dosing onwards. Similar to the hematological and associated morphological effects seen in the F0-generation, a non-adverse increased incidence and severity of extramedullary hematopoiesis in the spleen was present at in both sexes (up to moderate degree), along with higher spleen weight and with hematological changes. Other non-adverse changes in clinical chemistry parameters consisted of higher alanine and aspartate aminotransferase activity and total bilirubin (males), higher inorganic phosphate, urea and monocyte counts, lower glucose and cholesterol and longer prothrombin time (both sexes), higher potassium, lower urinary pH (both sexes), and higher specific gravity of the urine (females). Also, higher liver weights were recorded for both sexes, correlating to a non-adverse degree of hepatocellular vacuolation and hepatocellular hypertrophy for females (up to slight or minimal degree, respectively). Additionally, a non-adverse higher kidney weight was recorded for both sexes.


No test item-related changes were noted in any of the other F1-developmental post-weaning parameters investigated in this study (i.e., mortality, clinical observations, estrous cycle, sperm analysis, coagulation, splenic lymphocyte subpopulation analysis, macroscopy, neurohistopathology and brain morphometry).


 


Reproduction results – F1-generation


No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/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 and histopathological examination of reproductive organs).


 


Developmental results – F1-generation / F2-generation (pre-weaning)


No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).


No mortality occurred that was considered to be related to treatment with the test item. One control pup, one pup at 100 mg/kg bw/day, and four pups each at 300 and 1000 mg/kg bw/day were found dead or missing or were sacrificed for humane reasons. In addition, all pups from one litter at 1000 mg/kg bw/day were sacrificed after the dam was found dead. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.


No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e., litter size, gestation, live birth, viability, post-implantation and weaning indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, organ weights and macroscopic examination).


 


Developmental results – F2-generation (post-weaning)


No adverse developmental findings were observed in the post-weaning phase up to the highest dose level tested (1000 mg/kg bw/day).


One female each at 100 and 300 mg/kg bw/day were found dead or sacrificed in extremis during the post-weaning phase (likely) as the result of the gavage procedure.  


At 1000 mg/kg bw/day, a non-adverse temporarily lower weight gain and food consumption was recorded during the first week of dosing for both sexes, which became at least comparable to control levels as dosing progressed. Also, a non-adverse delayed onset of vaginal opening was recorded at 1000 mg/kg bw/day, similar to the change which occurred in the F1-generation. Mean body weight on the day at which vaginal opening was confirmed was higher than the control mean, which indicated that this change could not be ascribed to growth retardation. Means however remained within the historical control data range and the mean at 1000 mg/kg bw/day was similar to the mean at 100 mg/kg bw/day. Therefore, although a relationship to treatment with the test item could not be excluded, this was considered not to represent an adverse effect of the test item.


No test item-related changes were noted in any of the other F1-developmental post-weaning parameters investigated in this study (i.e., mortality, clinical observations and macroscopy).


 


In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following No Observed Adverse Effect Levels (NOAEL) of the test item were established:


- General toxicity (F0 and F1): NOAEL at least 1000 mg/kg bw/day


- Reproduction (F0 and F1): NOAEL at least 1000 mg/kg bw/day


- Developmental (F0, F1 and F2): NOAEL at least 1000 mg/kg bw/day


- Developmental neurotoxicity (F1): NOAEL at least 1000 mg/kg bw/day


 


2-Oral combined repeated dose and reproduction / developmental screening test OECD 422 (Huntington, 2010)


In a GLP study conducted according to OECD guideline 422, three groups each comprising five male and five female rats for the Toxicity subgroup and five male and ten female rats for the Reproductive subgroup received propylidynetrimethyl trimethacrylate at doses of 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and Reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume-dose.


There were two mortalities during the course of the study. One Reproductive subgroup female receiving 100 mg/kg bw/day was killed for welfare reasons on Day 22 of gestation due to dystocia. In addition, one Reproductive subgroup Control female (and consequently the litter) was killed for welfare reasons prior to dose administration on Day 4 of lactation. These isolated incidents were considered unrelated to treatment.


There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was also no effect of treatment on the food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study.


At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than Control during the two week pre-pairing period; females in this dose group showed similarly low weight gain during the first week of treatment only. Thereafter, for both sexes mean weight gain was essentially similar to Control, and the bodyweight gain of females during gestation and lactation was unaffected by treatment.


Oestrous cycle length, pre-coital interval and mating performance and fertility of the Reproductive subgroup females was unaffected by treatment. There was a suggestion of a minor shift towards a slightly longer gestation length among Reproductive subgroup females receiving 900 mg/kg bw/day.


Among the Toxicity subgroup animals, liver weights were slightly high in males and females receiving 900 mg/kg bw/day, and kidney weights were also high among females in this dose group. There were no associated haematological or biochemical changes, or macroscopic/microscopic abnormalities to explain the difference in the weight of the organs.


There were no macroscopic abnormalities and no test substance-related lesions at microscopic examination.


There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.


A statistically significant reduction in the mean number of implantation sites, associated with low litter size were observed at the high dose (900 mg/kg bw/d). In this screening study, the number of corpora lutea present for each animal was not determined. Thus, it cannot be assessed whether this is a spontaneous finding related to a reduction in the number of eggs available for fertilization or indicative of a treatment-related pre-implantation loss.


There was no effect on pre- or post-natal survival and on sex ratio at any dose level. At 900 mg/kg bw/day, mean male and female offspring bodyweights on Day 1 of age were higher than Control; these differences were attributed to the slightly lower litter size and slight shift in gestation length observed in this group. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment.


Based on the results of this study, it was concluded that the NOAEL for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.


 


 


 

Effects on developmental toxicity

Description of key information

Developmental toxicity of TMPTMA was studied in rat and rabbit species.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02-Sep-2014 to 18-Mar-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22nd January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
EC 2004/73 B31, dated April 29, 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
EPA 712-C-98-207, dated August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks at day 0 post coitum
- Weight at study initiation: 178 to 281 g at day 0 post coitum
- Fasting period before study: None
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands).
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 13/14) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed.
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 02-Sep-2014 (start of 7-day Acclimatization) To: 09-Oct-2014 (Last Necropsy)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The vehicle was pre-warmed to a temperature of approximately 40 °C. Propylidynetrimethyl trimethacrylate was weighed into a glass beaker on a tared precision balance and approximately 80 % of the warm vehicle was added (w/v). The mixture was homogenized using an electrical homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Stability and Storage of Dose Formulations: For at least 8 days at room temperature (20 ± 5 °C) based on stability results of Harlan Laboratories Study no. D84010 (Braun 2015, subchronic repeated oral toxicity

