Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproduction study is available. However, based on the result from a 90 -day oral OECD 408 study in rats the substance is classified as a Category 2 Reproductive Toxicant.

The oral (gavage) administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to male and female Wistar Han™:RccHan™:WIST strain rats at a dose level of 100 or 300 mg/kg bw/day resulted in toxicologically significant changes in sperm concentration and motility and an associated increase in sperm abnormalities. These were not reversible in males previously treated with 300 mg/kg bw/day after a twenty-eight day treatment-free period. It is therefore considered that a dose level of 10 mg/kg bw/day could be established as a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the male within the confines of this type of study. In contrast, there were no changes of toxicological significance in the females up to a dose level of 300 mg/kg bw/day, which could therefore be established as a NOAEL in the female within the confines of this type of study.

The draft results of an OECD 443 are available. These data have not been subject to detailed audit yet. In this study adverse effects omn fertility are observed.

Sperm analysis: Sperm parameters were considered to be unaffected by treatment up to 150 mg/kg/day. Statistically significant changes that distinguished males treated at 450 mg/kg/day from control animals are summarized below.Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.

450 mg/kg/day

- Lower percentage of motile sperm (0.50x; within historical control range)

- Lower percentage of progressive sperm (0.35x; within historical control range)

- Lower epididymal spermcount (0.82x; not reaching statistical significance, within historical control range)

- Lower number of cells with normalmorphology (mean of 81vs. 171 in the control group;below historical control range)

- Higher number of cells with a detached head (mean of 101vs. 3 in the control group;above historical control range)

- Lower number of cells with coiled tail (mean of 7vs. 23 in the control group; within historical control range)

- Presence of cells with other tail (mean of 7vs. 0 in the control group; within historical control range).

It should be noted that at the highest dose level tested (450 mg/kg/day) only 12 litters were available for evaluation, compared with 20, 23 and 25 litters in the control, 50 and 150 mg/kg/day groups, respectively.

Based on these draft results the substance is classified for effects on fertility in catergory 1b. The IUCLID dossier will be updated with the final report results as soon as these become available. The audited draft report will be available in December 2020. The final report is expected in Q1 2021.

Link to relevant study records
Reference
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
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
Study design according to TPE-D-2114440664-49-01/F
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
Condition:
Outbred, SPF-Quality.
Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Females (if applicable) nulliparous and non-pregnant: yes
Target Age at the Initiation of Dosing:
Males: approximately 6 weeks. Females: approximately 12-14 weeks.
Target Weight at the Initiation of Dosing:
Males: 100 to 200 g. Females: 200 to 250 g.
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals will be group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females will be cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males will be housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females will be individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females will be housed in Macrolon plastic cages (MIII type, height 18 cm). Pups will be housed with the dam.
The cages will contain appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and will be equipped with water bottles. The housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records.
Animals will be separated during designated procedures/activities. Each cage will be clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
Temperature: 18 to 24°C.
Humidity: 40 to 70%.
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated procedures).
Ventilation: At least 10 air changes per hour.
IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Test item dosing formulations (w/w) will be homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations will be prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.
Test item dosing formulations will be kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle will be continuously stirred until and during dosing. Adjustment will be made for specific gravity of the vehicle and test item. No correction will be made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance will be 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: 3.75, 37.5 or 112.5 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
- Lot/batch no. (if required): -
- Purity:-
Details on mating procedure:
Cohabitation/Mating Procedure – F0-Generation
Frequency:
Daily, after a minimum of 14 days of treatment. The mating period will consist of a maximum of 14 consecutive days.
Procedure:
Animals will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating will be confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated.
A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible, see section 13) will be used for re-mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples will be collected in weetk 1 of treatment. For all groups concentration will be measured. For group 2 and 4 homogeneity will be measured.

All samples to be analyzed will be transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.

Concentration and Homogeneity Analysis
Samples for Analysis: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis).
Sample Volume: Approximately 500 mg accurately weighed
Acceptance Criteria: For concentration, the criteria for acceptability will be mean sample concentration results within or equal to ± 10% for solutions of target concentration. For homogeneity, the criteria for acceptability will be a coefficient of variation (CV) of concentrations of ≤ 10% for each group.
Stability Analysis: Stability analyses will be performed in conjunction with the method development and validation study (Test Facility Study No. 20188240) to demonstrate if the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
The test item and vehicle will be administered to the appropriate animals by once daily oral gavage 7 days a week. Males will be 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 includes a minimum of ten weeks prior to mating and during the mating period. Females will be treated for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), 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 will not be dosed during littering. The dose volume for each animal will be based on the most recent body weight measurement. The doses will be given using a plastic feeding tube. The first day of dosing will be designated as Day 1 (exception: alternate animals used for replacement after Day 1 will assume the day of the animal being replaced). The dosing formulations will be stirred continuously during dose administration. A dose control system (DCS) will be used as additional check to verify the dosing procedure according to Standard Operating Procedures (Study Nos. 521667 will be used for DCS). Pups will not be treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Frequency of treatment:
daily
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
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)
Number of F1-animals expected: Approximately 1200 pups (100 litters x 12 pups).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a preliminary reproduction/developmental toxicity screening test with oral exposure of 1,1,3,3-Tetramethylbutyl Peroxyneodecanoate in rats (Test Facility Study No. 20188708), and in an attempt to produce graded responses to the test item. In this screening study, 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 which was considered to be test item-related. 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 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 controls) noted on PND 13. In the current study, the high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: The selected F0-animals and Cohort 1A animals will be fasted overnight with a maximum of
24 hours before blood sampling, but water will be available.
- Other: None
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Mortality/Moribundity Checks – F0-Generation Frequency: At least twice daily throughout the study. Procedure: Animals will be observed for general health/mortality and moribundity. Animals will not be removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
Frequency: During treatment, animals will be observed at least twice daily, up to the day prior to necropsy. These clinical observations will at least be conducted prior to dosing and after dosing.
Actual time before dosing will be reported as 0 hour afyer dosing and nominal time 06.00, actual time after dosing (0-30min) will be reported as 1 hour afyer dosing and nominal time 07.00. Nominal times will be used for computer registration only.

Animals will be observed for specific clinical signs. The time of onset, grade and duration of any observed signs will be recorded. Signs will be graded for severity and the maximum grade will be predefined at 1, 3 or 4. Grades will be 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) will be scored. In the data tables, the scored grades will be reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females will be weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females will be weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. In order to monitor the health status animals may be
weighed more often. This will be documented in the study raw data. Animals will be individually weighed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females will be 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.
Food consumption will be quantitatively measured.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Regular basis throughout the study. Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

OTHER:
Arena Observations
Frequency: Once before the first administration of the test item and at weekly intervals during the treatment period.
Procedure: Animals will be observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs will be recorded.
Oestrous cyclicity (parental animals):
Frequency: Daily vaginal lavage will be performed beginning 14 days prior to mating and during mating until evidence of copulation is observed. Vaginal lavage will continue for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage will also be taken to determine the stage of estrus. This will be done for all females, except for females that have to be euthanized in extremis or die spontaneously.
Procedure: Estrous stages will be determined by examining the cytology of vaginal lavage samples.
Sperm parameters (parental animals):
For all surviving males, the following assessments will be performed: Sperm samples will be taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility will be assessed from all samples. Sperm smears for morphological evaluation will be 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 will be recorded. Evaluation will be performed for all samples. One epididymis (right) will be removed, placed in labeled bags, and kept in the freezer at ≤ - 15°C. After thawing, the right epididymis will be weighed, homogenized and evaluated for sperm numbers. Evaluation will be performed for all samples. In the case of any abnormalities in the right epididymis, the right side organ will be fixed in modified Davidson's solution, and the left side organ will be used for evaluation of sperm numbers. If abnormalities are found in both epididymis, both these organs will be fixed in modified Davidson's solution and no evaluation of sperm numbers will be performed.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of [...] pups/litter ([...]/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 (if possible) will be selected. Selective elimination of pups, e.g. based upon body weight or AGD, will not be 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 are available, litters may be 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:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not performed

Mortality/Moribundity Checks – Cohorts 1A, 1B, 1C and 2A
Frequency: At least twice daily throughout the study. Procedure: Animals will be observed for general health/mortality and moribundity. Animals will not be removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations– Cohorts 1A, 1B, 1C and 2A
Frequency: During treatment, animals will be observed at least twice
daily, up to the day prior to necropsy. These clinical observations will at least be conducted prior to dosing. Procedure: Animals will be observed for specific clinical signs. The time of onset, grade and duration of any observed signs will be recorded. Signs will be graded for severity and the maximum grade will be predefined at 3 or 4. Grades will be 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) will be scored. In the data tables, the scored grades will be reported, as well as the percentage of animals affected in summary tables.

Arena Observations – Cohorts 1A, 1B, 1C and 2A
Frequency: Once on the day of weaning before dosing and thereafter
at weekly intervals during the treatment period. Procedure: Animals will be observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs will be recorded.

