1,1,3,3-tetramethylbutyl peroxyneodecanoate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In life period: 07 Jan - 27 Jul 2020

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 guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Study design according to ECHA TPE-D-2114440664-49-01/F

Test material

Test material information

additive 1

Reference substance name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

EC Number:

297-629-8

EC Name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

Cas Number:

93685-81-5

Molecular formula:

not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

isododecane

Constituent 1

Reference substance name:

1,1,3,3-Tetramethylbutyl peroxyneodecanoate (CAS Number: 51240-95-0)

IUPAC Name:

1,1,3,3-Tetramethylbutyl peroxyneodecanoate (CAS Number: 51240-95-0)

Test material form:

liquid

Remarks:

Clear colourless liquid

Details on test material:

Sponsor's identification: 1,1,3,3-Tetramethylbutyl peroxyneodecanoate (CAS Number: 51240-95-0)
Description: clear, colourless liquid
Purity: 69.2 %
Purity/Composition correction factor: no correction factor required
Batch number: 1911442793
Specific gravity/density: 0.8573
Physical Description: clear colourless liquid
Storage Conditions: in freezer (≤ -15°C)
Expiry date: 28 July 2020

Specific details on test material used for the study:

Specific gravity/density: 0.8573
Physical Description: clear colourless liquid
Storage Conditions: in freezer (≤ -15°C)
Purity/Composition correction factor: no correction factor required

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

Source: Charles River Deutschland, Sulzfeld, Germany.
At initiation of dosing, animals were 6-7 weeks old and weighed between 149 and 214 g (males) and between 115 and 163 g (females).

Sex:

male/female

Details on test animals or test system and environmental conditions:

On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm or type 2000P; 61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 22°C with an actual daily mean relative humidity of 45 to 76%. The values that were outside the targeted humidity range occurred for 14 days, were without a noticeable effect on the clinical condition of the animals and were considered not to have had any influence on the outcome of the study. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures .
During motor activity measurements, F1-Cohort 2A animals had no access to food for a maximum of 2 hours.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, F1-Cohort 2A animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

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. Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants that would interfere with the objectives of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
When preparing dosing formulations, the test item was weighed on ice directly from the freezer. The vehicle was added to the test item afterwards on the bench top (no need for acclimatization to room temperature). Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
Until Day 23, inclusive, dosing formulations were prepared daily as a suspension and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing.
From Day 24 onwards, dosing formulations were prepared weekly as a suspension, 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 5 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
The test substance were formulated in Arachis oil. This vehicle is selected based on a previous 90-day repeated toxicity study (Envigo Study No. 41501125) and in consultation with the Sponsor.
- Concentration in vehicle: 12.5, 37.5 or 112.5 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg

Details on mating procedure:

Cohabitation/Mating Procedure – F0-Generation
Frequency:
The mating period consisted of a maximum of 14 consecutive days.
Procedure:
Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating, after at least 10 weeks of treatment. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Remarks:

UPLC with UV detection

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected in week 1 of treatment. Concentration was measured for all groups, homogeneity and stability was measured for groups 2 and 4.
Dose formulation samples were also collected in week 11 and 22 of treatment. Concentration was measured for all groups, homogeneity was measured for groups 2 and 4.

Concentration Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if the mean sample concentration results of the suspensions were within or equal to ± 15% for suspensions of the target concentration.
Homogeneity Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <=10%.
Stability Analysis: Stability analyses performed previously in conjunction with the preliminary reproduction/developmental toxicity screening test in rats (Test Facility Study No. 20188708) demonstrated that the test item is stable in the vehicle when prepared and stored for at least 5 hours at room temperature under normal laboratory light conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20188708. In addition, at the first occasion in Week 1 of the treatment phase, the stability of the prepared formulation containing the test item was determined when stored over 8 days in a refrigerator. Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
 

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week. Males were treated for a minimum of 12 weeks, including 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating and post-mating period, up to and including the day before scheduled necropsy. This included a minimum of ten weeks prior to mating and during the mating period.
Females were treated 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing was designated as Day 1. The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
F0-animals were treated up to and including the day before necropsy.
Prior to weaning, pups were not treated directly but were potentially 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 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) were not dosed.

Frequency of treatment:

daily

Details on study schedule:

See above

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

450 mg/kg bw/day

No. of animals per sex per dose:

Number of F0-males: 100 (25 animals/group)
Number of F0-females: 100 (25 animals/group; nulliparous and non-pregnant)

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
In the screening study with 10 animals/sex/group, dose levels of 15, 150 and 450 mg/kg/day were tested. The rats of the control group received the vehicle, Arachis oil, alone. Males were treated for 10 weeks prior to mating, during mating, and up to termination (for 90 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-55 days). Females that failed to deliver pups were treated for 43 days.
The parental No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg/day based on kidney findings in males (i.e. pale discoloration and increased organ weight of the kidneys, together with correlating microscopic findings, including renal hyaline droplet accumulation, granular casts and tubular basophilia). Liver findings observed at 150 mg/kg/day (males) and 450 mg/kg/day (both sexes) were considered non-adverse as no degenerative changes were noted at the microscopic level. These findings included black brown discoloration of the liver of males at 150 and 450 mg/kg/day and increased liver weights in males and females at 450 mg/kg/day, correlating with centrilobular hepatocellular hypertrophy in males. Furthermore, a reduction of total T4 was observed in the high dose males. There were no correlating changes in serum TSH level, thyroid organ weight or any histopathological findings in the thyroid gland. However, possible adversity of the reduced serum level of total T4 could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL. The reproduction NOAEL was established at 150 mg/kg/day based on sperm findings. At the high dose (450 mg/kg/day), there were two males for which motile and progressive sperm was (nearly) absent, and an increased number of sperm cells with detached head. As these two males failed to sire, it was considered adverse. There was no microscopic correlate. An increased number of sperm cells with detached heads was also seen for other males at 450 mg/kg and for two males at 150 mg/kg, without any corroborative findings at the tissue level. In the absence of any effect on reproductivity, this was considered non-adverse. The developmental NOAEL was established at 150 mg/kg/day based on decreased body weights of pups at 450 mg/kg/day (0.86x of control) noted on PND 13.

Rationale for animal assignment (if not random): random

Fasting period before blood sampling for clinical biochemistry: The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Mortality/Moribundity Checks – F0-Generation Frequency: At least twice daily throughout the study. Procedure: Animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Frequency: During treatment, animals were observed at least twice daily, up to the day prior to necropsy. These clinical observations were at least conducted prior to dosing and after dosing.
Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 1, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHT:
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. Animals were weighed individually.

FOOD CONSUMPTION AND COMPOUND INTAKE:
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:
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. Subjective appraisal was maintained during the first 44 days of the pre-mating period. As a treatment-related effect on water consumption was suspected in treatment Week 7, quantitative measurement of water intake was introduced for all males and females from Day 45 of the pre-mating period onwards. Water consumption was measured overnight (24 ± 3 hours) twice weekly (for exceptions, see Appendix 16), except for males and females which were housed together for mating and for females without evidence of mating. For males this measurement continued up to and including the last Friday before scheduled necropsy. For 10 selected females/group (all groups) with evidence of mating measurement of water consumption continued during post-coitum on Days 1, 4, 7, 11, 14 and 17 post-coitum and during lactation on PND 1, 4, 7, 11, 14 and 17.

OTHER:
Arena Observations
Frequency: Once before the first administration of the test item and at weekly intervals during the treatment period.
Procedure: Animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. 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.

Litter observations:

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

PARAMETERS EXAMINED
To reduce variability among the litters, eight pups from each litter of equal sex distribution were selected. 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) is acceptable. In case less than 20 litters with at least 8 live pups/litter were available, litters were culled to a size which is aimed to be adequate for allocation of a sufficient number of pups into the respective cohorts.

GROSS EXAMINATION OF DEAD PUPS:
Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). 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. Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not performed

Mortality/Moribundity Checks – Cohorts 1A, 1B, 1C and 2A
At least twice daily throughout the study. 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.