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 25, 75 and 250 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 4 mL/kg body weight with a daily adjustment to the actual body weight
- Lot/batch no.: 453206485 (Source: Carl Roth GmbH + Co. KG, Karlsruhe/Germany, Expiry Date: 14-Jan-2016)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples were dispatched to the analytical laboratories internally (at room temperature) and stored frozen at -20 ± 5 °C until analysis.
The test item was used as the analytical standard.
Stock solutions of Propylidynetrimethyl trimethacrylate in acetonitrile were prepared for external calibration. For example, 34.77 mg of Propylidynetrimethyl trimethacrylate was weighed into a 50 mL volumetric flask and filled to about 75% of final volume with acetonitrile. The mixture was sonicated for at least five minutes and brought to volume with acetonitrile to yield a solution with a concentration of 695.4 µg/mL. Aliquots of this stock calibration solution were diluted with acetonitrile to obtain calibration solutions with nominal concentrations ranging from 10.43 to 99.44 µg/mL. On each occasion calibration solutions derived from two stock solutions were used for calibration.
Each sample was transferred into an appropriate volumetric flask. The sample vial was successively rinsed with at least two portions of tetrahydrofuran and the rinsings were combined in the volumetric flask. The flask was filled to about 75 % of the target volume with tetrahydrofuran and dissolution was achieved by sonication for at least five minutes. The flask was filled to the mark with tetrahydrofuran. Sample solutions were further diluted with acetonitrile into the calibration range.
Gas Chromatographic Conditions:
- Computerized System: EZ Chrom Elite; Agilent Technologies
- Column: BGB 1701 - BGB; 30 m x 0.25 mm; 0.25 µm
- Carrier Gas: Helium
- Temperature Gradient:
Rate [°C/min]; Temp. [°C]; Time [min]
0; 200; 0
10; 300; 5
- Running Time: 15 min
- Injector Temperature: 230 °C
- Injection Mode: Split ratio 5:1
- Flow: 1 mL/min (constant flow)
- Detector: FID; 250 °C (H2: 31; Air: 380; N2: 28 mL/min)
- Injection Volume: 2 µL
- Retention Time: Ca. 4.4 min
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) were higher than 0.99.
The Propylidynetrimethyl trimethacrylate peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no peak appeared at the retention time of Propylidynetrimethyl trimethacrylate and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.
The Propylidynetrimethyl trimethacrylate concentrations in the dose formulations ranged from 81.5 to 92.5 % with reference to the nominal and were within the accepted range of ±20 %. The homogeneous distribution of Propylidynetrimethyl trimethacrylate in the preparations was approved because single results found did not deviate more than 1.3 % from the corresponding mean and met the specified acceptance criterion of =15 %. Accordingly the effects were assigned to the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: Cohoused, After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- M/F ratio per cage: 1:1
- Length of cohabitation: After 11 days (08 to 18-Sep-2014) pairing of all test animals was successfully completed.
- Further matings: No
- Verification of same strain and source of both sexes: Yes, all animals belonged to the RccHan™: WIST(SPF)
- Proof of pregnancy: Vaginal plug observed or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: None
Duration of treatment / exposure:
15 days (during gestation period from day 6 to 20 post coitum)
Frequency of treatment:
Daily, at approximately 24 hour intervals
Duration of test:
21 days post coitum
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding study in Han Wistar rats, Harlan Laboratories non-GLP study no. D84021, using dose levels of 0, 100, 300 and 1000 mg/kg/day.
- Rationale for animal assignment: Computer-generated random algorithm
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Viability / Mortality observation were made twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- Time schedule: For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Gross macroscopic examination of all internal organs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Post mortem examination with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed and the data recorded.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions (embryonic): Yes
- Number of late resorptions (foetal): Yes
- Other: The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Foetuses were removed from the uterus by caesarean section, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. Microdissection technique (sectioning/dissection technique): Approximately one half of the foetuses from each litter were fixed in Bouin's fixative. They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol, carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.

The foetuses were sent to the contributing scientist for peer review at Huntingdon Life Sciences.
Statistics:
The following statistical methods were used to analyse food consumption, body weights, reproduction and skeletal examination:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.
Indices:
No indices were calculated as percentages were used.
Historical control data:
Tables summarizing the available historical control data from six studies performed at the test site (referenced 12/02, 12/03, 12/04, 13/01, 13/02 and 13/03) are given in Appendix V of the study report.
Historical control data were used in the discussion of foetal Visceral Abnormalities and Variations (incidence of the left-sided umbilical artery in group 4 marginally exceeded the upper range of the historical control data).
The number of foetal retinal folds observed in the control group was not in accordance with the occurrence reported in the historical control values where no retinal folds were observed. This could be explained by the fact that the source of the Bouin’s fixative for the present study was different from that used in studies that compiled the historical control data; this difference was considered to be a potential cause for the slight (but not clearly dose-related) increase in retinal folds noted (see Discussion of Retinal Folds observations, below).
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical symptoms or signs were observed at any dose level during the gestation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females survived until the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effects on mean absolute body weights were recorded.
The mean body weight gain was significantly lower (p<0.01) from day 9 p.c. in group 4 when compared to the controls (40 % in group 4 vs. 46 % in the controls on day 21).
The corrected body weight gain (corrected for the gravid uterus weight) was significant lower in group 4 (p<0.01) when compared to the controls (8.0 vs. 11.5 % in the controls on day 21). The effects on body weight gain and corrected body weight gain in group 4 were considered test item-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the food consumption during the study.
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were observed at any dose level during macroscopic examination.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
All females survived until the scheduled necropsy. No clinical signs were recorded in any group and the mean daily food consumption of all groups compared favourably. Although the mean absolute body weights were unaffected, a statistically significant lower mean body weight gain in group 4 (40 vs. 46 % in the controls on day 21 post coitum) and a statistically significant decreased corrected body weight gain (corrected for the gravid uterus weight) in group 4 (8.0 vs. 11.5 % in the controls on day 21) were considered to be test item-related. The reproduction data (post-implantation loss and mean number of foetuses per dam) was unaffected by treatment and no macroscopical findings were noted at any dose level.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Placenta weights were not affected by the treatment with the test item at any dose level. Mean placenta weights calculated on a litter basis were: 0.505 g, 0.485 g, 0.490 g and 0.495 g whereas calculated on an individual basis, they were 0.503 g, 0.483 g, 0.489 g and 0.495 g, both cited in order of ascending dose level. Mean values calculated on an individual basis were significantly lower in group 2 (p<0.05) when compared to the control. In the absence of a dose-response relationship, the difference was considered to be unrelated to the treatment.
Details on maternal toxic effects:
The statistically significant decrease in the implantation sites and increase in pre-implantation loss in group 2 was not test item-related since this was a single outlying value (female no. 48) and occurred before treatment started.
The relevant reproduction data (post implantation loss and number of foetuses per dam) were not affected by treatment with the test item.
Dose descriptor:
NOEL
Remarks:
(maternal toxicity)
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
(maternal toxicity)
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
no effects observed
Description (incidence and severity):
When compared with the controls, lower mean body weights of live foetuses were noted in group 4 (-6.1 % on litter basis and -8.2 % on individual basis). The difference attained statistical significance (p<0.01) and was considered to be test item-related. Both male and female foetuses in group 4 were significantly lower (all p<0.01) when compared with the controls.
The mean live foetus body weight of group 3 was similar to that of the control group.
The individual and litter mean body weight of live foetuses in group 2 was marginally lower (-2 %) than the controls; the reduction noted in mean individual foetus weights was statistically significant but unrelated to dose and therefore considered to be unrelated to the test item.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects on the sex ratio of the foetuses were noted in any group.
The proportion of male foetuses was 49.4, 54.7, 49.8 and 50.4 % in order of ascending dose level.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related findings were observed during external examination of the foetuses.
Umbilical hernia was recorded in one female foetus of group 3. A domed head, protruding tongue, and short and flexed forelimbs were recorded in one male foetus of group 4. These findings were recorded in single animals and therefore considered to be incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Bone and Cartilage Abnormalities and Variations
During skeletal examination of the foetuses, findings were noted in:
15 % examined foetuses (in 67 % litters) in group 1
15 % examined foetuses (in 57 % litters) in group 2
17 % examined foetuses (in 67 % litters) in group 3
17 % examined foetuses (in 60 % litters) in group 4
A small number of bone and cartilage abnormalities were noted in treated and control groups. The incidence of the findings in the treated groups was low, were not related to dose and considered to be of no toxicological relevance.
All bone and cartilage variations noted in treated groups were considered to be unrelated to the test item. The incidence of the findings in the treated groups was low and unrelated to dose.

Ossification and Supernumerary Ribs
There were no test item-related effects on the stage of development in any dose group.
The significantly increased incidence of incomplete ossification of sternabrae 5 or non-ossification of calcanei (bilateral) were considered likely to be a secondary result of the lower mean dam body weight gain and/or foetal body weights. These structures typically show high rates of non- or incomplete ossification in control rat foetuses and therefore a relationship with the test item treatment is considered unlikely.

Additional Cartilage Variations
All additional cartilage variations noted in treated groups were considered to be unrelated to the test item. The incidence of the findings in the treated groups was low and unrelated to dose.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the foetuses, findings were noted in:
63 % examined foetuses (in 100 % litters) in group 1
62 % examined foetuses (in 100 % litters) in group 2
57 % examined foetuses (in 100 % litters) in group 3
68 % examined foetuses (in 100 % litters) in group 4
No test item-related visceral abnormalities were noted. All abnormal changes were noted at low incidence and were unrelated to dose.
The high incidence of retinal folds noted in the foetuses of groups 3 and 4 was considered to be an artefact of fixation. All other changes were noted at low incidence and were unrelated to dose.
Although the incidence of the left-sided umbilical artery in group 4 marginally exceeded the upper range of the historical control data, the finding was noted at lower incidence in group 3 than in either group 2 or the control group. Therefore, this latter difference was considered to be of no toxicological relevance.
Details on embryotoxic / teratogenic effects:
The mean placenta weights of all groups were similar. The external examination of the foetuses showed no abnormalities of toxicological relevance and sex ratios were unaffected. Foetuses of group 4 dams had lower mean body weights (-6 % on litter basis and -8 % on individual basis). This was considered to be related to the treatment with test item.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Summary of Performance of Mated Females

Group
Dose (mg/kg)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

1 - 24

25 - 48

49 - 72

73 - 96

Number of mated females

24

24

24

24

Not pregnant (A)

3

3

3

4

Number of females with live foetuses at termination*

21

21

21

20

* Only dams with at least one live foetus at caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

(A) Females no. 4, 15, 24, 35, 43, 47, 49, 56, 61, 73, 78, 83, and 87 were not pregnant.