Body Weights – Cohorts 1A, 1B, 1C and 2A
Frequency: Weekly from weaning onwards. This will start on a specific date on which all pups are at least at PND 21. In addition, the body weight will be recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation.
In order to monitor the health status animals may be weighed more often. This will be documented in the study raw data.
Procedure: Animals will be individually weighed.

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

Water Consumption – Cohorts 1A, 1B, 1C and 2A Frequency: Regular basis throughout the study. Procedure: Water consumption will be monitored by visual inspection of the water bottles. If intergroup differences are noted, consumption may be assessed by weight.

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

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

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

Cohort 1A (20 animals/sex/group) The in-life procedures, observations, and measurements listed below will be performed for
the F1-females of Cohort 1A only, in addition to the procedures mentioned in section 16.1.

Estrous Cycle Determination – Cohort 1A
Frequency: First period: Daily vaginal lavage will be performed for all Cohort 1A females starting on the first day after onset of vaginal patency and will be minimally be continued until the first estrus is determined, in order to determine the time
interval between these two events. The estrus cycle data of the first period will not be reported. Data will be retained in the raw data. Second period: Daily vaginal lavage will be performed from PND 75 to 88. Procedure: Both periods: Estrous stages will be determined by examining the cytology of vaginal lavage samples.

Cohort 2A (10 animals/sex/group)
The in-life procedures, observations, and measurements listed below will be performed for the F1-animals of Cohort 2A only.

Acoustic startle response – Cohort 2A
Frequency: Once between PND 23-25. Procedure: Acoustic startle response (habituation) will be assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This will be performed in a sound-attenuated room. To the extent possible, treatment groups will be balanced across devices and the time of testing will be counterbalanced across dose group and sex. The animals
will be tested in sets of up to 3. The test sessions will consist of a five-minute acclimation period with a 65 ± 5-dB broadband
background white noise. The startle stimulus for each trial will be a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses will be recorded during the first 250 milliseconds following onset of the startle stimulus for each trial.
The test session will consist of 50 trials with an eight-second intertrial interval. Average response amplitude (AveN) will be analyzed in five blocks of 10 trials each.

Functional Observation Battery – Cohort 2A Frequency: Once between PND 63-75. Procedure: The Functional Observation Battery (FOB) tests will be conducted in the order of sequence indicated below and may be divided between several days. The detailed
clinical observations and locomotor activity will be conducted in separate room(s) specially equipped for these purposes. The other FOB tests may be conducted in the study room. 1. Detailed clinical observations: Detailed clinical observations consist of a number of tests conducted in- and out-side the home cage. To the extent possible, testing of animals will be counterbalanced
across dose groups. 2. Rectal temperature. Rectal temperature will be measured immediately after the detailed clinical observations. 3. Locomotor activity will be tested using the Kinder Scientific Motor Monitor System. Recording period will be one hour under normal laboratory light conditions. To the extent possible, treatment groups will be balanced across devices and the time of testing will be counterbalanced across dose groups. Total movements and ambulations will be 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 will be 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 will be recorded per animal as the mean of three measurements.

Body Weights – F1-Generation
Frequency: On PND 1, 4, 7, 13 and 21.
Procedure: Live pups will be individually weighed.

Sex – F1-Generation
Frequency: On PND 1, 4 and 13.
Procedure: Sex will be externally determined for all pups.

Anogenital Distance – F1-Generation
Frequency: On PND 1.
Procedure: Anogenital distance (AGD) will be measured for all live
pups. The AGD will be normalized to the cube root of body weight.

Areola/Nipple Retention – F1-Generation
Frequency: On PND 13.
Procedure: All males in each litter will be examined for the number of areola/nipples.

Postmortem examinations (parental animals):
All groupswill be subject to terminal body weight determination, necropsy, tissues collection and organ weighte determination.

Group 2 and 3 will get histopathology and histology of Target tissues, Reproductive tissues and gross lesions.
Group 1, 3, 4 and any unscheduled deaths will be subject to the attached list pdf with atttachments OECD443 (attachment C)

Unscheduled Deaths – F0-Generation
If an animal dies on study, a necropsy will be conducted and specified tissues will be saved, but not weighed. If necessary, the animal will be refrigerated to minimize autolysis. Animals may be euthanized for humane reasons as per Test Facility SOPs. These animals
will be deeply anaesthetized using isoflurane and subsequently exsanguinated. They will undergo necropsy, and specified tissues will be retained, but not weighed. The specified tissues which will be retained are mentioned in attached list pdf with atttachments OECD443 (attachment C)

Scheduled Euthanasia – F0-Generation
Animals surviving until scheduled euthanasia will have a terminal body weight recorded and will be deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies are summarized below: Males which sire: After successful mating and a minimum of 10 weeks of treatment. Males which fail to sire: At the end of the mating period and after a minimum of 10 weeks of treatment. Females which deliver: LD 23-25. Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27. Without evidence of mating: Approximately 24-26 days after the last day of the mating period. Females with total litter loss: Dams with no surviving pups will be euthanized within 24 hours after the last pup is found dead or missing. Except for females with total litter loss, all animals surviving to scheduled necropsy will be fasted overnight with a maximum of 24 hours before necropsy. Water will be available.

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

Sperm Analysis – F0-Generation
For all surviving males, the following assessments will be performed: Sperm samples will be taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility will be assessed from all samples. Sperm smears for morphological evaluation will be 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 will be recorded. Evaluation will be performed for all samples. One epididymis (right) will be removed, placed in labeled bags, and kept in the freezer at ≤ - 15°C. After thawing, the right epididymis will be weighed, homogenized and evaluated for sperm numbers. Evaluation will be performed for all samples. In the case of any abnormalities in the right epididymis, the right side organ will be fixed in modified Davidson's solution, and the left side organ will be used for evaluation of sperm numbers. If abnormalities are found in both epididymis, both these organs will be fixed in
modified Davidson's solution and no evaluation of sperm numbers will be performed.

Organ Weights – F0-Generation
The organs identified for weighing in the Tissue Collection and Preservation table in ATTACHMENT C will be weighed at necropsy for all scheduled euthanasia animals. Organ weights will not be recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs will be weighed together. Organ weight as a percent of body weight (using the terminal body weight) will be calculated.

Tissue Collection and Preservation – F0-Generation
Representative samples of the tissues identified in the Tissue Collection and Preservation table in ATTACHMENT C will be collected from all animals and preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.
Additional tissue samples may be collected to elucidate abnormal findings. For females which fail to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) will be fixed (if applicable), but will not be examined
Postmortem examinations (offspring):
Unscheduled Deaths – F1-Generation
Pups and fetuses may be euthanized for humane reasons as per Test Facility SOPs. Recognizable fetuses of females that die spontaneously or are euthanized in extremis will be examined externally and sexed (both externally and internally, if possible). Live fetuses will be euthanized by decapitation. Pups that are sacrificed in extremis, younger than 7 days, will be euthanized by decapitation. Pups sacrificed in extremis on or after PND 7 will be euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Pups found dead during the weekend can be fixed in identified containers containing 70% ethanol if not necropsied on the same day. Stillborn pups and pups found dead between birth and PND 13 will be sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy will be performed including sex determination (both externally and internally, if possible). Descriptions of all abnormalities will be recorded and may be collected and fixed in 10% buffered formalin at discretion of the Study Director. The stomach of pups not surviving to the scheduled necropsy date will be examined for the presence of milk, if possible. If possible, defects or cause of death will be evaluated.

Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) will be euthanized by decapitation. From two extra pups per litter, blood will be collected, if possible. Sex will be determined both externally and internally (if possible). Pups will be externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities will be recorded. External abnormalities may be collected and fixed in 10% buffered formalin at discretion of the Study Director.

TERMINAL PROCEDURES – F1-GENERATION FROM WEANING ONWARDS
All groupswill be subject to terminal body weight determination, necropsy, tissues collection and organ weighte determination except for 1C.

Cohort 1A will be fasted and euthanasia is scheduled day 89-95. Histology and histopathology: Full list for Gr. 1 and 4 Gross lesions and target tissues.
Cohort 1B euthanasia is scheduled day >97. No histopathology in first instance.
Cohort 1C euthanasia is scheduled after VP = vaginal patency, BPS = balanopreputial separation is positive.
Cohort 2A euthanasia is scheduled day 76-90. Cohort 2B euthanasia is scheduled day 21-22 Histology and histopathology: Neurohistopathology and morphometric analysis for Gr. 1 and 4.(Brain will be processed to block stage for all groups.) Target tissues for Gr. 2 and 3.