Clinical Observations– Cohorts 1A, 1B, 1C and 2A
During treatment, animals were observed at least twice daily, up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Arena Observations – Cohorts 1A, 1B, 1C and 2A
Once on the day of weaning before dosing 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.

Body Weights – Cohorts 1A, 1B, 1C and 2A
Weekly from weaning onwards. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. Animals were weighed individually.

Food Consumption – Cohorts 1A, 1B, 1C and 2A
Weekly from weaning onwards up to the day prior to scheduled necropsy. Food consumption was measured quantitatively.

Water Consumption – Cohorts 1A, 1B, 1C and 2A
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Vaginal Patency – Cohorts 1A, 1B, 1C and 2A
Daily for all females from PND 25 onwards. Examinations continued until vaginal patency was present. Vaginal patency (vaginal opening) was monitored by visual inspection of the vaginal area. Body weight was recorded on the day of acquisition of vaginal patency.

Balanopreputial Separation – Cohorts 1A, 1B, 1C and 2A
Daily for all males from PND 35 onwards. Examinations continued until balanopreputial separation was present. Balanopreputial separation (prepuce opening) was monitored by visual inspection of the genital area. Body weight was recorded on the day of acquisition of balanopreputial separation.

Stage of Estrus Determination – Cohorts 1A and 1B
On the day of scheduled necropsy, a vaginal lavage was taken. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously. Estrous stages were determined by examining the cytology of vaginal lavage samples.

Cohort 1A (20 animals/sex/group)
Estrous Cycle Determination – Cohort 1A
First period: Daily vaginal lavage was performed for all Cohort 1A females starting on the first day after onset of vaginal patency and was continued until the first estrus was determined, in order to determine the time interval between these two events. The estrus cycle data of the first period is not but will be retained in the raw data. Second period: Daily vaginal lavage was performed from PND 75 to 88. Estrous stages were determined by examining the cytology of vaginal lavage samples.

Cohort 2A (10 animals/sex/group)
Acoustic startle response – Cohort 2A
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. To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose group and sex. The animals weree 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 were 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 intertrial interval. Average response amplitude (AveN) was analyzed in five blocks of 10 trials each.
Functional Observation Battery – Cohort 2A
Once between PND 63-75. The Functional Observation Battery (FOB) tests were conducted in the order of sequence and was divided between several days. The detailed clinical observations and locomotor activity were conducted in separate room(s) specially equipped for these purposes. The other FOB tests were conducted in the study room.
1. Detailed clinical observations consisted of a number of tests conducted in- and out-side the home cage. To the extent possible, testing of animals was counterbalanced across dose groups.
2. Rectal temperature was measured immediately after the detailed clinical observations.
3. Locomotor activity was tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions. To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups. 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 (Series M4-10, Mark-10 Corporation).
7. Landing (hind) foot splay. This was recorded per animal as the mean of three measurements.

Body Weights – F1-Generation
On PND 1, 4, 7, 13 and 21. Live pups were weighed individually.
Sex – F1-Generation
On PND 1, 4 and 13. Sex was externally determined for all pups.
Anogenital Distance – F1-Generation
On PND 1. Anogenital distance (AGD) was measured for all live pups. The AGD was normalized to the cube root of body weight.
Areola/Nipple Retention – F1-Generation
On PND 13. All males in each litter were examined for the number of areola/nipples.

Postmortem examinations (parental animals):

All groups were subjected to terminal body weight determination, necropsy, tissues collection and organ weights determination.
Histopathological examination of target tissues, reproductive tissues and gross lesions was carried out of animals of groups 2 and 3 (low and mid dose).
Animals of groups 1 and 4 and any unscheduled deaths were subject as required by OECD443. - see attachment.

Unscheduled Deaths – F0-Generation
If an animal 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 were euthanized for humane reasons as per Test Facility SOPs. These animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained, but not weighed.

Scheduled Euthanasia – F0-Generation
For animals surviving until scheduled euthanasia terminal body weight was recorded; they were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- Males which sired: After successful mating and a minimum of 10 weeks of treatment.
- Males which failed to sire: At the end of the mating period and after a minimum of 10 weeks of treatment.
- Females which delivered: LD 23-25.
- Females which failed to deliver: With evidence of mating: Post-coitum Days 25-27. 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 were euthanized within 24 hours after the last pup was found dead or missing.
Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.

Necropsy – F0-Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Sperm Analysis – F0-Generation
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. 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.

Organ Weights – F0-Generation
The organs identified for weighing were weighed at necropsy for all scheduled euthanasia animals. 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) was calculated.

Tissue Collection and Preservation – F0-Generation
Representative samples of the tissues (OECD 443) were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

Postmortem examinations (offspring):

Unscheduled Deaths – F1-Generation
Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). 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 the single pup found dead on PND 14 a limited necropsy was performed including sex determination (both externally and internally, if possible).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood was collected, if possible. Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

TERMINAL PROCEDURES – F1-GENERATION FROM WEANING ONWARDS
A necropsy was conducted for animals that died on study, and specified tissues were collected. When necessary, animals were refrigerated before necropsy to minimize autolysis. Spare F1-animals which were not assigned to one of the cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital (Euthasol® 20%) or by exsanguinantion. 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). Descriptions of all external abnormalities were recorded.
For all animals, necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Organ weights were not recorded for animals found dead. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated. The organs identified for weighing and representative samples of the tissues mentioned in OECD 443 were weighed and collected.

Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 89-95, with a few exceptions (PND 85 or PND 99). 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 weighed and deeply anesthetized using isoflurane and subsequently exsanguinated.
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Cohort 1B
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

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

Cohorts 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 and no terminal body weight was recorded.
The animals were first anesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
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 was measured for all animals selected for neuropathology . Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
On PND 22-24, blood samples (1.0 mL) were collected between 7.00 and 10.30 a.m. from all animals by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure. Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).

Sperm Analysis – Cohort 1A
For all surviving males of Cohort 1A, the following assessments were performed: Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa 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.
As for animal Nos. 452 and 459 (Group 4), abnormalities were noted 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.
As for animal, 370 (Group 3) and 448 (Group 4), abnormalities were noted in both epididymides, both these organs were fixed in modified Davidson's solution and no evaluation of sperm numbers was performed.

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) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 µm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:
Parameter (Abbreviation) Unit Markers for identification
T-cells % Lymphoid cells CD3+/CD45RA-
T-helper cells % Lymphoid cells CD3+/CD4+/CD8-
T-cytotoxic cells % Lymphoid cells CD3+/CD4-/CD8+
B-cells % Lymphoid cells CD3-/CD45RA+
NK-cells % Lymphoid cells CD3-/CD161a+
Ratio T-helper cells/ T-cytotoxic cells (Th/Tc) - -
The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.
The % lymphoid cells of peripheral blood mononuclear cells (PBMC) will be determined using the Forward Scatter and Side Scatter.

Liver Enzyme Activity Measurement Ex Vivo – Cohort 1A
During necropsy at the end of treatment, a liver sample was collected from all Cohort 1A-males in the control and high dose group (Groups 1 and 4, respectively; 20 males/group) for liver enzyme activity measurements. This was done after weighing of the liver and prior to fixation. From each animal, the required liver samples for histopathological examination were taken first and preserved in fixative. Subsequently all remaining liver tissue (approximately 2-3 gram per male) was placed in individual, appropriately labeled, plastic containers and snap frozen in liquid nitrogen. Snap frozen samples were kept in liquid nitrogen or surrounded by dry ice until storage (for exceptions, see Appendix 16). The samples were stored in a freezer (≤ - 75°C) until dispatch on dry ice. The tissue sample containers were labeled with waterproof stick-on labels containing at least the following information: study code, animal number, matrix (L=Liver), time point (N=necropsy), biomarker research (BMR), scheduled sampling date. Labeled liver samples were transferred on dry ice to the In Vitro ADME (IVA) group at the Test Facility. After arrival at the In Vitro ADME (IVA) group, liver samples were stored at in a freezer (≤ - 75°C).
The following was measured:
- Determination of the Protein Concentration
- Determination of Cytochrome P450 (CYP450) Content
- Determination of the following enzymatic activities: 7-Pentoxyresorufin-O-Deethylation (PROD) Activity, Benzyloxyresorufin-O-Deethylation (BROD) Activity, and T4-Glucuronidation Activity
- UPLC-PDA-MS Analysis

Cohort 1B
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

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

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 and no terminal body weight was recorded.
The animals were first anesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
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 was measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. On PND 22-24, blood samples (1.0 mL) were collected between 7.00 and 10.30 a.m. from all animals by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure. Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).