Conclusions:
NOEL maternal toxicity 300 mg/kg bw/day, NOAEL maternal toxicity 1000 mg/kg bw/day or higher (body weight effects)
NOEL and the NOAEL prenatal development 300 mg/kg/day (lower mean foetal weights).
Executive summary:

The oral (gavage) prenatal developmental toxicity of the test item Propylidynetrimethyl trimethacrylate (CAS 3290-92-4) to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 414 (2001) guideline.

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 24 mated females per group were treated by gavage once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females were sacrificed on day 21 post coitum and the foetuses were removed by caesarean section.

All females survived until the scheduled necropsy. No clinical signs were recorded in any group, and the mean daily food consumption of all groups compared favourably. Although the mean absolute body weights were unaffected, a statistically significant lower mean body weight gain of the dams in group 4 (40 vs. 46 % in the controls on day 21 post coitum) and a statistically significant decreased corrected body weight gain (corrected for the gravid uterus weight) in group 4 (8.0 vs. 11.5 % in the controls on day 21) were considered to be test item-related. The reproduction data (post-implantation loss and mean number of foetuses per dam) was unaffected by treatment and no macroscopical findings were noted at any dose level.

The mean placenta weights of all groups were similar. The external examination of the foetuses showed no abnormalities of toxicological relevance and sex ratios were unaffected. Foetuses of group 4 dams had lower mean body weights (-6 % on litter basis and -8 % on individual basis). This was considered to be related to the treatment with test item.

In conclusion based on the slightly lower mean body weight gain, the NOEL (No Observed Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day, whereas the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 1000 mg/kg bw/day or higher. For prenatal development, the NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) were considered to be 300 mg/kg/day, based on the lower mean foetal weights noted at 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May 2017 - 04 August 2017
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:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Centre LAGO (Vonnas, France)
- Age: at the beginning of the treatment period, the animals were 18-20 weeks old
- Mean body weight: at the beginning of the treatment period, the animals had a mean body weight of 3772 g (range: 3180 g to 4580 g)
- Fasting period before study: no
- Housing: the animals were individually housed in noryl cages (Tecniplast, 4200 cm²)
- Diet: (SAFE, Augy, France) pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8 h/16 h.

IN-LIFE DATES: 03 July 2017 to 04 August 2017.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose (400-800 cps) / 0.5% Tween 80 in drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Justification for use and choice of vehicle: suitable formulation in the selected vehicle
- Concentration in vehicle: 33.3, 100 and 333.3 mg/mL
- Amount of vehicle: 3 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: a sample was taken from control and test item dose formulations and analyzed using the validated method
Stability / Homogeneity: The dose formulations containing the test item and prepared at 2 mg/mL and 333.3 mg/mL in 0.5% (w/v) carboxymethylcellulose and 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis were found to be homogeneous and stable after 5 and 9 days at room temperature and protected from light.
Details on mating procedure:
- Impregnation procedure: purchased time pregnant.
- Proof of pregnancy: vaginal plug (at the breeder's facility); referred to as Day 0 post-coitum.
Duration of treatment / exposure:
The dose formulations were administered daily from Day 6 to Day 28 p.c., inclusive.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results of a preliminary study on embryo-fetal development in New Zealand White rabbits where the test item was administered by the oral route from Day 6 to Day 18 p.c. at dose levels of 100, 300 or 1000 mg/kg/day.
At 100 mg/kg/day, no signs of maternal toxicity were noted and no effects on hysterectomy data or fetuses were observed.
At 300 mg/kg/day, no signs of maternal toxicity were noted. There were no significant signs of toxicity on the number of fetuses, the number of implantation sites or on post-implantation loss. Fetal examination did not reveal any malformations. Lower fetal body weight was recorded (34.5 g vs. 39.4 g in controls; p < 0.05).
At 1000 mg/kg/day, a body weight loss was recorded between Days 6 and 9 p.c. (-32 g), associated with lower food consumption (-12% vs. controls). Lower food consumption was observed again between Days 24 and 29 p.c. (-20% vs. controls), but there were no statistical differences on the body weight or food consumption at the end of the study when compared with control group. No significant signs of toxicity were observed on the number of fetuses, the number of implantation sites or post-implantation loss, and fetal examination did not reveal any malformation. Lower fetal body weight was recorded (34.0 g vs. 39.4 g in controls; p < 0.05).

Therefore, 1000 mg/kg/day was selected as the high dose level for the present study. The low dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: stratified procedure.
Maternal examinations:
MORBIDITY/MORTALITY:
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during treatment period, including weekends and public holidays.
Two females of the group 3, showing signs of poor clinical condition or having aborted, were humanely euthanized.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs (including evidence of abortion).

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c., and before premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: The quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 29 post-coitum.
- Examined: principal thoracic and abdominal organs.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early and late resorptions
- Number of dead and live fetuses
- Number of uterine scars
- Gross evaluation of placentas.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: all fetuses per litter
- Skeletal examinations: Yes: all fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other : fetal weight, fetal sex
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Historical control data:
Cf attached document
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See table 1.
There were no remarkable clinical observations in surviving animals.
In the 1000 mg/kg/day group, from Day 9 p.c. emaciated appearance was noted in female K31094. As this female had already lost 285 g (-8% of the initial body weight on Day 2 p.c.) and had nearly no food consumption (on Days 4 and 5 p.c.) during the pre-treatment period, a test item-related effect alone was considered to be unlikely.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths or abortions in the control, 100 or 1000 mg/kg/day groups.

In the 300 mg/kg/day group, there were two prematurely sacrificed females (K31050 and K31068):
-female K31050 was euthanized on humane grounds on Day 23 p.c. (emaciated appearance from Day 20 p.c., soft feces from Day 21 p.c. and hypoactivity together with abdominal breathing on Day 23 p.c.). This animal lost 21% of its body weight from Days 6 to 23 p.c. and nearly no food consumption was recorded on Days 15 to 19 p.c. At necropsy, this female (with 12 corpora lutea and 11 implantation sites: 4 dead fetuses and 7 live fetuses) showed edema on the pyloric region of the stomach mucosa, reddish depressed area(s) on the stomach mucosa and dilated colon with gas,
-female K31068 was euthanized due to abortion on Day 22 p.c. (blood and embryos in the bedding on Day 22 p.c.). At necropsy, this female (with 15 corpora lutea and 4 implatation sites, 1 uterine scar, 1 late resorption and 2 live fetuses) had no macroscopic post-mortem lesions.

These deaths were considered not to be test item-related as they were not dose-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 2.

When compared with controls, there were no effects on mean body weights or mean body weight changes at 100 or 300 mg/kg/day.

At 1000 mg/kg/day and when compared with controls, a slight mean body weight loss was noted over the first 3 days of treatment (-2 g vs. +27 g, not statistically significant), followed, from Day 12 p.c., by a lower mean body weight gain (statistically significant from Day 19 p.c. to Day 24 p.c.).
No statistically significant changes were observed in the final mean body weight of females. However, 4/22 females (K31078, K31093, K31094 and K31096) lost body weight during the gestation period from Day 6 p.c. to Day 29 p.c. Taking into account the amplitude of the body weight change over the whole treatment period ( 42%, p<0.05) and the body weight loss recorded in 4 females (corresponding to 18% of the size group), the decrease of body weight gain was considered to be test item-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 3.
When compared with controls, there were no statistical differences on mean food consumption at 100 or 300 mg/kg/day.