See Tissue Collection and Preservation table in the attached list pdf with atttachments OECD443 (attachment D-H)

If an animal dies on study, a necropsy will be conducted within 24 hours. If necessary, the animal will be refrigerated to minimize autolysis. Animals may be euthanized for humane reasons as per Test Facility SOPs. These animals will be deeply anaesthetized using
isoflurane and subsequently exsanguinated. Spare F1-animals which are not assigned to one of the Cohorts will be sacrificed between PND 22-24 by oral administration or intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Animals will be externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex will be determined (both externally and internally, if possible). Descriptions of all external abnormalities will be recorded. External abnormalities may be collected and fixed in 10% buffered formalin at discretion of the Study Director. For all animals, necropsy procedures will be performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, will be available. Tissues will be preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Additional tissue samples may be collected to elucidate abnormal findings. Organ weights will not be recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs will be weighed together. Organ weight as a percent of body weight (using the terminal body weight) will be calculated.

Cohort 1A
Scheduled necropsy of Cohort 1A will be conducted on PND 89-95. Cohort 1A animals surviving to scheduled necropsy will be deprived of food overnight (with a maximum of 24 hours) before necropsy, but water will be available. The animals will have a terminal
body weight recorded and will be deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals will be subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded. The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table in ATTACHMENT D will be weighed and collected.

Sperm Analysis – Cohort 1A
For all surviving males of Cohort 1A, the following assessments will be performed: Sperm samples will be taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility will be assessed from all samples. Sperm smears for
morphological evaluation will be 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 will be recorded. Evaluation will be performed for all samples. One epididymis (right) will be removed, placed in labeled bags, and kept in the freezer at ≤ - 15°C. After thawing, the left epididymis will be weighed, homogenized and evaluated for sperm numbers. Evaluation will be performed for all samples. In the case of any abnormalities in the right epididymis, the right side organ will be fixed in modified Davidson's, and the left side organ will be used for evaluation of sperm numbers. If abnormalities are found in both epididymis, both these organs will be fixed in modified Davidson's solution and no evaluation of sperm numbers will be performed.

Splenic Lymphocyte Subpopulation Analysis – Cohort 1A From 10 selected animals/sex/group2 of Cohort 1A, splenic lymphocyte subpopulation analysis will be performed at termination. If possible, one pup (male or female) will be selected per litter (20 litters in total). One half of the spleen will be kept on ice until splenic lymphocytes will be isolated using 70 µm cell strainers. The other half of the spleen will be preserved for histopathological evaluation. Splenocytes will be counted with the Coulter Counter Z1. The following subpopulations will be determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy: plenic Lymphocyte Parameters
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) will be determined using the Forward Scatter and Side Scatter.

Cohort 1B – Tissue Collection and Preservation
Scheduled necropsy of Cohort 1B will be conducted on ≥ PND 97. Cohort 1B animals will not be deprived of food overnight before necropsy. These animals will have a terminal body weight recorded and will be deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals will be subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded. The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table in ATTACHMENT E will be weighed and collected.

Cohort 1B - Additional Sample Collection for Liver Enzyme Activity
Measurements During necropsy at the end of treatment, a liver sample will be collected per animal (after weighing, prior to fixation and without interfering with potential histopathology) from all 20 control males and 20 high dose males in Cohort 1B as follows: Liver (left lateral lobe), approximately 2-3 gram per sample. The samples will be placed in individual, appropriately labeled, plastic containers and snap frozen in liquid nitrogen. Snap frozen samples should be kept in liquid nitrogen or surrounded by dry ice until storage. The samples must be stored at ≤ - 75°C at all times and must not be allowed to defrost until dispatch on dry ice. The tissue sample containers will be 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 time and sampling date. Labeled liver samples will be transferred on dry ice to the In Vitro ADME (IVA) group at the Test Facility. The Individual Scientist for Liver Enzyme Activity Measurement (ex vivo), J. Reinen, will be informed in advance.

Cohort 1B - Liver Enzyme Activity Measurement Ex Vivo
After arrival at the In Vitro ADME (IVA) group, liver samples will be stored at ≤ - 75°C.
Substrates and Reference Materials for Determination of the Enzyme Activities The table below summarizes the enzymatic activities, substrates and metabolites that will be investigated in this phase of the study.
Overview of Substrates and Metabolites used
Enzymatic Activity Substrate Metabolite
7-Pentoxyresorufin-O-dealkylation
(PROD) 7-Pentoxyresorufin (PR) Resorufin
Benzyloxyresorufin-O-dealkylation
(BROD) Benzyloxyresorufin (PR) Resorufin
T4-glucuronidation T4 T4-glucuronide

Stock and Spiking Solutions of the Substrate(s)
Stock solutions of the substrate(s) will be prepared at an appropriate concentration in a suitable solvent. Stock solutions will be aliquoted and stored in the freezer (≤ -15°C) and/or prepared freshly on the day of use. When stored in the freezer (≤ -15°C), stock solutions will
not be used for more than 1 month. Stock solutions of the substrate(s) will be diluted in an appropriate solvent to obtain spiking solutions in suitable vials. Spiking solutions will be prepared freshly on the day of use. Details regarding the exact procedure followed to prepare the substrate stock and spiking solutions will be specified in the study file and the Phase Report. Any residual volumes will be discarded unless otherwise requested by the Study Director.

Stock, Spiking and Standard Solutions of the Metabolite
For the PROD and BROD activity measurements, a calibration curve will be used to determine the amount of the resorufin metabolite formed. Stock solutions of the metabolite will be prepared at an appropriate concentration in a suitable solvent. Stock solutions will be stored in the refrigerator (2-8°C) until use. The stock solutions of the metabolite will be further diluted in an appropriate solvent to obtain spiking solutions which will be used to prepare metabolite standard solutions. Details regarding the exact procedure followed to prepare the metabolite stock, spiking and standard solutions will be specified in the study file and the Phase Report. Any residual volumes will be discarded unless otherwise requested by the Study Director.

Reference Materials
For the determination of the enzymatic activities, induced rat liver microsomes will be used as positive controls. Non-induced rat liver microsomes will be used as reference controls. Non-induced rat liver microsomes which have been inactivated in advance by boiling for 10 minutes will be used as negative controls. Details regarding the induced and control rat liver microsomes used during the study will be specified in the study file and the Phase Report.

Isolation of Liver Microsomes
Liver samples (approximately 2-3 grams) stored in the freezer (≤ -75°C) will be thawed at room temperature. Immediately after thawing, the liver samples will be placed on ice. The liver samples will be homogenized in ice-cold 0.1 M Tris-HCl buffer, pH 7.5 (3 mL/g liver)
containing 0.25 M sucrose, using a Potter-Elvehjem tube and Teflon pestle. The homogenate will be centrifuged for 30 minutes at 12,000 g (4°C) using a Beckman Avanti J-30 I Centrifuge (Beckman Coulter Nederland B.V., Mijdrecht, The Netherlands). The resulting
supernatant will be centrifuged at 105,000 g for 75 minutes (4°C). Subsequently, the microsomal pellet will be resuspended in 0.01 M Tris buffer pH 7.4 containing 3 mM EDTA. Microsomes will be stored in fixed aliquots in cryovials in the freezer (≤-75°C) until further
use. Prior to storage in the freezer (≤-75°C), microsomes will be frozen in liquid nitrogen. Any remaining liver sample will be stored in the freezer (≤-75°C) and will be discarded at finalization of the study.

Determination of the Protein Concentration
Protein concentrations will be measured in liver microsomes from each animal in duplicate by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a standard. Appropriate dilutions of the liver microsomes will be prepared in duplicate. Subsequently, a
fixed volume of a deoxycholate solution (supplied in a protein assay kit, Sigma) will be added after which the samples will be mixed immediately. After leaving the samples for 10 minutes at room temperature, a fixed volume of a trichloro acetic acid solution (also
supplied in the kit) will be added after which the samples will be mixed immediately. Samples will subsequently be centrifuged for 5 minutes at 10,000 rpm at room temperature. The supernatant will be removed after which the remaining pellet will be redissolved in a
fixed volume of Lowry Reagent Solution (also supplied in the kit) and incubated for 20 minutes at room temperature. Subsequently, a fixed volume of Folin & Ciocalteu’s phenol Reagent (also supplied in the kit) will be added after which the samples will be mixed
immediately. The color will be allowed to develop for 30 minutes. The solutions will then be transferred to a 96 well plate and the absorbance will be measured at 540 nm using a spectrophotometer (TECAN Infinite® M200 Pro, TECAN, Austria). The protein
concentration from the samples will be determined by comparison with the calibration curve of BSA using linear regression. If applicable, the deoxycholate-trichloro acetic acid precipitation step may be omitted. The exact procedure to determine the protein concentration will be specified study file and the Phase Report.