Histology – F0- and F1-Generation
Tissues mentioned in OECD 443 from a selection of the F0- and F1-animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).
Cohort 1A
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.
Cohorts 2A and 2B
In addition to the procedures described above, the entire brain from all Groups was processed to the block stage up front at the same time to avoid effects of fixation duration on morphometry. Sections of the brains of all Cohort 2A and 2B animals (all groups) were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet (LFB/CV). Both LFB/CV and HE stained sections of the brains from Cohort 2A and 2B animals (all Groups) were sent to Charles River Laboratories France Safety Assessment SAS and digitalized with a whole slide scanner. Subsequently, they were returned to the Test Facility for morphometric analysis. For morphometric (quantitative) analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections are obtained.
.
Histopathology – F0- and F1-Generation
All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported.

Animals of Cohorts 2A and 2B
Morphometric (quantitative) analyses of CNS tissues was performed for Cohort 2A and Cohort 2B animals of Groups 1 and 4. Analyses included measurements from neocortical, hippocampal, and cerebellar areas selected.

See also attachment

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The startle data set and the follicle count data set (at least 3 groups) was compared using an overall Kruskal-Wallis. Whenever, the overall test wais significant, the Wilcoxon Rank-Sum test wasill be applied to compare the treated groups to the control group.
Incidence
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test wais significant.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females , the remaining group values were calculated from the total number in each group.
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
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

Offspring viability indices:

See above

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant findings were observed up to 450 mg/kg/day.
Slight to moderate salivation was seen after dosing in males and females of all groups, including the control group. This finding of salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and relatively minor severity of the effect, its time of occurrence (i.e. after dosing) and the fact that both control and treated animals were affected similarly.
Other clinical signs noted during the treatment period included rales, hunched posture, piloerection, focal erythema, scabs, a wound, and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. Since they were observed at a low incidence and occurred in the absence of a dose-related response, they were considered signs of no toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females of the control group and two females at the low dose (50 mg/kg/day) died or were euthanized before scheduled necropsy. None of these deaths was considered to be caused by the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted in males and females up to 450 mg/kg/day.
For females at 450 mg/kg/day, a slightly higher body weight gain was observed during the first three weeks of premating. No toxicological relevance was attached to this finding, as changes compared to the concurrent control group were slight (ranging from 1.11-1.15x of control) and transient.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted in males and females up to 450 mg/kg/day.
In males at 150 and 450 mg/kg/day, a higher food intake relative to body weight was noted in pre-mating Week 6-7 (150 mg/kg/day), and Weeks 6-9 and Week 10-11 (450 mg/kg/day). No toxicological relevance was attached to this finding, as changes compared to the concurrent control group were slight (ranging from 1.05-1.08x of control) and transient only.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

A treatment-related effect on water consumption was suspected for F0-animals in treatment Week 7, based on subjective appraisal. Therefore, quantitative measurement of water consumption (twice weekly) was introduced from premating Day 45 onwards.
In males at 150 and 450 mg/kg/day, an increased absolute water consumption was observed during the remainder of the premating period and during the mating period (not always statistically significant). At both dose levels, mean of means was each 1.24x of control over Days 45-71 of the pre-mating period and each 1.23x of control during the mating period.
In females, absolute water consumption was increased at 450 mg/kg/day only, with a mean of means of 1.20x of control over pre-mating Days 45-71. During post-coitum, no clear difference in water consumption was observed between the groups. However, during lactation a trend towards higher water consumption was observed at the high dose level again (not always statistically significant). Mean of means was 1.14x of control over lactation Days 1-18.
Water consumption at 50 mg/kg/day (males and females) and 150 mg/kg/day (females) remained in the same range as controls.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology parameters were considered to be unaffected by treatment with the test item. Any statistically significant changes in hematology parameters were considered unrelated to treatment with the test item as changes were relatively slight, occurred in the absence of changes in correlating parameters, did not show a dose-related trend and/or were possibly caused by slightly low or high control means.
Coagulation parameters of treated rats were considered to be unaffected by treatment with the test item. The slightly decreased mean prothrombin time (PT) observed in males at all dose levels was considered to be caused by a slightly high control mean .

Historical control data hematology parameters for Wistar Han rats, F0-animals (period 2017-2019):
RDW (%) - F0-male: mean: 12.8; P5 - P95: 11.70 – 15.00 (n=86).
Haemoglobin (mmol/L) - F0-males: mean: 9.6; P5 - P95: 8.60 – 10.30 (n=86).
Lymphocytes (109/L) - F0-females: mean: 3.2; P5 - P95: 1.70 – 4.90 (n=77).
MCHC (mmol/L) - F0-females: mean 20.88; P5 - P95: 20.040 – 21.950 (n=87).
Historical control data coagulation parameters for Wistar Han rats, F0-males (period 2017-2019):
PT (s): mean: 17.6; P5 - P95: 15.60 – 19.30 (n=89).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses:
• Mean creatinine concentration was increased (1.10x of control) in males at 450 mg/kg/day.
• Mean glucose concentration was decreased (0.82x of control) in males at 450 mg/kg/day.
• Mean urea concentration was increased (1.22x of control) in females at 450 mg/kg/day.
• Mean inorganic phosphate concentration was increased (1.23x, 1.23x and 1.33x of control) in females at respectively 50, 150 and 450 mg/kg/day .
The slightly increased mean concentrations of sodium in males at 450 mg/kg/day (1.01x of control) and creatinine in females at 50, 150 and 450 mg/kg/day (1.13x, 1.12x and 1.15x of control, respectively) were considered to be the result of a slightly low control value .
Other statistically significant changes in clinical biochemistry parameters were considered not toxicologically relevant based on the low magnitude (< 5%) and/or direction of the change, or absence of a dose response relationship.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no differences noted between control and treated male and female rats in TSH and total T4 that were considered to be related to treatment with the test item.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in urine parameters distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses:
• Mean pH-value was decreased (pH: 6.2 vs. 6.6 in the control group) in males at 450 mg/kg/day.
• Mean numbers of epithelial cells were increased in males at 50, 150 and 450 mg/kg/day. The highest score of 3 was recorded in 6/10, 10/10 and 10/10 males of the 50, 150 and 450 mg/kg/day groups, respectively, compared to a maximum score of 2 in the control group).
• Mean volume of urine was increased (1.53x of control) in females at 450 mg/kg/day.

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings were noted in the heart, liver, and thyroid gland of males and females, and in the kidneys of males.

Heart: Cardiomyocyte vacuolation (often multifocal) was present in males starting at 150 mg/kg/day and in females starting at 50 mg/kg/day, both up to a moderate degree at 450 mg/kg/day. A higher incidence and severity of inflammatory cell infiltrate up to a moderate degree was present in males starting at 50 mg/kg/day; inflammatory cell infiltrate was present in females at 450 mg/kw/day at a minimal degree. A higher incidence of minimal myofiber necrosis was present in males at 50 mg/kg/day. In males at 150 or 450 mg/kg/day, myofiber necrosis was seen at a higher incidence (150 mg/kg/day) and at a minimal to slight degree (150 and 450 mg/kg/day).