At 1000 mg/kg/day and when compared with controls, mean food consumption was lower over the first 3 days of treatment and from Day 12 p.c. A statistically significant change was observed from Day 24 p.c. to Day 29 p.c. (-25%, p<0.05) that was below the lower limit of the Historical Control Data. Females with body weight loss were more impacted.
These differences in mean food consumption correlated with lower mean body weight gain were therefore considered to be test item related.
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:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See table 5.
Discolored area(s) in the stomach were observed in one female from each test item-treated group. Taking into account the absence of similar findings in the control group, a test item relationship was not excluded.

The other macroscopic observations (lungs with colored foci/nodules, liquid content in the abdominal cavity, cyst on the periovarian region and/or gall bladder bilobed/reduced in size) were not attributed to the test item as they were reported with no dose-relationship and/or are findings routinely observed in pregnant female rabbits of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
See table 4.
There were no effects on mean gravid uterus weight mean carcass weight.

At 1000 mg/kg/day and when compared with controls and Reference Control Data, on Day 29 p.c. a slightly lower mean net body weight change (not statistically significant) was noted. This correlated with lower mean body weight gain.
Females with body weight loss were more affected and value of one female (K31096: -947 g) was below the lower individual limit of the Reference Control Data [-678 g; +389 g].
Based on these correlations, the lower mean net body weight change was therefore considered to be test item-related.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 15.
In the 300 mg/kg/day group, one female was euthanized due to abortion on Day 22 p.c.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
When compared with controls or Historical Control Data, there were no test item-related effects on hysterectomy data.

In controls, mean number of corpora lutea was below the lower limit of the Historical Control Data leading to higher mean numbers of corpora lutea and implantations at 300 and 1000 mg/kg/day (p<0.05 and p<0.01, respectively), when compared with controls. Although these differences were statistically significant, they were considered to be fortuitous as corpora lutea appearance occurs before the treatment period.
The higher mean number of late resorptions observed in the 300 mg/kg/day group was considered to be of no toxicological importance as this difference from controls was not dose-related.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No dams with total resorption.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
The higher mean number of late resorptions observed in the 300 mg/kg/day group was considered to be of no toxicological importance as this difference from controls was not dose-related.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
Number of dams with dead fetuses = 1 at 100 mg/kg/day only, not dose-related.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
No early deliveries.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
See table 15.
Dose descriptor:
NOAEL
Remarks:
for systemic maternal parameters
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: net body weight change
Dose descriptor:
NOAEL
Remarks:
for reproduction parameters
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
From 100 mg/kg/day and when compared with controls, male and female fetuses had a slight to markedly lower mean body weight (-8% to -23% vs. controls). Female fetuses were more affected than male fetuses at 300 (males: -10% and females: -14% vs. controls) and 1000 mg/kg/day (males: -21% and females: -23% vs. controls), but mean fetal body weight values were within the range of the Historical Control Data.
The lower mean fetal body weight was considered to be related to the test item and adverse at 1000 mg/kg/day taking into account the amplitude of the difference.

Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See table 6.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
At 300 and 1000 mg/kg/day and when compared with controls, a slightly, not statistically significant, higher mean percentage of male fetuses was noted (59.0% and 59.4%, respectively, vs. 50.2% in controls). These differences were considered to be related to the test item but of minor toxicological importance as they were of minor amplitude and close to the upper limit of the Historical Control Data.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the mean litter values of fetal body weight were decreased when compared with controls and outside the HCD. These decreases at 1000 mg/kg/day were considered as adverse.
Description (incidence and severity):
n/a
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Variations
See table 8.
In the 1000 mg/kg/day group and when compared with controls, statistically significant, higher fetal (12/212 fetuses) and litter incidences (5/22 litters) of yellowish/greenish coloration of the amniotic fluid (p<0.001 and p<0.05, respectively) were observed. Nine out of twelve affected fetuses showed the lowest fetal weight of the litters. Taking into account the amplitude of the incidences (litter and fetal) of this non adverse finding, it was considered to be test item-related.

Other external variations (i.e. local edema on abdomen and paw hyperflexion) were considered to be unrelated to the test item treatment as they were not dose-related and/or were observed with a similar incidence in control and test item-treated groups.


Malformations
See table 9.
In the 1000 mg/kg/day group, three fetuses from three litters showed external malformations:
-fetus K31086-01 (fetal body weight: 23.1 g): ectrodactyly, left malpositioned digit and gastroschisis. This fetus also had skeletal malformations (i.e. left tibia misshapen, 2 absent metatarsal bones and left digits hindpaw and scoliosis),
-fetus K31083-11 (fetal body weight: 37.3 g): omphalocele,
-fetus K31094-01 (fetal body weight: 23.3 g): ombilical hernia. This fetus also had visceral malformations (i.e. marked dilated cerebral ventricle, bilateral).
These findings, mainly affecting the abdominal wall (mid-line defects: gastroschisis, omphalocele or ombilical hernia), were observed in three malformed fetuses from three different litters and two of these fetuses showed low fetal body weight.

In the 300 mg/kg/day group, there was no external malformation.
In the 100 mg/kg/day group, one malformed fetus: K31030-02 with cleft lip was present.
In the control group, there was no external malformation.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Cartilage
See table 12.
When compared with controls and/or Historical Control Data, there were higher litter and/or fetal incidences of fetuses with sternebra(e) cartilage present and/or misshapen, and metacarpal bone(s) cartilage present from 100 mg/kg/day and/or talus cartilage present at 1000 mg/kg/day. These findings were associated with a delayed ossification as described in the § Variations [i.e. unossification or incomplete ossification of hemisternebra(e), unossification or incomplete ossification of 6th sternebra(e), unossification or incomplete ossification of 1st metacarpal bone, unossification or incomplete ossification of talus] and were considered as test item treatment-related, but they are not considered to be adverse.

The other findings [i.e. cartilage of rib(s) branched, cartilage of forepaw median phalanx present, and/or cartilage of metatarsal bone absent; and cartilage of forepaw proximal phalanx present which is not reported in summary table] were not dose related, noted without a statistically significant occurrence and/or were within or close to the range of the Historical Control Data. Therefore a test item relationship was considered to be unlikely.

Variations
See table 13.
In the 1000 mg/kg/day group and when compared with controls and/or Historical Control Data, there were dose-related higher fetal and/or litter incidences of fetuses with incomplete ossification of interparietal, unossification or incomplete ossification of hemisternebra(e), unossification or incomplete ossification of 6th sternebra(e), unossification or incomplete ossification of 1st metacarpal bone, unossification or incomplete ossification of talus. These findings, associated with higher fetal and litter cartilage findings (see § Cartilage) and mainly observed with a statistically significant occurrence, were therefore considered to be test item-related but not adverse.

In the 300 mg/kg/day group and when compared with controls and Historical Control Data, there were higher fetal and/or litter incidences of fetuses with unossification or incomplete ossification of hemisternebra(e).
These dose-related findings, associated with higher fetal and litter cartilage findings (see § Cartilage), were therefore considered to be test item-related but not adverse.
There were also higher fetal and litter incidences of fetuses with incomplete ossification of interparietal,
incomplete ossification of 6th sternebra(e) and unossification of 1st metacarpal bone. A relationship to the test item could not be ruled out for these dose-related variations, although their incidences were within the range of the Historical Control Data.

In the 100 mg/kg/day group and when compared with controls, there were higher litter incidences of fetuses with unossification of 1st metacarpal bone and/or incomplete ossification of hemisternebra(e). A relationship to the test item could not be ruled out for these dose-related variations which were also associated with higher fetal and litter cartilage findings (see § Cartilage), although their incidences were within the range of the Historical Control Data.

The other variations [i.e. supernumerary 13th or 14th rib(s), knobby rib(s), incomplete ossification of forepaw median phalanx and/or unossified forepaw median phalanx; and unossified interparietal, misshapen sternebra(e), ribs ossification point on 13th thoracic vertebra(e), and/or unossified pubis which are not reported in the summary table] were not dose-related, noted without a statistically significant occurrence and/or were within or close to the range of the Historical Control Data. Therefore a test item relationship was considered to be unlikely.


Malformations
See table 14.
There was no test item-related increase in the frequency of skeletal malformations.