Determination of Cytochrome P450 (CYP450) Content
The cytochrome P450 (CYP) concentration will be calculated from the CO-reduced difference spectrum according to the method of Omura and Sato (1964). The difference spectra of liver microsomal preparations will be measured using a spectrophotometer (UV
1800 double beam UV/visible spectrophotometer, Shimadzu, ‘s-Hertogenbosch, The Netherlands). Each microsomal preparation will be measured at least once. At first the baseline of the microsomal preparations will be recorded. Subsequently, CO will
be bubbled carefully through the sample for approximately 30 seconds. The spectrum of the CO-saturated samples will also be recorded. Reduction of samples will be effected by adding dithionite. The spectrum of reduced samples will be measured and the CYP concentration
will be calculated from the difference between the spectra of the saturated and reduced samples.

Determination of the Enzymatic Activity
-Pentoxyresorufin-O-Deethylation (PROD) Activity 7-Pentoxyresorufin-O-deethylation (PROD) activity will be determined according to the method of Burke et al. (1977), adapted for the use in 96-well plates and a microplate reader (TECAN Infinite® M200 Pro, TECAN, Austria). Incubation mixtures will be prepared by mixing liver microsomes with 0.1 M potassium phosphate buffer pH 7.8 and pentoxyresorufin. The reaction mixtures will be pre-incubated in the microplate reader for 1 minute at 37±1°C and the reaction will be started by the addition of NADPH. The formation of resorufin will be detected fluorometrically (excitation 535 nm, emission 588 nm) by comparison with a resorufin calibration curve. Details regarding the exact procedure followed to prepare the incubation mixtures and the
exact reaction conditions will be specified in the study file and Phase Report.

Benzyloxyresorufin-O-Deethylation (BROD) Activity
Benzyloxyresorufin-O-deethylation (BROD) activity will be determined according to the method of Burke et al. (1977), adapted for the use in 96-well plates and a microplate reader (TECAN Infinite® M200 Pro, TECAN, Austria). Incubation mixtures will be prepared by
mixing liver microsomes with 0.1 M potassium phosphate buffer pH 7.8 and benzyloxyresorufin. The reaction mixtures will be pre-incubated in the microplate reader for 1 minute at 37±1°C and the reaction will be started by the addition of NADPH. The formation of resorufin will be detected fluorometrically (excitation 535 nm, emission 588 nm) by comparison with a resorufin calibration curve. Details regarding the exact procedure followed to prepare the incubation mixtures and the exact reaction conditions will be specified in the study file and Phase Report.

T4-glucuronidation Activity
Incubation mixtures will be prepared on ice in duplicate by mixing 0.1 M Tris-HCl buffer pH 7.4 with liver microsomes and UGT reaction mixtures A and B. After shaking, the mixtures will be pre-incubated for 15 minutes on ice. Subsequently, the mixtures will be pre-
incubated for 5 minutes at 37±1°C in a water bath. The reaction will be started by the addition of the appropriate T4 spiking solution. After incubation at 37±1°C for a defined time period, the samples will be transferred to ice and a protein precipitation solution will be
added to precipitate proteins. Subsequently, a sample pre-treatment procedure will be used to remove precipitated proteins and to prepare the samples for analysis by UPLC-PDA-MS. Details regarding the exact procedure followed to prepare the substrate incubation mixtures,
the exact reaction conditions and the sample pre-treatment procedure used before sample analysis will be specified in the study file and Phase Report.

Cohort 1C
Scheduled necropsy of Cohort 1C will be conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals will not be deprived of food overnight before necropsy. Terminal body weight will not be recorded. The animals will be deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals will be subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded. In case of macroscopic abnormalities, gross lesions will be preserved in the most appropriate fixative together with the identification marks (see ATTACHMENT F).

Cohort 2A and 2B
Scheduled necropsy of Cohort 2A will be conducted on PND 76-90. Scheduled necropsy of Cohort 2B will be conducted on PND 21-22. The animals will not be deprived of food overnight before necropsy. Terminal body weight will be recorded. The animals will first be anaesthetized using isoflurane and will be 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 will be subjected to a limited examination, with special attention being paid to the reproductive organs. After perfusion, the cranium will be removed, exposing the brain. The skull including the brain is placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull. The fixed brains will be removed and weighed, and the length and maximum width of the brain will be measured for all animals selected for neuropathology (Length of the brain: on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum. Width of the brain: pituitary region. Measurements are conducted using a digital caliper. ) Subsequently, the brain will be fixed in 10% buffered formalin together with selected PNS tissues.
Representative samples of the tissues identified in the Tissue Collection and Preservation table in ATTACHMENT G will be collected.

Cohort Surplus
Scheduled necropsy of Cohort Surplus will be conducted on PND 22-24. Cohort Surplus animals will not be deprived of food overnight before necropsy. Terminal body weight will be recorded. On PND 22-24, blood samples (1.0 mL) will be collected between 8.00 and 11.30 a.m. from all animals by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Blood samples will be collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4), see section 17.1. All animals will be subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded. The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table in ATTACHMENT H will be weighed and collected.
Statistics:
see attached 'OECD 443 statistics'
Reproductive indices:
General Reproduction Data – F0-Generation
Frequency: Daily from the mating period onwards.
Procedure: Male number paired with, mating date, confirmation of pregnancy, and delivery day will be recorded. Palpation and/or body weight measurement may be used to aid in confirmation of pregnancy. The females will be allowed to litter normally. Postnatal
day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as Lactation Day (LD) 0
for the dam and PND 0 for the offspring, and used for recording of delivery. Females that are littering will be left undisturbed. Cage debris of pregnant females will be examined for evidence of premature delivery. Signs of difficult or prolonged parturition will be recorded, if applicable.
Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, will be recorded, if applicable.
Offspring viability indices:
See above
Clinical signs:
no effects observed
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 similary.

Other clinical signs noted during the treatment period included rales, hunched posture, piloerection, local 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
Description (incidence):
Two females in the control group and two females at the low dose (50 mg/kg/day) did not survive until scheduled necropsy:
- Control Female No. 114 had to be euthanized for humane reasons on Day 29 of the premating period. She presented with piloerection from premating Day 24 onwards, hunched posture from premating Day 26 post-dose onwards (Days 7-8 as well), and ptosis and a lean appearance on premating Day 29. The growth of Female No. 114 was severely retarded during the first 3 weeks of treatment (i.e. only 12% body weight gain from Days 1-22), followed by 5% body weight loss from Days 22-29. For comparison, for all remaining control females a body weight gain of 35-58% was recorded from Days 1-29. At necropsy, the following gross findings were observed: emaciation, esophagus with white mucous and soft white-grey content, collapsed lungs, gastro-intestinal tract extended with gas. A full necropsy is performed and the outcome of histopathological examination is awaited.
- Control Female No. 103 had a total litter loss on lactation Day 6 and was euthanized.
- Female No. 134 (50 mg/kg/day) was found dead on the morning of premating Day 7. There were no findings during clinical observations on the days before her death. Since this female was severely cannibalised, no cause of death could be established.
- Female No. 144 (50 mg/kg/day) died in the animal facility after collection of blood on the day of scheduled necropsy (lactation Day 24). Its death was considered to be related to complications of the anaesthesia.
Body weight and weight changes:
no effects observed
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 minor, but statistically significantly 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 only.
Food consumption and compound intake (if feeding study):
no effects observed
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 females at 150 and 450 mg/kg/day, a lower food intake relative to body weight was noted in pre-mating Week 6 (150 mg/kg/day), and Weeks 6-8 and Week 10 (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, and/or occurred in the absence of a dose-related trend.

Description (incidence and severity):
A treatment-related effect on water consumption was suspected for F0-animals based on subjective appraisal. Therefore, quantitative measurement of water consumption twice weekly was introduced for all animals (both sexes) from premating Day 45 onwards.

In males at 150 and 450 mg/kg/day, an increased water consumption (absolute) was observed during the remainder of the premating period and during the mating period (not always statistically significant). When compared to the control group, mean of means was 1.24x (both dose levels) over Days 45-71 of the pre-mating period and 1.23x and 1.19x, respectively, during the mating period. Changes did not always reach statistical significance and occurred in the absence of clear dose-proportionality.
In females, absolute water intake was increased at 450 mg/kg/day only. Over pre-mating Days 45-71, it reached a mean of means of 1.20x of control. 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 (reaching statistical significance over lactation Days 4-5 and 11-12 only). Mean of means was 1.14x of control over lactation Days 1-18.

Note: A relatively high water intake was noted for Group 3 males in cage 12 from Days 3-4 of the mating period. Possibly a mistake was made when correcting for the number of males present in this cage. This will be checked when preparing the draft report.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in haematology parameters were noted in males and females up to 450 mg/kg/day.
The following statistically significant changes in haematology parameters distinguished treated animals from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Mean number of lymphocytes was slightly increased in females at 450 mg/kg/day (1.35x; within historical control range).
- Mean red cell distribution width (RBW) was slightly increased in males at 150 and 450 mg/kg/day (1.04x and 1.07x, respectively; within historical control range).
- Mean haemoglobin concentration was slightly decreased in males at 450 mg/kg/day (0.97x; marginally outside historical control range).
- Mean corpuscular hemoglobin concentration (MCHC) was slightly decreased in females at 450 mg/kg/day (0.78x; within historical control range).
All these statistically significant changes were considered to be of no toxicological relevance as they were marginally only, were observed in the absence of changes in correlating parameters and/or remained within the range considered normal for rats of this age and strain.
The statistically significant changes in absolute numbers of neutrophils and eosinophils observed in males at 150 mg/kg/day and females at 50 mg/kg/day, respectively, were considered to be unrelated to treatment with the test item as they occurred in the absence of a treatment-related distribution.