Liver: Hepatocellular hypertrophy, mainly centrilobular, was present in males at 450 mg/kg/day at a minimal degree, and in females starting at 150 mg/kg/day up to a slight degree at 450 mg/kg/day. This finding correlated with the higher liver weights and, in some instances, with the macroscopic enlarged liver.

Thyroid Gland: A higher incidence of follicular cell hypertrophy was noted in males at 450 mg/kg/day up to a slight degree, and in females starting at 150 mg/kg/day at a minimal to slight degree. A higher incidence (both sexes) and severity (males only) of colloid alteration was present at 450 mg/kg/day, at a minimal (females) to slight (males) degree.

Kidneys:
Hyaline droplet accumulation was present at a higher incidence and severity in males starting at 50 mg/kg/day up to a marked degree. Tubular basophilia was observed at a higher incidence and severity in males starting at 50 mg/kg/day up to a moderate degree. Granular casts were noted in males starting at 50 mg/kg/day up to a marked degree. Tubular degeneration was present in males starting at 50 mg/kg/day up to a slight degree. This combination of findings in the kidney is also known as alpha2u-globulin nephropathy. It is likely correlated with the increased kidney weight and with the observed macroscopic kidney findings (enlarged and pale discolored ).

There were no other relevant histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was one female treated at 450 mg/kg/day (No. 200) with a small oligodendroglioma in the brain. This rare neoplastic lesion in a single animal was considered to be a spontaneous alteration and unrelated to the treatment with the test item.

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 unaffected by treatment with the test item. Most females had regular cycles of 4 to 5 days, except for one female in each of the control group (No. 102), at in the 150 mg/kg/day group (No. 170), and in the 450 mg/kg/day group (No. 186) which were observed with an irregular cycle of respectively 2, 3 or 8 days. All these females had normal litters. Given their incidental nature and in the absence of a correlation to pregnancy status, these findings did not indicate a relation with treatment. For one control female (No. 125) cycle regularity in the premating phase could not be determined as it had only one complete estrous cycle (of 5 days). Also this control female had a normal litter.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Changes in motility and morphology of sperm were noted at 450 mg/kg/day.
Mean percentages of motile sperm (0.50x of control) and progressive sperm (0.35x of control) had markedly decreased in males at 450 mg/kg/day. Samples of 2/25 males contained neither motile nor progressive sperm, and samples of another 4/25 males did not contain any progressive sperm. In addition, mean numbers of spermatozoa with a normal morphology had decreased at 450 mg/kg/day (81 vs. 171 in the control group ); more cells showed a detached head (101 vs. 3 in the control group) or other tail (7 vs. 0 in the control group). The observed decrease in sperm with coiled tail (7 vs. 23 in the control group) was attributed to the increase of sperm with other abnormalities. Sperm concentration (epididymal sperm count) was considered to be unaffected by treatment up to 450 mg/kg/day.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

In the control group, there was 1/25 couple with total litter loss, 1/25 couple where the female had implantation sites only and 1/25 couple where the female was euthanized in extremis on pre-mating Day 29. In the 50 mg/kg/day treated group, there was one female found dead on pre-mating Day 7. There were 4/24 couples of the 50 mg/kg/day treated group and 11/25 couples of the 450 mg/kg/day treated group that were not pregnant. There were 2/25 couples of the 450 mg/kg/day treated group that did not mate.

No abnormalities were seen in the reproductive organs (examined for all control group and 450 mg/kg/day treated males and females).

Spermatogenesis Staging: 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.

Mating index for both males and females was slightly decreased at 450 mg/kg/day. Mating was confirmed for all females, except for two females in the 450 mg/kg/day group. This resulted in a slightly lower mating index (both sexes) of 92% in the 450 mg/kg/day group, compared to 100% in each the control, 50 and 150 mg/kg/day group.

Fertility index for both males and females was markedly decreased at 450 mg/kg/day.The fertility index (both sexes) was 100, 83, 100 and 52% for the control, 50, 150 and 450 mg/kg/day groups, respectively. The number of non-pregnant females versus mated females was 0/24, 4/24, 0/25 and 11/23 in the control, 50, 150 and 450 mg/kg/day groups, respectively.

Details on results (P0)

Precoital time was considered not to be affected by treatment with the test item. Most females showed evidence of mating within the first 4 days of cohabitation. For one control female, three females at 50 mg/kg/day, one female at 150 mg/kg/day and two females at 450 mg/kg/day it took 12-14 days before mating could be confirmed. These longer precoital times are occasionally encountered in this type of study and in the absence of any dose-related occurrence this was considered not to be unrelated to treatment with the test item.

Number of implantation sites was not affected by treatment with the test item. Mean numbers of implantation sites were 11.3, 11.1, 12.2 and 12.1 for the control, 50, 150 and 450 mg/kg/day groups, respectively. One control female (No. 115) had two implantation sites only (and no offspring). In addition, one female each at 50 and 150 mg/kg/day (Nos. 150 and 167) was noted with three implantation sites. As this occurred in the absence of a dose response relationship, it was considered to be unrelated to treatment.

Gestation index and duration of gestation were not affected by treatment with the test item. Except for one control female (No. 115) with two implantations only, all pregnant females had live offspring, resulting in a gestation index of 96% in the control group and 100% for all groups treated with the test item.

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.

The total number of offspring born compared to the total number of uterine implantations was not affected by treatment with the test item. Post-implantation survival index was 94, 93, 91 and 97% for the control, 50, 150 and 450 mg/kg/day groups, respectively. For one control female (No. 112), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 21-23 days of lactation. No toxicological relevance was attached to this finding

Litter size was not affected by treatment with the test item. Mean numbers of living pups at first litter check were: 10.6, 10.2, 11.0 and 11.6 in the control, 50, 150 and 450 mg/kg/day groups, respectively.

Sex ratio was considered unaffected by treatment with the test item. At 150 mg/kg/day, the male/female sex ratio was statistically significantly higher than the control ratio (55/45 vs. 43/57 in the control group). However, this was considered to reflect normal biological variation, also given the absence of changes in other supportive parameters such as anogenital distance. Therefore, it was considered not to be related to treatment with the test item.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

>= 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects on reproduction

3

Dose descriptor:

NOAEL

Effect level:

< 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: increased kidney weight and histopathological kidney changes pointing at alpha-2u nephropathy, a male-rat-specific syndrome with no relevance to man

4

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slight increase in incidence of cellullar infilltrate and myofiber necrosis in males (compared to controls) and minimal cardiomyocyte vacuolation in 2/25 females at 50 mg/kg bw/day

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed among pups that were considered to be related to treatment with the test item. Fissures with bleeding were noted for one pup at 50 mg/kg/day (Litter No. 138) at first litter check only. No toxicological relevance was attributed to this isolated finding. The nature and incidence of other clinical signs observed remained within the range considered normal for pups of this age, and were therefore regarded not to be toxicologically relevant.

No toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Slight salivation was seen after dosing in males and females of all groups, including the control group, on almost all treatment days. This finding of salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and relatively minor severity of the effect, its time of occurrence (i.e. after dosing) and the fact that both control and treated animals were affected similarly.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was unaffected by treatment with the test item. Live birth index was 95% for the control group and 99% for all groups treated with the test item.

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not affected by treatment with the test item. Viability index was 98, 98, 99 and 100% for the control, 50, 150 and 450 mg/kg/day groups, respectively.

The number of live offspring on Day 21 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. Weaning index was 99, 99, 100 and 99% for the control, 50, 150 and 450 mg/kg/day groups, respectively.