In the 300 and 1000 mg/kg/day group and when compared with controls, there were higher fetal and litter incidences of fetuses with split interparietal and fetuses with fused sternebra(e).
At 1000 mg/kg/day, litter (but not fetal) incidence of fused sternebra(e) was statistically significantly higher than that of the control group and higher than the maximal incidence of the Historical Control Data. Both findings are consistent with a developmental delay in ossification and could be considered as a normal step leading to definitive formation of the bone (De Sesso and Scialli, 2018). They were therefore considered as skeletal variations (not adverse) and not as malformations.
In the 1000 mg/kg/day group, multi-malformed fetus K31086-01 (fetal body weight: 23.1 g) showed scoliosis, misshapen left tibia and absence of two metatarsal bones and two digits in the left hindpaw. This fetus also had external malformations (i.e. ectrodactyly, malpositioned digit and gastroschisis).
The other malformations [i.e. split interparietal, absent interparietal, fused rib(s)] observed in the 100, 300 and/or 1000 mg/kg/day groups were of isolated occurrence (this could include split interparietal that was not associated to any other cranial malformation at 100 mg/kg/day), not dose-related and/or were within or close to the range of the Historical Control Data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
See table 10.
There was no test item-related increase of the frequency of soft tissue variations.

Soft tissue variations were observed without a dose relationship (i.e absent brachiocephalic trunk), with an isolated low incidence (i.e. small kidney, dilated renal pelvis and liquid contents in cranial cavity) and/or with a litter incidence lower than controls (i.e. small gall bladder) and/or with an incidence lower than the Historical Control Data (i.e. dilated renal pelvis). Therefore they were considered to be unrelated to the test item treatment.

Other soft tissue variations, not presented in this summary table, were observed without a dose relationship and were considered to be unrelated to the test item treatment.


Malformations
See table 11.
There was no test item-related increase in the frequency of soft tissue malformations.

In the 1000 mg/kg/day group, two fetuses from two different litters showed malformations (K31094-01: marked dilated cerebral ventricle; K31080-03: ventricular septum defect and dilated aortic arch) that were not recorded in the control group and/or in the Historical Control Data.

In the 300 mg/kg/day group, three fetuses from three different litters showed malformations. Fetus K31056 03 showed marked dilated cerebral ventricle together with small cerebrum and fetus K31057-07 had small lung. Small lung is present in the Historical Control Data.
Fetus K31059-08 showed absent aortic arch and dilated pulmonary trunk. Both malformations are present in the Historical Control Data.

In the 100 mg/kg/day group, there was no visceral malformation.

In the control group, one litter contained one malformed fetus (K31007-03 with absent gall bladder).
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
other: fetal weight
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: vertebra
Description (incidence and severity):
See 'Overall remarks, attachments'
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Clinical signs

Dose-level (mg/kg/day)

0

100

300

1000

Emaciated appearance

0

0

0

1

Blood in the bedding

0

0

2

0

Cutaneous lesions (tail)

0

1

0

0

Abnormal growth of teeth

1

0

0

1

Difficulty during gavage administration

2

0

0

0

No remarkable observations

21

23

20

22

 

Table 2: Body weight and body weight change

Dose-level (mg/kg/day)

0

100

300

1000

Mean body weight (g)

 

 

 

 

Day 6 p.c.

3770

-

3748
(-1)

3806
(+1)

3806
(+1)

Day 29 p.c.

4128

-

4082
(-1)

4142
(0)

4015
(-3)

Mean body weight change (g)

 

 

 

 

Days 6-9 p.c.

+27

+27

+31

-2

Days 9-12 p.c.

+44

+52

+45

+47

Days 12-15 p.c.

+90

+82

+71

+54

Days 15-19 p.c.

+72

+60

+41

+64

Days 19-24 p.c.

+95

+93

+98

+40*

Days 24-29 p.c.

+30

+19

+11

+6

Days 6-29 p.c.

+358

-

+334

(-7)

+331

(-8)

+209*

(-42)

p.c.: post-coitum.

-: not applicable.

Statistical significance: *: p<0.05.

( ): in brackets, percentage difference vs. controls.


 

Table 3: Food consumption

Dose-level (mg/kg/day)

0

100

300

1000

Days 6 to 9 p.c.

177

-

169
(-5)

183
(+3)

160
(-10)

Days 9 to 12 p.c.

175

-

181
(+3)

184
(+5)

174
(-1)

Days 12 to 15 p.c.

166

-

163
(-2)

160
(-4)

142
(-14)

Days 15 to 19 p.c.

195

-

182
(-7)

174
(-11)

165
(-15)

Days 19 to 24 p.c.

173

-

168
(-3)

159
(-8)

146
(-16)

Days 24 to 29 p.c.

118

-

107
(-9)

107
(-9)

89*
(-25)

p.c.: post-coitum.

-: not applicable.

Statistical significance: *: p<0.05.

( ): in brackets, percentage difference vs. controls.

Table 4: Net body weight change

Dose-level (mg/kg/day)

0

100

300

1000

Gravid uterus weight

473.6

497.8

520.4

467.0

 

-

(+5)

(+10)

(-1)

Carcass weight

3654.3

3583.9

3621.1

3547.8

 

-

(-2)

(-1)

(-3)

Net body weight change from Day 6 p.c.

-116.1

-164.3

-189.4

-258.1

-: not applicable.

( ): in brackets, percentage difference vs. controls.


 

Table 5: Macroscopic examination

Dose-level (mg/kg/day)

0

100

300

1000

Lungs: brownish colored focus(i)

1

0

0

0

Lungs: colored nodule(s)

0

0

0

1

Abdominal cavity: liquid contents

1

0

0

1

Stomach: blackish or reddish colored depressed area(s)

0

1

0

1

Stomach: whitish colored raised area(s)

0

0

1

0

Connective tissue, periovarian region:
serous cyst(s)

3

1

1

2

Gall bladder: reduced in size

0

0

0

2

Gall bladder: bilobed

0

0

1

1

No necropsy observations

20/24

23/24

19/22

18/24

 

Table 6: Hysterectomy data

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Number of females with live fetuses
at termination

22

20

20

22

159

Mean number of corpora lutea per animal

10.4

11.9

12.3*

12.7**

[11.4-12.8]

Mean number of implantations per animal

9.0

9.9

10.9*

11.0**

[9.5-11.4]

Mean pre-implantation loss (%)

14.1

15.5

10.4

13.2

[10.5-17.9]

Mean number of live fetuses per animal

8.1

9.1

9.1

9.6

[8.5-10.6]

Dead fetuses (%)

0.0

0.5

0.0

0.0

[0.00-1.00]

Mean number of implantation scars

0.0

0.0

0.0

0.0

/

Mean number of early resorptions

0.4

0.3

0.5

0.3

/

Mean number of late resorptions

0.5

0.6

1.4**a

1.0

/

Mean post-implantation loss (%)

9.4

7.9

16.0

11.7

[3.9-20.5]

Statistical significance: *: p<0.05 and **: p<0.01.

a: statistically significant for total number of late resorptions.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016); [Range of study means: min.; max.].

/: not reported in HCD.


 

Table 7: Fetal body weight and sex

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Mean fetal body weight

40.0

-

36.7*
(-8)

35.2**
(-12)

30.9#
(-23)

[27.6; 48.7]

Mean fetal male body weight

39.5

-

36.2

(-8)

35.5*

(-10)

31.2#

(-21)

[28.0; 50.9 ]

Mean fetal female body weight

40.0

-

37.0

(-8)

34.6**

(-14)

30.9#

(-23)

[17.2; 49.0]

Mean percentage of male fetuses

50.2

53.9

59.0

59.4

[47.4; 58.3]

-: not applicable.

Statistical significance: *: p<0.05, **: p<0.01 and #: p<0.001.

(): in brackets percentage difference vs. controls.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016); [Range of litter means: min.; max.].

Table 8: External variations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

2 (9.1)

2 (10.0)

3 (15.0)

7 (31.8)

11 (6.9)(b)

Fetuses affected, n (%)

2 (1.1)

2 (1.1)

3 (1.6)

15** (7.1)

14 (0.9)(b)

Amniotic fluid: abnormal color, F (L) %

0 (0)

0 (0)

0 (0)

5.7#(22.7*)

0 (0)

Local edema,F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0 (0)

Paw: hyperflexion,F (L) %

1.1 (9.1)

1.1 (10.0)

1.1 (10.0)

2.4 (22.7)

0 (0)

F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

Statistical significance: *: p<0.05, **: p<0.01 and #: p<0.001 for number of fetuses or litters.