Coagulation parameters of treated rats were considered not to have been affected by treatment.
The slightly decreased mean prothrombin time (PT) observed in males from 50 mg/kg/day onwards (0.94x, 0.94x and 0.92x at 50, 150 and 450 mg/kg/day, respectively) was considered to be unrelated to treatment with the test item as this change occurred in the absence of a treatment-related distribution. All mean values remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses. Toxicological evaluation of these changes will follow after receipt of the histopathology report.
- Mean alanine transaminase (ALAT) activity was decreased in males at 450 mg/kg/day (0.77x; within historical control range).
- Mean total protein concentration was marginally increased in males at 450 mg/kg/day (1.03x; within historical control range).
- Mean albumin concentration was marginally increased in males at 450 mg/kg/day (1.04x; within historical control range).
- Mean urea concentration was decreased in males at 450 mg/kg/day and increased in females at 450 mg/kg/day (0.83x and 1.22x, respectively; within historical control range).
- Mean creatinine concentration was increased in males at 450 mg/kg/day and in females at 50, 150 and 450 mg/kg/day (1.10x in males, and 1.13x, 1.12x and 1.15x, respectively, in females; within historical control range).
- Mean glucose concentration was decreased in males at 450 mg/kg/day (0.82x; within historical control range).
- Mean bile acid concentration was decreased in males at 150 and 450 mg/kg/day (0.45x and 0.46x, respectively; both within historical control range).
- Mean sodium concentration was marginally increased in males at 450 mg/kg/day (1.01x; within historical control range).
- Mean potassium concentration was increased in females at 150 and 450 mg/kg/day (1.11x at both dose levels; at the upper limit of historical control range).
- Mean inorganic phosphate concentration was increased in females at 50, 150 and 450 mg/kg/day (1.23x, 1.23x and 1.33x, respectively; all within historical control range).

The statistically significant decrease of mean total bilirubin concentrations observed in males at 50 mg/kg/day (0.90x; within historical control range) was considered to be unrelated to treatment with the test item as it occurred in the absence of a treatment-related distribution.
Note: In the tables with clinical biochemistry parameters the albumin/globulin ratio is included. This is not a default parameter and will therefore be removed in the next version.
Description (incidence and severity):
The following statistically significant changes 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 in males at 450 mg/kg/day (0.94x; above historical control range).
- Mean score for epithelial cells was increased in males at 50, 150 and 450 mg/kg/day (i.e. score 3 in all treated groups, compared to score 2 in the control group; within historical control range).
- Mean volume of urine was increased in females at 450 mg/kg/day (1.53x; within historical control range).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were unaffected by treatment with the test item.
Most females had regular cycles of 4 to 5 days. An irregular cycle of 2, 3 or 8 days was noted for one female in each the control group (No. 102), 150 mg/kg/day group (No. 170), and 450 mg/kg/day group (No. 186). All these females with irregular cycle had normal litters. Given their incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Sperm parameters were considered to be unaffected by treatment up to 150 mg/kg/day.
Statistically significant changes that distinguished males treated at 450 mg/kg/day from control animals are summarized below. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
450 mg/kg/day
- Lower percentage of motile sperm (0.50x; within historical control range)
- Lower percentage of progressive sperm (0.35x; within historical control range)
- Lower epididymal spermcount (0.82x; not reaching statistical significance, within historical control range)
- Lower number of cells with normal morphology (mean of 81 vs. 171 in the control group; below historical control range)
- Higher number of cells with a detached head (mean of 101 vs. 3 in the control group; above historical control range)
- Lower number of cells with coiled tail (mean of 7 vs. 23 in the control group; within historical control range)
- Presence of cells with other tail (mean of 7 vs. 0 in the control group; within historical control range).

Note: Results from stage dependent qualitative evaluation of spermatogenesis in the testis are pending.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycle:
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. An irregular cycle of 2, 3 or 8 days was noted for one female in each the control group (No. 102), 150 mg/kg/day group (No. 170), and 450 mg/kg/day group (No. 186). All these females with irregular cycle had normal litters. Given their incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Mating Index
Mating index for both males and females was 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 mating index (both sexes) of 100, 100, 100 and 92% for the control, 50, 150 and 450 mg/kg/day groups, respectively.
Precoital time:
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 absence of any dose-related occurrence this was considered not to be related to treatment with the test item.

Number of implantation sites:
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. All mean values remained within the normal range.
Fertility Index
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.
Thyroid hormone analysis:
There were no differences noted between control and treated rats that were considered to be related to treatment with the test item
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Critical effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (nominal)
System:
male reproductive system
Treatment related:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
Note: Clinical signs observed in animals that died before scheduled necropsy are summarized under Mortality.

No toxicologically relevant findings were observed up to 450 mg/kg/day.
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 similar.
Other clinical signs noted during the treatment period included rales, hunched posture, piloerection, ptosis, diarrhea, chromodacryorrhoea (eye), scabs, wounds, alopecia, and a bent tail. 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 / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were seven unscheduled deaths in the F1-generation. These deaths were considered to be unrelated to treatment with the test item.

0 mg/kg/day:
- Female No. 525 in Cohort 1A was found dead on Day 38 of the treatment period (pre-dose). This female presented with rales in the second week of treatment (Days 9-11), but not shortly before its death. Necropsy findings included cannibalism and lungs that failed to collapse.
- Female No. 567 in Cohort 1C was found dead on Day 9 of the treatment period (pre-dose). The day before its death this female was noted with gasping. Gross findings at necropsy included isolated grey-white foci on different lobes of the liver and a single dark red focus on the thymus.

50 mg/kg/day:
- Male No. 299 in Cohort 1A was found dead on Day 34 of the treatment period (post-dose). Before its death, there were no relevant findings at clinical observations. Gross findings at necropsy included perforation of the esophagus and body cavity containing tan, watery cloudy fluid.
- Male No. 311 in Cohort 1B was found dead on Day 47 of the treatment period (pre-dose). This male had been observed with rales on several days over Weeks 3-7 and squeaking on treatment Day 39 (post-dose) and Day 40 (pre-dose). It displayed a growth that was delayed until Week 6, followed by 13% body weight loss over Weeks 6-7. Necropsy findings included beginning autolysis and lungs that failed to collapse.

150 mg/kg/day:
- Male No. 376 in Cohort 1A was found dead on Day 16 of the treatment period (pre-dose). This male had been noted with rales after treatment on Day 4 only. Slight salivation was observed from Day 3 onwards. Body weight gain was decreased when compared to the remaining males in this mid dose group. No macroscopic abnormalities were observed at necropsy.
- Male No. 389 in Cohort 1B was found dead on Day 13 of the treatment period (pre-dose). Before its death this male was noted with rales on treatment Days 10 and 11. At necropsy, cryptorchism of the right testis was observed.
- Female No. 681 in Cohort 1A was found dead on treatment Day 35 (pre-dose). There were no toxicological relevant findings during in-life. This female was severely cannibalized, preventing any investigation of cause of death.

450 mg/kg/day:
- All animals in the high dose group survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gains were noted in males and females up to 450 mg/kg/day.
In general, lower mean body weights were noted in males of the 50 and 450 mg/kg/day groups when compared with the concurrent control group. Changes were relatively slight and did not always reach statistical significance. In addition, statistically significantly lower absolute body weights were observed in males at 150 mg/kg/day on Days 1 and 71. Body weight gain in the test item-treated groups were in the same range as for the control group, except for body weight gain in males at 150 mg/kg/day which was statistically significantly higher on Day 8. In absence of a dose-related distribution, none of these changes was considered to be related to treatment with the test item.
For females at 450 mg/kg/day a slightly, but statistically significantly lower absolute body weight was observed on Day 15. In the subsequent period, a slight, but statistically significantly increase in body weight gain was observed over Days 22-71 (ranging from 1.07-1.11x control; not always statistically significant). This slightly higher body weight gain did not result in significant changes in absolute body weight between females in the high dose and control group.