A total of seven F1-animals that were dosed in the post-weaning phase were found dead during Days 7-45 of treatment, mostly pre-dose. This concerned 2/120, 2/100 and 3/120 animals of the control, 50 and 150 mg/kg/day groups, respectively, whereas all 60 animals of the high dose group (450 mg/kg/day) survived until scheduled necropsy. For none of these deceased animals, the exact cause of death could be identified. However, based on the nature of findings at the clinical observations (rales, gasping) and/or necropsy (perforation of the esophagus and body cavity containing tan, watery cloudy fluid, lungs not collapsed), it was most likely suspected that the cause of death of these relatively young animals was related to the gavage administration procedure. Overall, the mortality incidence recorded over the dose groups did not show a dose-related trend, and none of the F1-animals that survived until scheduled necropsy showed any lesions in the respiratory tract.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in body weight of pups were noted during lactation. A trend towards lower mean body weights (both sexes) was observed in pups of the 450 mg/kg/day group from PND 4 onwards. Changes were relatively slight (down to 0.93x of control in males (on PND 13) and 0.92x of control in females (on PND 7); not statistically significant) and partial recovery was observed until the end of the weaning period (0.95x and 0.94x of control in males and females (on PND 21), respectively). All mean values remained within the available historical control range. Therefore, this change was considered not to be toxicologically relevant.

In females at 450 mg/kg/day, a slightly higher mean body weight gain was noted from Day 22 onwards (not always statistically significant). When compared to the concurrent control group, differences were relatively slight only (≤ 10 %) and did not result in any clear change in absolute body weight. Most likely it was caused by the slightly lower body weight in this high dose group (- 6%) at start of the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption (before and/or after correction for body weight) were recorded across the dose groups. Any statistically significant changes in food consumption (absolute or relative to body weight) observed during treatment were considered not related to treatment with the test item as a dose-related trend was absent and/or changes were not consistently recorded with continuing treatment.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean number of red blood cells was decreased (0.94x of control) in males at 450 mg/kg/day. This change occurred together with decreased mean concentrations of hemoglobin (0.93x of control) and hematocrit (0.94x of control). Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
The slightly shorter mean prothrombin time (PT) observed in males at 450 mg/kg/day was considered not to represent a change of toxicological relevance based on the direction of the change and its minimal magnitude. The slightly longer mean prothrombin time (PT) observed in females at 50 mg/kg/day was considered unrelated to treatment as no dose-related response was evident.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean glucose concentration was decreased (0.74x of control) in males at 450 mg/kg/day. Mean cholesterol concentration was increased (1.37x and 1.16x of control) at 450 mg/kg/day in males and females, respectively (not statistically significant in females).
The increased sodium concentration observed in males at 450 mg/kg/day was considered not to represent a change of toxicological relevance based on its minimal magnitude.
In the absence of a dose-related response, the decreased glucose concentration noted in females at 50 and 450 mg/kg/day was considered not to be test item-related.

Culled pups: serum T4 levels in male and female pups culled at PND 4 were considered unaffected by treatment.
Cohort Surplus: Increased serum levels of both TSH and total T4 (2.21x and 1.49x of control, respectively) were noted in female pups at 450 mg/kg/day. Based on the magnitude of change, a test item related effect could not be excluded. However, as the mean values for both parameters remained within the available historical control range , and thyroid organ weight was unaffected, this was considered not adverse.
Cohort 1A: Mean TSH concentrations were increased at 450 mg/kg/day (2.46x and 2.02x of control) in males and females, respectively (not statistically significant in females). Based on the magnitude of change, a possible relation to treatment with the test item could not be excluded. However, as the mean value in both sexes remained within the available historical control data , it was considered not adverse. No changes in serum levels of total T4 were observed following treatment up to 450 mg/kg/day (both sexes).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Mean specific gravity was increased (1.01x of control) in males at 450 mg/kg/day. Although the relative change in the mean value was minimal, urines from 5/10 high dose males had a specific gravity that was above the highest control value (1.041-1.046 vs. 1.038).
Mean urinary pH was decreased (pH: 6.3 and 6.1 vs. 6.7 in the control group) in males at 150 and 450 mg/kg/day, respectively.
Mean number of epithelial cells was increased in males at 50, 150 and 450 mg/kg/day. The highest score of 3 was recorded in 3/9, 7/9 and 6/10 males of the 50, 150 and 450 mg/kg/day groups, respectively (compared to a maximum score of 2 in the control group). Statistical significance was reached in the 150 and 450 mg/kg/day groups.

Sexual maturation:

no effects observed

Description (incidence and severity):

Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrus, and time between vaginal opening and first estrus in females were considered not to be affected by treatment with the test item. At 150 mg/kg/day, mean time to balanopreputial separation (BPS) was increased compared to the control group (42.9 days vs. 41.4 days in controls). In the absence of a dose-related response, this apparent later onset of balanopreputial separation at the mid dose was regarded unrelated to treatment with the test item.
At the individual level, one male (No. 339) at 50 mg/kg/day was noted that did not show balanopreputial separation before PND 58. For all remaining males in this study, balanopreputial separation was recorded between PND 38-50. Furthermore, for one female (No. 617) at 50 mg/kg/day, it took 12 days between vaginal opening and first estrus. All other females in this study presented with first estrus after 1-8 days. No toxicological relevance was attached to these delays as they were isolated findings at the low dose level only.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

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

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 450 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Cohort Surplus: no relevant changes in organ weights were noted.

Cohort 1A (PND 85-99): There was a dose-related increase in mean heart weight in males and females (absolute and relative to body weight) starting at 150 mg/kg/day. There was a higher mean liver weight in males and females (absolute and relative to body weight) at 450 mg/kg/day. In males, there was a dose-related increase in mean kidney weight (absolute and relative to body weight) starting at 50 mg/kg/day, and a dose-related increase in mean spleen weight (relative to body weight) at 150 and 450 mg/kg/day. In females, there was a statistically significant higher mean thymus weight (absolute) at 450 mg/kg/day. The individual values were all within the normal range of biological variation.

Cohort 1B (≥ PND 97): There was an increased mean liver weight (absolute and relative to body weight) in females at 50 and 150 mg/kg/day. In males, mean liver weight (relative to body weight) was higher at 150 mg/kg/day. In addition, there was a dose-related increase in mean kidney weight (absolute and relative to body weight) in males at 50 and 150 mg/kg/day. It should be noted that no Cohort 1B animals were available in the 450 mg/kg/day group for evaluation.
The increased mean thyroid gland weight (absolute) noted in females at 50 mg/kg/day, was regarded unrelated to treatment as it occurred in the absence of a dose-related response.

Fixed brain weights at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2A) were considered not affected by treatment with the test item.
Cohort 2B (PND 21-22): The decrease in brain weight in females at 50 mg/kg/day (absolute) was considered incidental based on a lack of change in the higher dose groups and the absence of a dose response. In addition, although there was a significant increase in brain weight (relative to body weight) in females treated at 450 mg/kg/day, it was considered to be related to the slightly lower terminal body weight and was therefore not considered to be test item-related.
Cohort 2A (PND 76-90): There were no changes in fixed brain weights of males or females that were considered to be related to treatment with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups sacrificed before weaning or spare pups that were considered to be related to treatment with the test item. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Cohort 1C (males: ≥ PND 35; females: ≥ PND 25): Note: No Cohort 1C animals are were available in the 50 and 450 mg/kg/day groups. There were no macroscopic abnormalities in Cohort 1C animals treated at 150 mg/kg/day that were considered to be related to treatment with the test item.

Cohort 2B (PND 21-22): There were no test item-related gross observations.

Cohort Surplus (PND 22-24): There were no test item-related gross observations.