(b): mean incidence.


 

Table 9: External malformations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

0 (0)

1 (5.0)

0 (0)

3 (13.6)

6 (3.8)(b)

Fetus affected, n (%)

0 (0)

1 (0.6)

0 (0)

3 (1.4)

6 (0.4)(b)

Affected Fetuses/Litter, Mean%

0.0

0.6

0.0

5.4

/

Cleft lip, F (L) %

0 (0)

0.6 (5.0)

0 (0)

0 (0)

0 (0)

Digit: ectrodactyly,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Digit: malpositioned,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Trunk: gastroschisis,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0.5 (5.3)(a)

Trunk: omphalocele,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Trunk: ombilical hernia,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0.4 (4.0)(a)

F: fetal incidence, L: litter incidence.

/:not reported in HCD; HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence; (b): mean incidence.

Table 10: Soft tissue variations

Dose-level (mg/kg/day)

0

100

300

1000

HCD(a)

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Cranial cavity: liquid contents, F (L) %

0 (0)

0 (0)

0.5 (5.0)

0.5 (4.5)

0 (0)

Gall bladder: small, F (L) %

2.2 (18.2)

1.1 (10.0)

1.1 (5.0)

4.2 (13.6)

0.6 (5.6)

Kidney: dilated renal pelvis,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

1.7 (16.0)

Kidney: small kidney,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Brachiocephalic trunk: absent,F (L) %

0 (0)

2.2 (15.0)

1.1(10.0)

2.4 (13.6)

4.0 (29.2)

F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence.


 

Table 11: Soft tissue malformations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

1 (4.5)

0 (0)

3 (15.0)

2 (9.1)

8 (5.0)(b)

Fetus affected, n (%)

1 (0.6)

0 (0)

3 (1.6)

2 (0.9)

8 (0.5)(b)

Affected Fetuses/Litter, Mean%

0.8

0.0

2.1

5.3

/

Brain: small cerebrum, F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0 (0)

Brain: marked dilated cerebral ventricle,F (L) %

0 (0)

0 (0)

0.5 (5.0)

0.5 (4.5)

0 (0)

Heart: ventricular septum defect,

 F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Lungs: small,F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0 (0)

Gall bladder: absent,F (L) %

0.6 (4.5)

0 (0)

0 (0)

0 (0)

0.6 (5.6)(a)

Vessels: dilated aortic arch,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0.4 (4.0)(a)

Vessels: absent aortic arch, F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0.5 (5.3)(a)

Vessels: dilated pulmonary trunk,

F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0.5 (5.3)(a)

F: fetal incidence, L: litter incidence.

/:not reported in HCD; HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence; (b): mean incidence.


 

Table 12: Skeletal examinations

Cartilage

Dose-level (mg/kg/day)

0

100

300

1000

HCD(a)

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Cartilage of sternebra(e): present, F(L) %

34.1 (81.8)

41.7 (85.0)

47.8*(95.0)

49.1**(95.5)

15.7 (62.5)

Cartilage of sternebra(e): misshapen, F(L) %

0 (0)

0 (0)

0 (0)

1.4 (9.1)

0 (0)

Cartilage of rib(s): branched, F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Cartilage of metacarpal bone(s): present,F(L) %

6.7 (27.3)

7.2 (50.0)

9.9 (55.0)

20.3#(54.5)

4.5 (33.3)

Forepaw: cartilage of median phalanx present,F(L) %

4.5 (22.7)

15.0**(55.0)

6.6 (50.0)

12.3**(45.5)

6.3 (29.2)

Cartilage of talus: present,

F(L) %

0 (0)

0 (0)

1.1 (10.0)

4.7** (31.8**)

0 (0)

Cartilage of metatarsal bone absent,F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence.

Statistical significance: *: p<0.05 , **: p<0.01, #: p<0.001 for number of fetuses or litters.

Table 13: Skeletal variations

Dose-level (mg/kg/day)

0

100

300

1000

HCD(a)

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Interparietal: incomplete ossification, F(L) %

1.1 (9.1)

4.4 (30.0)

4.4 (35.0)

4.7*(36.4)

4.1 (36.8)

Hemisternebra(e): unossified,

F(L) %

2.8 (13.6)

1.7 (15.0)

5.5 (30.0)

6.1 (50.0*)

2.9 (16.7))

Hemisternebra(e): incomplete ossification, F(L) %

2.2 (9.1)

1.7 (15.0)

4.9 (30.0)

6.6 (50.0**)

3.5 (22.2)

6thsternebra(e): incomplete ossification, F(L) %

2.2 (18.2)

2.8 (20.0)

4.4 (30.0)

8.5**(27.3)

6.9 (42.1)

6thsternebra(e): unossified, F(L) %

0 (0)

0 (0)

0.5 (5.0)

0.9 (9.1)

0.4 (4.2)

Supernumerary 13thrib(s), F(L) %

50.8 (86.4)

70.6#(100.0)

65.4**(100.0)

62.7*(90.9)

81.2 (100.0)

Knobby rib(s), F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Supernumerary 14thrib(s), F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Metacarpal bone: incomplete ossification,1stmetacarpal(s),F(L) %

5.6 (22.7)

6.1 (40.0)

6.6 (35.0)

14.2**(45.5)

8.4 (61.1)

Metacarpal bone: unossified
1stmetacarpal(s),F(L) %

1.1 (9.1)

2.2 (20.0)

3.3 (30.0)

9.4#(45.5*)

7.4 (33.3)

Forepaw: incomplete ossification median phalanx,F(L) %

3.4 (22.7)

10.6*(45.0)

6.0 (40.0)

9.4*(40.9)

10.0 (44.0)

Forepaw: unossified median phalanx,F(L) %

1.1 (9.1)

5.6*(30.0)

2.2 (15.0)

4.2 (22.7)

3.8 (20.8)

Talus: unossified, F(L) %

0 (0)

0 (0)

0.5 (5.0)

2.4 (18.2)

0.5 (5.6)

Talus: incomplete ossification, F(L) %

0 (0)

0 (0)

0.5 (5.0)

2.8*(22.7*)

0.6 (5.6)

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence.

Statistical significance: *: p<0.05, **: p<0.01, #: p<0.001 for number of fetuses or litters.

Table 14 : Skeletal malformations

Dose-level (mg/kg/day)

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

8 (36.4)

4 (20.0)

10 (50.0)

15 (68.2)

46 (28.9)(b)

Fetus affected, n (%)

10 (5.6)

4 (2.2)

15 (8.2)

21 (9.9)

56 (3.6)b)

Affected Fetuses/Litter, Mean%

6.4

2.5

7.7

13.7

/

Supraoccipital: split,F(L) %

0.6 (4.5)

0 (0)

0 (0)

0 (0)

0 (0)

Interparietal: split,F(L) %

1.1 (9.1)

1.7 (15.0)

2.2 (15.0)

2.8 (27.3)

1.2 (11.1)(a)

Interparietal: absent,F(L) %

1.7 (13.6)

0 (0)

1.6 (15.0)

0.9 (4.5)

3.4 (31.6)(a)

Lumbar hemivertebra:
supernumerary,F(L) %

0.6 (4.5)

0 (0)

0 (0)

0 (0)

0 (0)

Spine: scoliosisF(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Sternebra(e): fused,F(L) %

2.2 (13.6)

0.6 (5.0)

3.8 (30.0)

5.7 (45.5*)

2.2 (20.8)(a)

Rib(s): fused,F(L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0.9 (8.3)(a)

Tibia: misshapen,F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Metatarsal(s): absent, F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Hindpaw: absent digit(s),

 F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

/:not reported in HCD; HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence; (b): mean incidence.

Statistical significance: *: p<0.05 for number of litters.

Table 15 : Pregnancy status

Dose-level (mg/kg/day)

0

100

300

1000

Mated dams, n

24

24

24

24

Pregnant females alive at term, n

22

20

20

22

Non pregnant dams, n

2

4

2

2

Prematurely euthanized dams (poor clinical condition), n

0

0

1

0

Found dead dams, n

0

0

0

0

Aborted dams, n

0

0

1

0

Dams with live fetuses on Day 29p.c.

