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Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guidelineopen allclose all
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).

Test material

Constituent 1
Chemical structure
Reference substance name:
Propylidynetrimethyl trimethacrylate
EC Number:
221-950-4
EC Name:
Propylidynetrimethyl trimethacrylate
Cas Number:
3290-92-4
Molecular formula:
C18H26O6
IUPAC Name:
2,2-bis[(methacryloyloxy)methyl]butyl methacrylate (non-preferred name)

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

open allclose all
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

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

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.

Effect levels (F1)

open allclose all
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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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.

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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