Food consumption and compound intake (if feeding study):
no effects observed
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 450 mg/kg/day, a statistical significant increase in relative food consumption was noted in treatment Weeks 8 and 9. No toxicological relevance was attached to this finding, as changes compared to the concurrent control group were very slight (ranging from 1.04-1.08x of control) and values for individual cages remained in the normal range of biological variation. It should be noted that throughout the observation period the numbers of cages available with animals at 450 mg/kg/day was approximately half of the numbers available in the control, 50 and 150 mg/kg/day groups.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in haematology parameters were noted in males and females up to 450 mg/kg/day.
The following statistically significant changes in haematology parameters distinguished treated animals from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Mean number of red blood cells (RBC) was slightly decreased in males at 450 mg/kg/day (0.94x; within historical control range).
- Mean haemoglobin concentration was slightly decreased in males at 450 mg/kg/day (0.93x; just below historical control range).
- Mean haematocrit concentration was slightly decreased in males at 450 mg/kg/day (0.94x; just below historical control range).
- Mean corpuscular hemoglobin concentration (MCHC) was slightly decreased in males at 150 mg/kg/day (0.98x).
- Mean number of white blood cells (WBC) was increased in females at 150 and 450 mg/kg/day (1.41x at both dose levels; within historical control range).
- Mean number of lymphocytes was slightly increased in females at 50, 150 and 450 mg/kg/day (1.52x, 1.62x and 1.57x, respectively; within historical control range).
- Mean number of large unstained cells (LUC) was marginally increased in females at 450 mg/kg/day.
All these statistically significant changes were considered to be unrelated to treatment with the test item as they were marginally only, were caused by a relatively low control value, occurred in the absence of a dose-related response and/or remained within the range considered normal for rats of this age and strain.
Note: In the tables with haematology parameters two values are reported for numbers of lymphocytes. This will be corrected in the next version.

Coagulation parameters of treated rats were considered not to have been affected by treatment.
The slightly decreased mean prothrombin time (PT) observed in males at 450 mg/kg/day (0.95x) was considered not to be toxicological relevant as this change compared with the control group was very slight only (without reaching statistical significance) and the mean remained within the range considered normal for rats of this age and strain.
The slightly increased mean prothrombin time (PT) observed in females at 50 mg/kg/day (1.08x) was considered unrelated to treatment as no dose-related distribution was evident.
Description (incidence and severity):
The following changes (statistically significant unless stated otherwise) distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses. Toxicological evaluation of these changes will follow after receipt of the histopathology report.
- Mean glucose concentration was decreased in males at 450 mg/kg/day (0.74x; within historical control range) and females at 50 and 450 mg/kg/day (0.85x and 0.83x; within historical control range).
- Mean cholesterol concentration was increased in males and females at 450 mg/kg/day (1.37x and 1.16x, respectively; not reaching statistical significance in females; both means within historical control range).
- Mean sodium concentration was marginally increased in males at 450 mg/kg/day (1.01x; within historical control range).
Note: In the tables with clinical biochemistry parameters the albumin/globulin ratio is included. This is not a default parameter and will therefore be removed in the next version.

Thyroid hormone analysis:
Serum T4 and TSH levels in male and female pups of Cohort Surplus at PND 22 were unaffected by treatment with the test item.
Data from Cohort 1A (PND 89-95): not yet available
Description (incidence and severity):
The following statistically significant changes distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Mean specific urine gravity was marginally increased in males at 450 mg/kg/day (1.01x; above historical control range).
- Mean pH value was decreased in males at 150 and 450 mg/kg/day (0.94x and 0.91x, respectively; below historical control range).
- Mean score for epithelial cells was increased in males at 150 and 450 mg/kg/day (i.e. score 3 in both groups, compared to score 1 in the control group; above historical control range).

In females, there were no differences noted in urinalysis parameters between control and treated rats that were considered to be related to treatment with the test item.
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 statistically significantly increased compared to the control group (42.9 days vs. 41.4 days in controls). In 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.
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.
Description (incidence and severity):
The following necropsy findings distinguished treated from control animals. Statistical significant changes are indicated in bold.

Cohort 1A (PND 89-95):
Kidneys
- Enlargement:
0/20, 0/19, 1/19, 3/20 males in the control, 50, 150 and 450 mg/kg/day groups, respectively.
- Pale/greenish discoloration:
0/20, 3/19, 7/19, 13/20 males in the control, 50, 150 and 450 mg/kg/day groups, respectively.
Liver
- Enlargement:
0/20, 0/19, 1/19, 4/20 males in the control, 50, 150 and 450 mg/kg/day groups, respectively.
- Black-brown discoloration:
0/20, 1/19, 0/19, 6/20 males in the control, 50, 150 and 450 mg/kg/day groups, respectively.
0/19, 1/20, 0/19, 2/20 females in the control, 50, 150 and 450 mg/kg/day groups, respectively.
Epididymides
- Nodules:
0/20, 0/19, 0/19, 4/20 males in the control, 50, 150 and 450 mg/kg/day groups, respectively.

Cohort 1B (PND ≥97):
Note: No Cohort 1B animals are available in the 450 mg/kg/day group.

Kidneys
- Pale discoloration:
0/19, 3/19, 12/19 males in the control, 50 and 150 mg/kg/day groups, respectively.
Liver
- Enlargement:
0/19, 1/19, 2/19 males in the control, 50 and 150 mg/kg/day groups, respectively.
0/20, 0/19, 1/20 females in the control, 50 and 150 mg/kg/day groups, respectively.

Cohort 1C (After VO/BPS positive):
Note: No Cohort 1C animals are available in the 50 and 450 mg/kg/day groups.
There were no macroscopic abnormalities in Cohort 1C animals that were considered to be related to treatment with the test item.

Cohort 2A (PND 76-90):
Kidneys
- Enlargement:
0/10, 4/10, 4/10, 7/10 males in the control, 50, 150 and 450 mg/kg/day groups, respectively.

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

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

All remaining macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see table below
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: sperm analysis
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects

Organ weights – F0

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

 

Males

 

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

17**

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; significant changes shaded in grey

 

Females

 

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

10**

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; significant changes shaded in grey

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 – F1 1A-Generation Males and Females

 

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<0.01; significant changes shaded in grey

 

Note: At the individual level, relatively low organ weights of testes, epididymides, prostate and seminal vesicles were recorded in Cohort 1A Male No. 370 (150 mg/kg/day) which correlated with the necropsy finding ‘reduced in size’.

Note: At the individual level, an unrealistic high organ weight was noted for the ovaries of Cohort 1A Female No. 771 (450 mg/kg/day). Tables will be corrected before issuing the draft report.

 

Cohort 1B

Note: No Cohort 1B animals are available in the 450 mg/kg/day group.

 

Mean Percent Organ Weight Differences from Control Groups – F1 1B-Generation Males and Females

 

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

Note: At the individual level, an unrealistic high organ weight was noted for the ovaries of Cohort 1A Female No. 771 (450 mg/kg/day). Tables will be corrected before issuing the draft report.

 

Brain dimensions – F1

Cohort 2A (PND 76-90):not yet available

Cohort 2B (PND 21-22):not yet available

 

Brain weight – F1

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.

Note: At the individual level, an unrealistic highfixed brain weightwas noted for Cohort 2A Male No. 252 (control). Tables will be corrected before issuing the draft report.

 

Cohort 2B (PND 21-22):

There were fixed brain weight changes as indicated in the table below.

Mean Percent Fixed Brain Weight Differences from Control Groups – F1 2B-Generation Males and Females

 

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.

Reproductive/Developmental Data - Post-Weaning

 

Sexual Maturation – F1 (Cohorts 1A, 1B, 1C and 2A)

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 statistically significantly increased compared to the control group (42.9 days vs. 41.4 days in controls). In 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. 

Estrous Cycle Determination (Cohort 1A):

Length and regularity of the estrous cycle were not affected by treatment with the test item. All females had regular cycles of 4 to 5 days.

Sperm analysis (Cohort 1A):

Sperm parameters were considered to be unaffected by treatment up to 150 mg/kg/day.

Statistically significant changes that distinguished males treated at 450 mg/kg/day from control animals are summarized below. (Relative) changes in mean values as compared to the concurrent control group are indicated between parentheses.

450 mg/kg/day

- Lower percentage of motile sperm (0.67x; within historical control range)

- Lower percentage of progressive sperm (0.48x; within historical control range)

- Lower epididymal spermcount (0.69x; not reaching statistical significance, within historical control range)

- Lower number of cells with normalmorphology (mean of 100vs.182 cells in the control group; within historical control range)

- Higher number of cells with a detached head (mean of 74vs.2 cells in the control group;above historical control range)

- Lower number of cells with coiled tail (mean of 3vs.12 cells in the control group; within historical control range)

- Higher mean number of cells with an abnormal neck (mean of 19vs. 1 cell in the control group;above historical control range).

 

Note: Results from stage dependent qualitative evaluation of spermatogenesis in the testis are pending.

 

Splenic Lymphocyte Subpopulation Analysis – F1 (Cohort 1A -PND 89-95)

There were no test item-related effects on splenic lymphocyte subpopulations observed.

 

Histopathology – F0/F1:

(including brain morphometry (Cohorts 2A and 2B) and quantitative evaluation of follicles (Cohort 1A)):

not yet available(expected two weeks before unaudited draft report date (04 Dec 2020)).