Cohort 1A (PND 85-99): An enlarged liver was present in 1/19 and 4/20 males at 150 and 450 mg/kg/day, respectively. The enlarged liver correlated with the higher liver weights and with the hepatocellular hypertrophy in males at 450 mg/kg/day. Black-brown (or red-brown in a single male) discoloration of the liver was noted in 6/20 males and 2/20 females treated at 450 mg/kg/day. There was no clear microscopic correlate for this finding, although there was pigment deposition in males and females at 450 mg/kg/day that sometimes coincided with the macroscopic finding. Enlarged kidneys were observed in 1/19 and 3/20 males at 150 and 450 mg/kg/day, respectively. Pale or greenish discoloration of the kidneys was noted in 3/19, 7/19 and 13/20 males at 50, 150 and 450 mg/kg/day, respectively. Both gross kidney findings were likely associated with the alpha2u-globulin nephropathy starting at 50 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Cohort 1B (≥ PND 97): Note: No Cohort 1B males and females were available in the 450 mg/kg/day groups. An enlarged liver was present in 0/20, 1/19, 2/19 males and 0/20, 0/20, 1/20 females in the control, 50 and 150 mg/kg/day groups, respectively. Pale discoloration of the kidneys was noted in 0/20, 3/19, 12/19 males in the control, 50 and 150 mg/kg/day groups, respectively.

Histopathological findings:

no effects observed

Description (incidence and severity):

Brain dimensions (length and width of brain) at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2B) were considered not to have been affected by treatment up to 450 mg/kg/day.
In the F1-animals (Cohort 2A), there were no test item-related effects on the H&E or Luxol Fast Blue/Cresyl Violet stained sections of brain or peripheral nerves in the control or high dose group males or females.
There was no evidence of changes in brain morphometry in test item-treated animals at either PND 21-22 (Cohort 2B) or PND 76-90 (Cohort 2A).

See further at P1

Developmental neurotoxicity (F1)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 2A:
A lower Average Response Amplitude (both as overall mean response (0.76x of control), and during the individual five test blocks (0.79, 0.71, 0.75, 0.75 and 0.75x of control for Blocks 1, 2, 3, 4 and 5, respectively; reaching statistical significance for blocks 1-3 only)).
A lower Maximum Response Amplitude (both as overall mean (0.76x of control) and during the individual five test blocks (0.75, 0.73, 0.73, 0.77 and 0.79x of control for Blocks 1, 2, 3, 4 and 5, respectively; reaching statistical significance for Blocks 1-3 and overall response).
These findings in Average Response Amplitude and Maximum Response Amplitude were considered to be related to treatment with the test item; however, all means remained within the available historical control range.
Historical control data for Wistar Han rats (period 2017-2021):
Cohort 2A males - Acoustic Startle Response - Average response amplitude Ave (N):
Block 1: mean = 0.152; P5-P95 = 0.0900 - 0.2490 (n=60)
Block 2: mean = 0.139; P5-P95 = 0.0705 - 0.2100 (n=60)
Block 3: mean = 0.131; P5-P95 = 0.0705 - 0.1995 (n=60)
Block 4: mean = 0.130; P5-P95 = 0.0705 - 0.2100 (n=60)
Block 5: mean = 0.123; P5-P95 = 0.0705 - 0.2000 (n=60)
Mean over Means = 0.134; P5-P95 = 0.0804 - 0.2000 (n=60)
Latency Time to Achieving the Maximum Response Amplitude was unaffected in males up to 450 mg/kg/day. Values remained in the same range as for controls. No changes were noted in males up to 150 mg/kg/day and females up to 450 mg/kg/day. All treated groups showed an acoustic startle response that was similar to the concurrent control group.

Detailed clinical observations revealed no symptoms that were considered to be related to treatment with the test item. All clinical signs, including vocalisation in one female at 450 mg/kg/day, were within the normal range of behavioural findings for this type of study.

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

Motor activity of animals treated up to 450 mg/kg/day (both sexes) was considered not affected by treatment. All groups showed a similar motor activity habituation profile with in general a decreasing trend in activity over the duration of the test period.

Functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were not affected by treatment with the test item. A statistically significantly lower mean foot splay value was recorded for males at 150 mg/kg/day. In the absence of a dose-related response, this variation was considered not to represent an effect of the test item.

Fixed brain weights at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2A) were considered not affected by treatment with the test item. The lower mean brain weight in females at 50 mg/kg/day (absolute) was considered incidental based on a lack of change in the higher dose groups and the absence of a dose response.
The higher mean brain weight (relative to body weight) in females treated at 450 mg/kg/day was considered to be related to the slightly lower terminal body weight and was therefore considered not test item-related.
Brain dimensions (length and width of brain) at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2A) were considered not to have been affected by treatment up to 450 mg/kg/day.
There were no findings noted in the H&E or Luxol Fast Blue/Cresyl Violet stained sections of brain (Cohorts 2A and 2B) or peripheral nerves (Cohort 2A) in the control or high dose group males or females at either PND 21-22 (Cohort 2B) or PND 76-90 (Cohort 2A) that were considered to be test item-related.
There was no evidence of changes in brain morphometry in test item-treated animals at either PND 21-22 (Cohort 2B) or PND 76-90 (Cohort 2A).

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Cohort 1A: Splenic lymphocyte subpopulations were considered unaffected by treatment with the test item. The lower subpopulation of natural killer (NK) cells in males at 50 mg/kg/day was considered not to represent an effect of the test item, as this shift occurred in the absence of a dose related trend.

Details on results (F1)

Liver Microsomal Protein Concentration and CYP Content

Mean total liver microsomal protein content was increased in males of the 450 mg/kg/day group as compared to control males (1.24x of control).

Mean microsomal CYP content was increased in males of the 450 mg/kg/day group as compared to control males (1.42x of control).

Mean PROD activity was significantly increased in liver microsomes from males of the 450 mg/kg/day group compared to control males when activity was expressed either per mg protein (18.8x of control) or per nmol CYP (12.8x of control). The incubations performed with BNF- and PB-induced male Sprague-Dawley RLM (positive control) showed significant formation of resofurin while the inactivated male Sprague Dawley RLM (negative control) did not show any resorufin formation. These results demonstrate that the incubation assay was performed correctly.

Mean BROD activity was significantly increased in liver microsomes from males of the 450 mg/kg/day group as compared to control males when activity was expressed either per mg protein (22.7x of control) or per nmol CYP (15.4 x of control). The incubations performed with BNF- and PB-induced male Sprague-Dawley RLM (positive control) showed significant formation of resofurin while the inactivated male Sprague Dawley RLM (negative control) did not show any resorufin formation. These results demonstrate that the incubation assay was performed correctly.

Mean T4-glucuronidation activity was increased in liver microsomes from males of the 450 mg/kg/day group compared to control males (2.4x of control). The incubations performed with 3-MC-induced male Sprague-Dawley RLM (positive control) and the non-induced male Sprague-Dawley (reference control) both showed a significant formation of T4-glucuronide while the inactivated male Sprague-Dawley RLM (negative control) did not show any T4-glucuronide formation. These results demonstrate that the incubation assay was performed correctly.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: slight increase in incidence of cellullar infilltrate and myofiber necrosis in males (compared to controls) and minimal cardiomyocyte vacuolation in 4/20 males and 3/19 females at 50 mg/kg bw/day

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

< 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: increased kidney weight and histopathological kidney changes pointing at alpha-2u nephropathy, a male-rat-specific syndrome with no relevance to man

3

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: no developmental (neuro)toxicity observed.

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The following statistically significant changes distinguished treated from control animals. Mean percent weight differences from control group are indicated.

 

Males (P0)

 

Dose level (mg/kg):	50 mg/kg/day	150 mg/kg/day	450 mg/kg/day
Body Weight	3	-1	-1
Heart (males)
Absolute	11**	14**	27**
Relative to body weight	9	15**	29**
Liver (males)
Absolute	5	6	17**
Relative to body weight	3	7**	18**
Kidneys (males)
Absolute	8	18**	27**
Relative to body weight	6	19**	28**
Spleen (males)
Absolute	6	6	11*
Relative to body weight	3	7	12**

*: P<0.05; **: P<0.01

 

Females (P0)

 

Dose level (mg/kg):	50 mg/kg/day	150 mg/kg/day	450 mg/kg/day
Body Weight	0	0	1
Heart (females)
Absolute	-2	11**	20**
Relative to body weight	-2	11**	18**
Liver (females)
Absolute	-3	8	9*
Relative to body weight	-3	8*	7
Thymus (females)
Absolute	15	-5	30**
Relative to body weight	16	-4	30**

*: P<0.05; **: P<0.01

Note: At the individual level, unrealistic high values were noted for the epididymides of Male No. 87 (Group 4) and spleen of Female No. 172 (Group 3). Tables will be corrected before issuing the draft report.