22

20

20

22

n: number

Conclusions:
The test item, Propylidynetrimethyl trimethacrylate, was administered by gavage, once daily from Days 6 to 28 p.c. inclusive, to mated female New Zealand White rabbits at dose levels of 100, 300 or 1000 mg/kg/day.

According to the results obtained in this study:
-The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the lower body weight gain, lower net body weight change and lower food consumption observed at 1000 mg/kg/day.
-The NOAEL for embryo-fetal development was considered to be 300 mg/kg/day based on adverse effects on fetal weight associated with increased incidences of external malformations at 1000 mg/kg/day.
Executive summary:

The objective of this prenatal developmental toxicity study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day before scheduled hysterectomy [Day 6 to Day 28 post-coitum (p.c.) inclusive].


 


Methods


The test item was administered to three groups of 24 mated female New Zealand White rabbits, once daily from Day 6 to Day 28 p.c., by gavage, at dose levels of 100, 300 or 1000 mg/kg/day (groups 2 to 4). An additional group of 24 mated females received the vehicle, 0.5% Carboxymethylcellulose (400-800 cps) / 0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions, and acted as the control group (group 1). A constant dosage volume of 3 mL/kg/day was used.


 


The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c. animals were euthanized and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. Macroscopic lesions were sampled and preserved in a fixative. Live fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.


 


Results


Chemical analysis of dose formulations: the test item concentrations in the administered dose formulations analyzed infirst and last weeks of treatmentremained within an acceptable range of variations (-1.3% to +8.0%) when compared with the nominal values (± 15% of the nominal concentrations).


 


Pregnancy status: in the control, 100, 300 and 1000 mg/kg/day groups, there were 22, 20, 20 and 22 females with live fetuses at hysterectomy, respectively.


 


Mortality/abortion: there were no test item-related unscheduled deaths or abortions in any group.


 


Clinical signs: there were no remarkable clinical observations in surviving animals.


 


Body weight, body weight change, net body weight change and food consumption:at 1000 mg/kg/day and when compared with controls, there were lower body weight change over the whole treatment period (-42%, p<0.05),net body weight change on Day 29p.c.(-258.1 gvs. -116.1 g in control animals)and food consumption (down to -25% on Days 24 to 29p.c., p<0.05).During the gestation period (from Day 6p.c.to Day 29p.c.), four out of twenty-two females showed body weight loss together with decreased food consumption and low net body weight change values.


Taking together, these findings were considered to be adverse at 1000 mg/kg/day.


 


Maternal terminal examination: at necropsy, discolored area(s) were observed in the stomach of one female in each dose levels group.Taking into account the absence of similar findings in the control group, a test item treatment relationship cannot be excluded.


There were no effects on mean gravid uterus weight or mean carcass weight.


 


Hysterectomy data: there were no test item-related effects on hysterectomy data in surviving dams on Day 29p.c.


 


Fetal body weight and percentage of male fetuses:


From 100 mg/kg and when compared with controls, lower mean fetal boy weights were observed but all values were within the HCD. At 1000 mg/kg/day, the mean litter value of fetal body weight was decreased when compared with controls and outside the HCD. This decrease at 1000 mg/kg/day was considered as adverse.


 


At 300 and 1000 mg/kg/day, a minimally, not statistically significant, higher percentage of male fetuses was observed (59.0% and 59.4%, respectively,vs.50.2% in controls). This non-adverse percentage was close to the upper limit of the Historical Control Data (58.3%).


 


Fetal examinations:


External examination


At 1000 mg/kg/day and when compared with controls, there were test item-related higher fetal and litter incidences of external variations, namely abnormal color of amniotic fluid [5.7% (p<0.001) and 22.7% (p<0.05)vs.0% in controls, respectively].


Three fetuses from three different litters also had ectrodactyly, malpositioned digit and gastroschisis (fetus K31086-01 F), omphalocele (fetus K31083-11 M) and umbilical hernia (fetus K31094-01 M). Although the fetal and litter incidences of these malformations mainly impacting the abdominal wall were similar to the Historical Control Data, a relationship to the test item cannot be ruled out.


 


Soft tissue examination


There were no test item-related variations or malformations at soft tissue examinationat any dose level.


 


Cartilage and skeletal examinations


When compared with controls and/or Historical Control Data, test item-related findings of delayed ossification were noted with higher litter and/or fetal incidences from 100 mg/kg/day [i.e.unossification of 1st metacarpal bone and/or incomplete ossification of hemisternebra(e)], from300 mg/kg/day [i.e.incomplete ossification of interparietal,unossification of hemisternebra(e), and/or incomplete ossification of 6th sternebra(e)] and at 1000 mg/kg/day[i.e.unossification of 6th sternebra(e), incomplete ossification of 1st metacarpal bone and/or unossification or incomplete ossification of talus].


 


When compared with controls and/or Historical Control Data, there were increased fetal and litter incidences of fetuses with fused sternebra(e) at 300 (3.8% and 30.0%vs.2.2% and 13.6% in controls, respectively) and 1000 mg/kg/day [5.7 and 45.5 (p<0.05), respectively], and split interparietalat 300 (2.2% and 15.0%vs.1.1% and 9.1% in controls, respectively) and1000 mg/kg/day (2.8% and 27.3%).These test item-related findings were rather considered as variations (non-adverse findings) asthey were consistent with a developmental delay in ossification.


 


Onefetus (K31086-01 F), showing external malformations, also had skeletal malformations (tibia misshapen, absent digits hindpaw and scoliosis).


 


Conclusion


The test item,Propylidynetrimethyl trimethacrylate, was administered by gavage, once daily from Days 6 to 28 p.c.inclusive, to mated female New Zealand White rabbits at dose levels of 100, 300 or 1000 mg/kg/day.


 


According to the results obtained in this study:


.         The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the lower body weight gain, lower net body weight change and lower food consumption observed at 1000 mg/kg/day.


.         The NOAEL for embryo-fetal development was considered to be 300 mg/kg/day based on adverse effects on fetal weight associated with increased incidences of external malformations at 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
This study is reliable, performed with OECD guidelines.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

First key study: Developmental study in rats (Braun 2015):

The oral (gavage) prenatal developmental toxicity of the test item Propylidynetrimethyl trimethacrylate (CAS 3290-92-4) to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 414 (2001) guideline.

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 24 mated females per group were treated by gavage once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females were sacrificed on day 21 post coitum and the foetuses were removed by caesarean section.

All females survived until the scheduled necropsy. No clinical signs were recorded in any group, and the mean daily food consumption of all groups compared favourably. Although the mean absolute body weights were unaffected, a statistically significant lower mean body weight gain of the dams in group 4 (40 vs. 46 % in the controls on day 21 post coitum) and a statistically significant decreased corrected body weight gain (corrected for the gravid uterus weight) in group 4 (8.0 vs. 11.5 % in the controls on day 21) were considered to be test item-related. The reproduction data (post-implantation loss and mean number of foetuses per dam) was unaffected by treatment and no macroscopical findings were noted at any dose level.

The mean placenta weights of all groups were similar. The external examination of the foetuses showed no abnormalities of toxicological relevance and sex ratios were unaffected. Foetuses of group 4 dams had lower mean body weights (-6 % on litter basis and -8 % on individual basis). This was considered to be related to the treatment with test item.

In conclusion based on the slightly lower mean body weight gain, the NOEL (No Observed Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day, whereas the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 1000 mg/kg bw/day or higher. For prenatal development, the NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) were considered to be 300 mg/kg/day, based on the lower mean foetal weights noted at 1000 mg/kg/day.

First supporting study: Dose-range finding study in rats (Kaiser 2014):

The oral (gavage) prenatal developmental toxicity of the test item Propylidynetrimethyl trimethacrylate (CAS 3290-92-4) to Wistar rats was investigated in a non-GLP dose range-finding study similar to the OECD TG 414 (2001) protocol.

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 8 mated females per group were treated by gavage with the test item once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). Using corn oil as vehicle a standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was administered. Control animals were dosed with the same volume of vehicle alone.

The treatment with the test item did not result in any mortalities. No clinical signs were observed. No effects on food consumption were recorded. At necropsy, no macroscopic findings related to the treatment with the test item were found.

No findings were noted during external examination of foetuses. No effects on sex ratio and body weights of foetuses were recorded.