 

Conclusions:
Based on these draft results, that show adverse effect on sperm and reproductive outcome, the substance is classified for effects on fertility in catergory 1b. The IUCLID dossier will be updated with the final report results as soon as these become available. The audited draft report will be available in December 2020. The final report is expected in Q1 2021.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Species:
rat
Quality of whole database:
K1 GLP study. Please note this information is based on a draft report and applicable to the effects observed on sperm and fertility. The overal NOAEL for the final study needs to be determined.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The oral administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to pregnant rats by gavage during gestation Days 5 to 19, at dose levels of 30, 175 or 1000 mg/kg bw/day resulted in an adverse effect on body weight gain and food consumption at the high dose level.  Although some improvement was evident, cumulative body weight gains for females treated with 1000 mg/kg bw/day remained lower than controls throughout the dosing period with body weight gain adjusted for the contribution of gravid uterus also being markedly lower than controls.  Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 175 mg/kg bw/day within the confines of this type of study.

At 1000 mg/kg bw/day, fetal and litter weights were marginally lower than control with skeletal examination identifying a number of treatment-related variants.  These observations were, however, considered to be due to maternal toxicity which results in slight reduction in fetal growth rather than an indication of a direct effect of the test item on fetal growth and development.  The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day whilst the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was deemed to be 175 mg/kg bw/day within the confines of this type of study.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 04 August 2015 Experimental Completion Date: 01 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification : 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0)
Physical State/Appearance:Clear colorless liquid
Chemical Name:1,1,3,3-Tetramethylbutyl peroxyneodecanoate
Chemical Formula:C18H36O3
Purity:69.2%
Batch Number:1503447025
Label : TRIGONOX 423-C70 1,1,3,3-tetramethylbutyl peroxyneodecanoate 1kg Below -15°C Batch/lot: 1503447025 Expiry Date 14 June 2015
Date Received : 17 June 2015
Storage Conditions : Stored at approximately -20 °C, used/formulated under ambient conditions
Expiry Date : 15 April 2016
No correction for purity was made.
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Females prior to Day 3 of gestation
- Weight at study initiation: 187 - 285g
- Fasting period before study: No
- Housing: Individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 04 August to 01 December 2015
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Oral gavage
- Concentration in vehicle: 0, 7.5, 43.8, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) at Harlan Laboratories Ltd., Shardlow.

Stock solutions of the test item in acetonitrile were prepared for external standard calibration at a concetration of 1 mg/mL. Aliqupots of stock standard solutions were used to prepare working standard solutions in aceytonitrile with concentration of 0.1 mg/mL. On each occasion standard solutions derived from two standard stock solutions were used for calculation.

The fomulations received were extracted with acetonitrile. An aliquot of the test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile, ultra sonicated and centrifuged for 10 minutes. Where necessary sample solutions were further diluted with acetonitrile to achieve working concentrations. The test item concentration in the test samples was determined by gas chromatography (GC) using the external standard technique.
The accuracy and linearity determinations were determined under study number 41501125.

The formulations were found to comprise test item in the range of 98% to 103% and, thus, the required content limit of ±10% with reference to the nominal content was met. The results obtained during study number 41501125 indicate the accurate use of the test item in Arachis Oil BP as vehicle during this stiudy. The formulations were found to be homogeneoulsy prepared and sufficient formulation stability under storage conditions was proven.
The detection system was found to have acceptable linearity. The analytical system had acceptable recoveries of test item in vehicle. The method of analysis was validated and proven to be suitable for use.
Details on mating procedure:
The experimental animals were obtained time-mated from the supplier.
Duration of treatment / exposure:
The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 30, 175 or 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil BP) to serve as a control.
Frequency of treatment:
Daily
Duration of test:
Experimental Starting Date: 04 August 2015
Experimental Completion Date: 01 December 2015
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - 0 mg/mL
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Low - 7.5 mg/mL
Dose / conc.:
175 mg/kg bw/day (actual dose received)
Remarks:
Intermediate - 43.8 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High - 250 mg/mL
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Yes
- Cage side observations checked in Table 2 and Appendix 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Yes

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Visual inspection

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Ovaries and uteri
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Other:
- Dead fetus

All implantations and viable fetuses were numbered according to their intrauterine position.

Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [all per litter]
Statistics:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
Most caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Computerized sysyems used:
R Environment for Statistical Computing


Historical control data:
Historical control data used for comparison purposes for fetal examination findings and uterus weights.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations for any of the animals throughout the study.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At all dose levels, females showed a dose-related reduction in group mean body weight gains over Days 5 to 6 of gestation with most animals from the 1000 mg/kg bw/day showing actual body weight losses. Subsequent recovery was evident for females receiving 30 or 175 mg/kg bw/day with periodic body weight gains for these animals remaining comparable with controls from Day 6 of gestation. Females treated with 1000 mg/kg bw/day also showed improvement in body weight performance following the initial affect such that body weight gains over Days 7 to 14 of gestation were comparable with controls; however, further reduction in body weight gain was observed over Days 14 to 17 of gestation resulting in lower cumulative body weight gains for these animals throughout the treatment period. The overall body weight gain for these females was approximately 21% lower than controls with body weight gain adjusted for gravid uterus contribution also being markedly lower than controls. The effect on body weight development for the 1000 mg/kg bw/day females was considered to be of toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was marked reduction in food consumption at the start of dosing for females given 1000 mg/kg bw/day. Gradual improvement was apparent, however, dietary intake for these animals remained statistically significantly lower than controls up to Day 17 of gestation. Taken together with the effect on body weight development at this dose level, this observation was considered to be of toxicological significance.
There was no effect of treatment on food intake at 30 or 175 mg/kg bw/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group mean gravid uterus weight for females treated with 1000 mg/kg bw/day was also slightly lower than controls; however, it is worth noting that whilst 4/24 control females showed gravid uterus weights slightly above the historical data ranges the corresponding values for most females treated with 1000 mg/kg bw/day were within these ranges. Group mean body weight gain when adjusted for contribution from gravid uterus for these females was also markedly lower than controls and with most individual values below the historical control data ranges, this finding was deemed to be of toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
For all dose groups, there were no statistically significant treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities. At 1000 mg/kg bw/day, a number of skeletal anomalies achieved statistical significance. These included incomplete ossification of occipital (supra-occipital) region, thoracic centrum and sternbrae, no ossification of metacarpal, and caudal vertebrae - less than 4 ossified. These observations are minor variants and although group mean values were outside the historical background data ranges, they were deemed likely due to a slightly slower fetal growth rate resulting from maternal toxicity rather than an indication of a direct effect of the test item on fetal development. Any other skeletal findings at all dose levels were considered to be within normal biological variation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was considered to be no detrimental effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and post-implantation losses at 30, 175 or 1000 mg/kg bw/day.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of late deaths for the 1000 mg/kg bw/day dose group was slightly higher than controls but the intergroup difference did not achieve statistical significance and with one litter (Female 86) from this dose group showing four late deaths this observation was deemed unlikely to be of any toxicological significance.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All animals (24) in all dose groups, including control, became pregnant.
Other effects:
no effects observed
Details on maternal toxic effects:
The oral administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to pregnant rats by gavage during gestation Days 5 to 19, at dose levels of 30, 175 or 1000 mg/kg bw/day resulted in an adverse effect on body weight gain and food consumption at the high dose level. Although some improvement was evident, cumulative body weight gains for females treated with 1000 mg/kg bw/day remained lower than controls throughout the dosing period with body weight gain adjusted for the contribution of gravid uterus also being markedly lower than controls. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 175 mg/kg bw/day within the confines of this type of study.
Key result
Dose descriptor:
NOAEL
Effect level:
<= 175 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
placenta
Description (incidence and severity):
Total placental weight for females at 1000 mg/kg bw/day was statistically significantly lower than controls (p<0.05). The corresponding values for the 30 or 175 mg/kg bw/day dose groups were comparable with controls.
Abnormalities:
effects observed, non-treatment-related
Localisation:
uterus
Description (incidence and severity):
The uterus of Female 77 at 1000 mg/kg bw/day was filled with yellow fluid but this female was pregnant at necropsy. This is an isolated finding, therefore unlikely to be related to treatment.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, group mean fetal weights were marginally lower than controls which resulted in slightly lower litter weight for this dose group. These findings were deemed likely due to maternal toxicity rather than a direct effect of treatment on fetal growth and development
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 1000 mg/kg bw/day, group mean fetal weights were marginally but statistically significantly lower than controls (p<0.001 for males, p<0.01 for females and p<0.001 for combined) resulting in statistically significantly lower litter weight (p<0.01).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was considered to be no detrimental effect of maternal treatment on litter data as assessed by live litter size.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was considered to be no detrimental effect of maternal treatment on litter data as assessed by sex ratio.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, group mean fetal weights were marginally but statistically significantly lower than controls (p<0.001 for males, p<0.01 for females and p<0.001 for combined) resulting in statistically significantly lower litter weight (p<0.01).
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, the number of small fetuses was slightly higher than controls, however, the mean percentage affected remained within the historical control data ranges and as such this finding was considered to be of no toxicological significance. Of other external findings, neither the type, incidence nor distribution indicated any obvious effect of maternal treatment on fetal development at any dose level
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal Examination of fetuses/litters revealed a number of statistically significant anomalies in relation to controls (p<0.01). At 1000 mg/kg bw/day, these included incomplete ossification of occipital (supra-occipital) region (p<0.01), thoracic centrum (p<0.01) and sternbrae (p<0.05), no ossification of metacarpal (p<0.05), and caudal vertebrae - less than 4 ossified (p<0.001); group mean values for these anomalies were above the historical control data ranges and as such these intergroup differences were deemed to be treatment-related. These resulted in a higher percentage of the fetuses affected for the 1000 mg/kg bw/day dose group (p<0.01).