The following statistically significant changes distinguished treated from control animals. Mean percent weight differences from control group are indicated.

 

Mean Percent Organ Weight Differences from Control Groups – Males and Females (Cohort 1A)

	Males			Females
Dose level (mg/kg/day):	50	150	450	50	150	450
Body weight	-2	-4	-4	5	4	3
Heart
Absolute	4	13**	19**	6	14**	30**
Relative to body weight	7	19**	24**	1	10**	26**
Liver
Absolute	3	0	16**	5	8	20**
Relative to body weight	6	4	21**	0	4	16**
Kidneys
Absolute	8*	14**	22**	2	7	5
Relative to body weight	9**	19**	27**	-2	2	2
Spleen
Absolute	2	7	13	6	-2	7
Relative to body weight	4	12*	17**	0	-5	3

  *: P<0.05; **: P<p,p** p<0.01

 

Mean Percent Organ Weight Differences from Control Groups – Males and Females (Cohort 1B)

	Males			Females
Dose level (mg/kg/day):	50	150	450	50	150	450
Body weight	-4	-5	n.a.	0	0	n.a.
Liver
Absolute	-1	3	n.a.	8*	10**	n.a.
Relative to body weight	3	8**	n.a.	8**	10**	n.a.
Kidneys
Absolute	8*	14**	n.a.	3	0	n.a.
Relative to body weight	13**	18**	n.a.	3	1	n.a.

  *: P<0.05; **: P<0.01; significant changes shaded in grey

Mean Percent Fixed Brain Weight Differences from Control Groups – Males and Females (Cohort 2B)

	Males			Females
Dose level (mg/kg/day):	50	150	450	50	150	450
Body weight	-2	-8	-10	-4	-6	-10
Brain
Absolute	4	2	-2	-4*	-1	0
Relative to body weight	5	9	9	0	4	10*

  *: P<0.05, significant changes shaded in grey

The fixed brain weight change observed in females at 450 mg/kg/day was considered to be related to the lower terminal body weight and was therefore not directly test item-related. In absence of a dose-related distribution, no toxicological relevance was attached to the fixed brain weight change noted in females at 50 mg/kg/day.

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following No Observed Adverse Effect Levels (NOAELs) of 1,1,3,3-Tetramethylbutyl Peroxyneodecanoate were established:

General toxicity (F0):
- Kidneys: no NOAEL could be established based on adverse changes in kidneys (males) at 50, 150 and 450 mg/kg/day. The kidney changes were considered related to alpha 2u-globulin nephropathy, a male-rat-specific syndrome with no relevance to man.
- Heart: In the heart, the sequence of findings was considered to start with vacuolation of the cardiomyocytes (often multifocal), leading to cell damage and the recruitment of inflammatory cells and, in some instances, resulting in myofiber necrosis. Based on a slight increase in incidence of cellullar infiltrate and myofiber necrosis in males (compared to controls) and minimal cardiomyocyte vacuolation in 2/25 females at 50 mg/kg/day, in fact no NOAEL could be set for heart findings; however, in view of the limited number of animals and the limited severity at 50 mg/kg bw/day, and taking the dose-response relationship into account, the NOAEL was considered close to 50 mg/kg/day.

Reproductive toxicity (F0 and F1):150 mg/kg/day; mainly based on the reduced fertility index (F0) and reduced sperm quality (F0 and F1) at 450 mg/kg/day.

Developmental toxicity (F1) (until weaning on PND 21): At least 450 mg/kg/day.

Developmental toxicity (F1) (post-weaning; at adult age), General toxicity:
- Kidneys: no NOAEL could be established based on adverse changes in kidneys (males) at 50, 150 and 450 mg/kg/day. The kidney changes were considered related to alpha 2u-globulin nephropathy, a male-rat-specific syndrome with no relevance to man (see at F0).
- Heart: Based on minimal cardiomyocyte vauolation in 4/20 males and 3/19 females at 50 mg/kg/day, in fact no NOAEL could be set for heart findings; however, in view of the limited number of animals and the limited severity at 50 mg/kg bw/day, and taking the dose-response relationship into account, the NOAEL was considered close to 50 mg/kg/day.

Developmental neurotoxicity (F1- Cohorts 2A and 2B): At least 450 mg/kg/day.

Developmental immunotoxicity (F1 - Cohort 1A): At least 450 mg/kg/day.

Executive summary:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of 1,1,3,3-Tetramethylbutyl Peroxyneodecanoate 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 when present.

In addition, the study was expected to provide information on the effects of 1,1,3,3-Tetramethylbutyl Peroxyneodecanoate on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were evaluated: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation.

Furthermore, the information obtained from the developmental neurotoxicity assessments would characterize potential effects in those systems.

Finally, ex vivo liver enzyme induction (CYP and UGT) was determined using liver microsomes from control and high dose males in Cohort 1A.

The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg/day, based on the results of a preliminary reproduction/developmental toxicity screening test in rats with oral exposure to 1,1,3,3-Tetramethylbutyl Peroxyneodecanoate (Test Facility Study No. 20188708).

F₀-males (25/group) were treated for 12-13 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy.
F₀-females (25/group) were treated for a minimum of 15 weeks, including 10 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. F₀-females which failed to deliver or had a total litter loss were treated for 14-15 weeks.

From weaning onwards (PND 21), F₁-animals of Cohorts 1A, 1B, 1C and 2A were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 2-4 weeks). The F₁-animals of Cohort Surplus, Cohort 2B and Spares (not assigned to one of the cohorts) were not dosed.

Chemical analyses of formulations were conducted at three occasions during the study to assess accuracy and homogeneity. In addition, stability over 8 days in the refrigerator was determined at the first occasion. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test formulations prepared were confirmed to be stable for at least 8 days when stored in the refrigerator. Stability for at least 5 hours at room temperature under normal laboratory light conditions at concentrations bracketing those used in the present study was demonstrated previously.

For the F₀-generation, the following parameters and endpoints were evaluated in this study: mortality/ moribundity, clinical signs, body weight, food consumption, water consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined for the F₀‑generation: 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).

For the F₁-generation, the following parameters and endpoints 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 and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations, neurohistopathological examinations, morphometric analysis, and ex vivo liver enzyme induction (CYP and UGT) using liver microsomes from control and high dose males in Cohort 1A.

F₀-Generation - Parental Results

Two females of the control group and two females of the low dose group (50 mg/kg/day) died or were euthanized before scheduled necropsy. One of these control females was euthanized due to a total litter loss (Lactation Day 6). None of these deaths was considered to be caused by the test item.

Test item-related microscopic alterations, considered adverse, were noted in the heart and kidneys of F₀-males and/or F₀-females starting at 50 mg/kg/day:

Heart: morphological findings were noted in both F₀-males and F₀-females at all doses, but at 50 mg/kg/day these findings were present at a low incidence and minimal severity. The sequence of findings was considered to start with vacuolation of the cardiomyocytes, leading to cell damage and the recruitment of inflammatory cells and, in some instances, resulting in myofiber necrosis. In addition, a higher mean heart weight (absolute and relative to body weight) was seen in F₀-males and F₀-females at 150 and 450 mg/kg/day. The cardiomyocyte vacuolation, consisting of (often multifocal) accumulation of well demarcated, round small clear vacuoles, has been described as part of a degenerative process and was therefore considered adverse. The increased inflammatory cell infiltrate and myofiber necrosis were also considered adverse.  