In group 4, the body weight gain and corrected body weight gain of the dams was slightly decreased. The post-implantation loss and the number of embryonic resorptions were slightly increased. The number of foetuses (in relation to the number of implantations) was slightly decreased. These effects were considered test item-related. No effects of the treatment with the test item were recorded in groups 2 and 3.

In conclusion based on the results of this study, nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day are considered to be suitable for the subsequent main study for effects on embryo-foetal development in the Han Wistar rat.

Second supporting study : developmental study in rats (Serota 1982):

Test compound (TMPTMA) was evaluated for tolerance in pregnant rats to determine dose levels for a subsequent teratology screening study. Test substance was administered by gavage at 500, 2500 and 5000 mg/kg/day (6 inseminated females per dose) from Days 6-15 of gestation. The dose of 5000 mg/kg/day was highly toxic for females; mortality, severe clinical signs, decrease of bodyweight and macroscopic changes were observed. As the dose of 2500 mg/kg/day was well tolerated by females, this dose was selected to evaluate the embryo/fetal toxicity and teratogenic effects of Propylidynetrimethyl trimethacrylate. Test substance was administered by gavage from Days 6-15 of gestation to 22 females.

Compound-related maternal toxicity was observed: decreased survival, decreased body weight gains, increased incidence of clinical signs, and an increased incidence of gross pathology findings. Foetotoxic effects were observed and consisted of effects on resorptions, fetal viability, fetal weights and fetal lengths. The decrease in mean gravid uterine weights was directly attributed to the foetotoxic effect. Those effects on the fetuses considered to be directly related to the maternal toxicity of the test compound. An increase in the incidence of visceral variants (23%) was observed when compared to control (3%). A higher than control number of fetuses exhibiting skeletal variants was observed. The incidence was found to be significant for skeletal variants, lagging ossification, skull closure less than 75% and angulated ribs. The types of visceral and skeletal variants observed to be increased over control are suggestive as resulting from the maternal toxicity noted.

The Registrants consider that it is not possible to conclude on the developmental toxicity of Propylidynetrimethyl trimethacrylate in rats based on Serota’s studies based on the following arguments: Only one group tested at the maximum tolerated dose of 2500 mg/kg/day; High incidence of maternal death (5/22 females); Severe decreased maternal body weight during days 6-20 of pregnancy (-20 g for treated females vs +87 g in controls); Only 17 females evaluated for fetal examinations in a context of severe maternal toxicity; No NOAEL could be set.

Second key study: Developmental study in rabbits (Papineau 2018):

The objective of this prenatal developmental toxicity study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day before scheduled hysterectomy [Day 6 to Day 28 post-coitum (p.c.) inclusive]. The test item was administered to three groups of 24 mated female New Zealand White rabbits, once daily from Day 6 to Day 28p.c., by gavage, at dose levels of 100, 300 or 1000 mg/kg/day (groups 2 to 4). An additional group of 24 mated females received the vehicle, 0.5% Carboxymethylcellulose (400-800 cps) / 0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions, and acted as the control group (group 1).A constant dosage volume of 3 mL/kg/day was used.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29p.c.animals were euthanized and submitted to a macroscopicpost-mortemexamination. Hysterectomy was performed and the numbers ofcorpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. Macroscopic lesions were sampled and preserved in a fixative. Live fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

In the control, 100, 300 and 1000 mg/kg/day groups, there were 22, 20, 20 and 22 females with live fetuses at hysterectomy, respectively.

There were no test item-related unscheduled deaths or abortions in any group. There were no remarkable clinical observations in surviving animals.

At 1000 mg/kg/day and when compared with controls, there were lower body weight change over the whole treatment period (-42%, p<0.05),net body weight change on Day 29 p.c.(-258.1 g vs. -116.1 g in control animals) and food consumption (down to -25% on Days 24 to 29 p.c., p<0.05).During the gestation period (from Day 6 p.c.to Day 29p.c.), four out of twenty-two females showed body weight loss together with decreased food consumption and low net body weight change values. Taking together, these findings were considered to be adverse at 1000 mg/kg/day.

At necropsy, discolored area(s) were observed in the stomach of one female in each dose levels group. Taking into account the absence of similar findings in the control group, a test item treatment relationship cannot be excluded.

There were no effects on mean gravid uterus weight or mean carcass weight. There were no test item-related effects on hysterectomy data in surviving dams on Day 29 p.c.

From 100 mg/kg and when compared with controls, lower mean fetal boy weights were observed but all values were within the HCD. At 1000 mg/kg/day, the mean litter value of fetal body weight was decreased when compared with controls and outside the HCD. This decrease at 1000 mg/kg/day was considered as adverse.

At 300 and 1000 mg/kg/day, a minimally, not statistically significant, higher percentage of male fetuses was observed (59.0% and 59.4%, respectively, vs. 50.2% in controls). This non-adverse percentage was close to the upper limit of the Historical Control Data (58.3%).

At 1000 mg/kg/day and when compared with controls, there were test item-related higher fetal and litter incidences of external variations, namely abnormal color of amniotic fluid [5.7% (p<0.001) and 22.7% (p<0.05) vs.0% in controls, respectively].

Three fetuses from three different litters also had ectrodactyly, malpositioned digit and gastroschisis (fetus K31086-01 F), omphalocele (fetus K31083-11 M) and umbilical hernia (fetus K31094-01 M). Although the fetal and litter incidences of these malformations mainly impacting the abdominal wall were similar to the Historical Control Data, a relationship to the test item cannot be ruled out.

There were no test item-related variations or malformations at soft tissue examinationat any dose level.

When compared with controls and/or Historical Control Data, test item-related findings of delayed ossification were noted with higher litter and/or fetal incidences from 100 mg/kg/day [i.e.unossification of 1st metacarpal bone and/or incomplete ossification of hemisternebra(e)], from300 mg/kg/day [i.e.incomplete ossification of interparietal,unossification of hemisternebra(e), and/or incomplete ossification of 6th sternebra(e)] and at 1000 mg/kg/day [i.e.unossification of 6th sternebra(e), incomplete ossification of 1st metacarpal bone and/or unossification or incomplete ossification of talus].

When compared with controls and/or Historical Control Data, there were increased fetal and litter incidences of fetuses with fused sternebra(e) at 300 (3.8% and 30.0%vs.2.2% and 13.6% in controls, respectively) and 1000 mg/kg/day [5.7 and 45.5 (p<0.05), respectively], and split interparietalat 300 (2.2% and 15.0%vs.1.1% and 9.1% in controls, respectively) and1000 mg/kg/day (2.8% and 27.3%).These test item-related findings were rather considered as variations (non-adverse findings) asthey were consistent with a developmental delay in ossification. One fetus (K31086-01 F), showing external malformations, also had skeletal malformations (tibia misshapen, absent digits hindpaw and scoliosis).

In conclusion, According to the results obtained in this study:  The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the lower body weight gain, lower net body weight change and lower food consumption observed at 1000 mg/kg/day. The NOAEL for embryo-fetal development was considered to be 300 mg/kg/day based on adverse effects on fetal weight associated with increased incidences of external malformations at 1000 mg/kg/day.

Third supporting study : dose-rande finding study in rabbits (Chalmey 2017):

The test item, was administered to time-mated female New-Zealand White rabbits, by oral route, once daily, from Days 6 to 28p.c., at dose levels of 100, 300 or 1000 mg/kg/day.

At 100 mg/kg/day, there were no adverse findings on maternal parameters orat examination of the fetuses.

At 300 mg/kg/day, there were no toxicity findings on maternal parameters; the mean fetal body weight was reduced when compared with controls.

At 1000 mg/kg/day, in average, the females lost weight between Days 6 and 9p.c.and the mean food consumption was slightly reduced in dams. However, there were no effects on dam mean body weight. The fetal body weight was reduced when compared with controls.

Therefore, on the basis of the experimental conditions and results of this study, the dose of 1000 mg/kg/day was considered to be below the Maximum Tolerated Dose in pregnant rabbits and could be selected as the highest dose to be tested in a further main study.

Justification for classification or non-classification

Based on the available data and taking into account the NOAELs set at 1000 mg/kg/day in the OECD 443 study, no classification for fertility and development is required for TMPTMA according to the CLP Regulation (EC) N° 1272-2008. 

Additional information