Kidneys: alpha_2u-globulin nephropathy (consisting of a higher incidence and severity of hyaline droplet accumulation, tubular basophilia and tubular degeneration, and granular casts in the outer medulla, representing accumulation of cell debris) was noted in F₀-males starting at 50 mg/kg/day (sometimes correlating with the gross enlarged and/or pale discolored kidneys, and a higher kidney weight). Based on the degenerative nature, this male-specific pathologic syndrome was considered adverse within the context of this study; however, as it is a rat-specific finding, it has no relevance to man. Additional kidney-related changes in males consisted of a higher water consumption (up to a moderate degree) at 150 and 450 mg/kg/day, a higher mean plasma creatinine concentration (although slight in magnitude) and a lower mean urinary pH-value at 450 mg/kg/day, as well as a higher number of epithelial cells in urine of males of all treatment groups (i.e. starting at 50 mg/kg/day). In females, a higher water intake (up to a moderate degree) was noted at 150 and 450 mg/kg/day.

F₀-Generation - Reproductive Results

The fertility index was markedly lower at 450 mg/kg/day (100, 83, 100 and 52% for the control, 50, 150 and 450 mg/kg/day groups, respectively). Test item-related effects on male fertility were considered the underlying cause of this significant lower number of pregnant females in the high dose group. Analysis of sperm taken at scheduled necropsy revealed severe effects on sperm from high dose F₀-males, i.e. mean percentages of motile sperm and progressive sperm were markedly decreased in males at 450 mg/kg/day. Notably, samples of 2/25 F₀-males contained neither motile nor progressive sperm, and no progressive sperm was found in the samples of another four F₀-males. In addition, mean numbers of spermatozoa with normal morphology were lower at 450 mg/kg/day; more spermatozoa with detached head or other tail were noted at this high dose. Epididymal sperm count was unaffected by treatment up to 450 mg/kg/day, and there was no microscopic correlate in the male reproductive organs (incl. spermatogenic profiling in the testis). Female fertility appeared to be unaffected by treatment with the test item.

F0-Generation / F1-Generation (Pre-Weaning) - Developmental Results

No developmental toxicity was observed up to and including the highest dose level tested (450 mg/kg/day).

F₁-Generation (Post-Weaning) - Developmental Results

A total of 2/120, 2/100 and 3/120 F₁-animals of the control, 50 and 150 mg/kg/day groups, respectively, were found dead during Days 7-45 of treatment, mostly pre-dose; these deaths were considered not test item-related. All 60 F₁-animals of the high dose group (450 mg/kg/day) survived until scheduled necropsy.

Similar to the F₀-generation, adverse morphological findings were noted in the heart and kidneys of F₁-males and/or F₁-females (Cohort 1A) starting at 50 mg/kg/day. In addition, adverse findings were observed in the liver of F₁-animals (both sexes) at 450 mg/kg/day:

Heart: a higher incidence and/or severity of cardiomyocyte vacuolation was seen in F₁-males and F₁-females starting at 50 mg/kg/day and inflammatory cell infiltrate was observed in F₁-males starting at 150 mg/kg/day and in F₁-females at 450 mg/kg/day, and a higher incidence of myofiber necrosis was noted in F₁-males and F₁-females at 450 mg/kg/day (the vacuolation alone or the combination of findings was often seen together with a higher heart weight). It should be noted that findings were present at a low incidence and minimal severity at 50 mg/kg/day. However, similar to the F₀-generation, all these heart findings in the F₁-generation were considered adverse (for details, see F₀-Generation - Parental Results

Kidneys: alpha_2u-globulin nephropathy (consisting of an increased incidence and severity of hyaline droplet accumulation, tubular basophilia and tubular degeneration, and granular casts) was seen in F₁-males starting at 50 mg/kg/day (often seen together with the gross enlarged and/or pale or greenish discolored kidney, and the higher kidney weight). Based on its degenerative nature, this rat-male-specific pathologic syndrome was considered adverse within the context of this study. Analysis of urinalysis parameters revealed several changes related to male kidney function that were considered test item-related: Specific gravity of the urine was higher in F₁-males at 450 mg/kg/day. Although the relative change in the mean value was minimal, urines from 5/10 high dose F₁-males had a specific gravity that was above the highest control value. In addition, a dose-related lower urinary pH was noted in F₁-males at 150 and 450 mg/kg/day, and higher numbers of epithelial cells were counted in F₁-males from 50 mg/kg/day onwards. In view of the adverse morphological kidney lesions observed in F₁-males of all treatment groups, these changes in urinary parameters were considered adverse. However, as alpha_2u-globulin nephropathy is a male-rat-specific pathologic syndrome, it has no relevance to man.

Liver: pigment deposition was noted in the lumen of intrahepatic bile ducts (Maltese cross) of F₁-males and F₁-females at 450 mg/kg/day (sometimes correlating with macroscopic black-brown discoloration of the liver). As this finding may indicate abnormalities in the chemical steps that lead to heme, it was considered adverse. A possible correlation to the lower mean number of red blood cells, together with lower mean concentrations of hemoglobin and hematocrit observed in high dose F₁-males is likely. It is unknown whether the mechanism for the deposition of the pigment in the liver of the rat is different from the mechanisms known for men. A possible relationship between the morphological changes in the liver and the observed higher mean plasma cholesterol concentration in F₁-males and F₁-females at 450 mg/kg/day could not be excluded.

Also similar to the F₀-generation, sperm analysis revealed findings on motility and morphology of sperm in F₁-males (Cohort 1A) at 450 mg/kg/day that were considered test item-related. Mean percentages of motile sperm and progressive sperm were markedly decreased. Notably, samples of 2/20 F₁-males contained neither motile nor progressive sperm, and no progressive sperm was found in the samples of another two F₁-males. In addition, mean numbers of cells with a detached head or with an abnormal neck were higher. Different from F₀-males, also epididymal sperm count was significantly lower in these high dose F₁-males. All these changes in sperm parameters were considered adverse. No test item-related effects were noted at histopathological examination of the male reproductive organs (incl. spermatogenic profiling in the testis).

F₁-Generation (Post-Weaning) - Developmental Neurotoxicity

No test item-related morphological changes were noted in the central nervous system (CNS) and/or peripheral nervous system (PNS) of animals of Cohorts 2A and 2B. Functional tests in Cohort 2A animals did not reveal any adverse effects of treatment on the nervous system.

F₁-Generation (Post-Weaning) - Developmental Immunotoxicity

No test item-related findings were noted on the developmental immunotoxicity endpoints (i.e. splenic lymphocyte subpopulations, lymphoid histopathology and organ weights) investigated in Cohort 1A animals.

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 (NOAELs) of 1,1,3,3-Tetramethylbutyl Peroxyneodecanoate were established: 

General Toxicity (F₀ and F₁): No NOAEL could be established based on adverse changes in kidneys (males) and heart (males and females) at 50, 150 and 450 mg/kg/day. Notes: The kidney changes were considered related to alpha_2u-globulin nephropathy, a male-rat-specific syndrome with no relevance to man. In the heart, the sequence of findings was considered to start with vacuolation of the cardiomyocytes (often multifocal), leading to cell damage and the recruitment of inflammatory cells and, in some instances, resulting in myofiber necrosis. At 50 mg/kg/day, cardiomyocyte vacuolation was seen in a limited number of animals and at a minimal degree only (F₀-generation: 2/24 females; F₁-generation: 4/20 males and 3/19 females), as such the NOAEL for heart findings was close to 50 mg/kg/day. 

Reproductive Toxicity (F₀ and F₁): 150 mg/kg/day; mainly based on effects on the fertility index (F₀) and reduced sperm quality (F₀ and F₁) at 450 mg/kg/day.

Developmental Toxicity (F1) (Until Weaning on PND 21): At least 450 mg/kg/day.

Developmental Toxicity (F1) (Post-Weaning): See at General Toxicity_.

Developmental Neurotoxicity (F₁): At least 450 mg/kg/day.

Developmental Immunotoxicity (F₁ - Cohort 1A): At least 450 mg/kg/day.