Fetuses/litters from 175 mg/kg bw/day dose group also showed statistically significantly higher incomplete ossification of thoracic centrum (p<0.05) although group mean value remained within the background data ranges and as such this observation was considered to be within normal biological variation.

Additionally, the skeletal anomaly of thoracic centrum – dumb bell shaped was statistically significantly higher for fetuses/litters from the 175 and 1000 mg/kg bw/day dose group when compared with controls (p<0.01); a dose-relationship was apparent but group mean values were within the background data ranges and as such these observations were considered to be incidental. Other statistically significant intergroup differences included interparietal (unossified areas) for the 30 and 175 mg/kg bw/day dose groups (p<0.05), incomplete ossification of hyoid and ossification of ventral arch for the 1000 mg/kg bw/day dose group (p<0.01) but corresponding mean values were lower than controls and these findings were considered to be incidental. Any other intergroup differences did not achieve statistical significance and group mean values were usually within the background data ranges.
Visceral malformations:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus from a control litter appeared to be a conjoined twin showing a number of malformations including additional tail and forelimb, malpositioned and bulging eyes, open eye macroglossia, malpositioned pinnae, encephalocoele and full body oedema; these findings were deemed incidental.
Key result
Dose descriptor:
NOEL
Effect level:
<= 175 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Dose descriptor:
NOAEL
Effect level:
<= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: forelimb
skeletal: sternum
skeletal: vertebra
Description (incidence and severity):
Skeletal Examination of fetuses/litters revealed a number of statistically significant anomalies in relation to controls (p<0.01). At 1000 mg/kg bw/day, these included incomplete ossification of occipital (supra-occipital) region (p<0.01), thoracic centrum (p<0.01) and sternbrae (p<0.05), no ossification of metacarpal (p<0.05), and caudal vertebrae - less than 4 ossified (p<0.001); group mean values for these anomalies were above the historical control data ranges and as such these intergroup differences were deemed to be treatment-related. These resulted in a higher percentage of the fetuses affected for the 1000 mg/kg bw/day dose group (p<0.01).

Additionally, the skeletal anomaly of thoracic centrum – dumb bell shaped was statistically significantly higher for fetuses/litters from the 175 and 1000 mg/kg bw/day dose group when compared with controls (p<0.01); a dose-relationship was apparent but group mean values were within the background data ranges and as such these observations were considered to be incidental. Other statistically significant intergroup differences included interparietal (unossified areas) for the 30 and 175 mg/kg bw/day dose groups (p<0.05), incomplete ossification of hyoid and ossification of ventral arch for the 1000 mg/kg bw/day dose group (p<0.01) but corresponding mean values were lower than controls and these findings were considered to be incidental. Any other intergroup differences did not achieve statistical significance and group mean values were usually within the background data ranges.
Developmental effects observed:
yes
Lowest effective dose / conc.:
175 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Table1     Summary of Female Performance

Category

Number of Females at Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Initial Group Size

24

24

24

24

Pregnant

24

24

24

24

 


 

Table2     Summary Incidence of Daily Clinical Observations

Dose Level (mg/kg bw/day)

Number of Animals

Clinical Observations

0 (Control)

24

No Abnormalities Detected

30

24

No Abnormalities Detected

175

24

No Abnormalities Detected

1000

24

No Abnormalities Detected

 


 

Table 3     Group Mean Body Weight Values

Dose Level (mg/kg bw/day)

 

Body Weight (g) at Day of Gestation

3

5

6

7

8

11

14

17

20

0 (Control)

mean

232.8

243.8

248.9

253.8

257.3

275.7

295.1

326.0

367.4

sd

22.0

24.3

24.6

24.8

25.7

26.2

28.8

32.7

36.0

n

24

24

24

24

24

24

24

24

24

30

mean

235.2

247.9

250.8

255.9

261.0

279.3

299.0

328.3

373.0

sd

19.2

22.3

20.7

21.4

20.7

21.5

22.8

26.6

27.7

n

24

24

24

24

24

24

24

24

24

175

mean

235.4

249.9

251.5

257.5

263.5

281.5

301.7

331.5

375.5

sd

16.9

17.6

18.8

17.4

17.4

19.6

20.8

23.3

24.5

n

24

24

24

24

24

24

24

24

24

1000

mean

234.0

248.8

243.7

245.3

252.8

270.4

287.9

305.8*

346.4*

sd

21.0

20.9

20.0

18.9

20.1

23.5

24.1

25.9

30.5

n

24

24

24

24

24

24

24

24

24

 


 

Table 4     Group Mean Body Weight Change Values

Dose Level (mg/kg bw/day)

 

Body Weight Change (g) during Days of Gestation

3 to 5

5 to 6

6 to 7

7 to 8

8 to 11

11 to 14

14 to 17

17 to 20

0 (Control)

mean

11.0

5.1

5.0

3.5

18.4

19.4

30.9

41.4

sd

7.6

5.3

2.7

3.8

4.2

3.7

6.1

6.0

n

24

24

24

24

24

24

24

24

30

mean

12.7

2.9

5.1

5.1

18.3

19.7

29.3

44.8

sd

6.6

5.7

4.2

4.8

3.0

3.5

6.8

5.8

n

24

24

24

24

24

24

24

24

175

mean

14.5

1.6

6.0

6.0

18.0

20.1

29.8

44.0

sd

5.2

6.8

5.6

5.1

5.7

3.6

4.1

4.9

n

24

24

24

24

24

24

24

24

1000

mean

14.8

-5.1***

1.6*

7.5

17.6

17.5

17.9***

40.6

sd

5.2

4.9

4.7

5.9

7.6

6.0

6.4

11.3

n

24

24

24

24

24

24

24

24

 

Dose Level (mg/kg bw/day)

 

Cumulative Body Weight Change (g) from Day 5 of Gestation

6

7

8

11

14

17

20

0 (Control)

mean

5.1

10.0

13.5

31.9

51.3

82.2

123.6

sd

5.3

6.3

7.4

7.9

9.5

13.2

16.5

n

24

24

24

24

24

24

24

30

mean

2.9

8.0

13.1

31.4

51.1

80.4

125.1

sd

5.7

6.3

6.1

6.4

7.8

11.6

14.2

n

24

24

24

24

24

24

24

175

mean

1.6

7.6

13.6

31.6

51.8

81.6

125.6

sd

6.8

5.1

5.6

7.7

9.3

11.0

13.5

n

24

24

24

24

24

24

24

1000

mean

-5.1***

-3.5***

4.0***

21.6***

39.1***

57.0***

97.6***

sd

4.9

6.6

7.1

10.7

12.3

15.9

20.6

n

24

24

24

24

24

24

24


 

Table 5     Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

Dose Level (mg/kg bw/day)

 

Body Weight (g) on Days of Gestation

Body Weight Change (g) during Days of Gestation

Gravid Uterus Weight
(g)

Adjusted
Body Weight (g)
 Day 20

Adjusted
Body Weight Change (g)
5-20

5

20

5-20

0 (Control)

mean

243.8

367.4

123.6

83.113

284.3

40.5

sd

24.3

36.0

16.5

11.341

29.7

12.5

n

24

24

24

24

24

24

30

mean

247.9

373.0

125.1

84.197

288.8

40.9

sd

22.3

27.7

14.2

9.787

27.1

12.4

n

24

24

24

24

24

24

175

mean

249.9

375.5

125.6

83.782

291.8

41.8

sd

17.6

24.5

13.5

6.764

22.8

12.3

n

24

24

24

24

24

24

1000

mean

248.8

346.4

97.6

73.972**

272.4

23.7***

sd

20.9

30.5

20.6

11.928

21.6

13.9

n

24

24

24

24

24

24

 


 

Table 6     Group Mean Food Consumption Values

Dose Level (mg/kg bw/day)

 

Food Consumption (g/rat/day) between Days of Gestation

3 - 5

5 - 8

8 - 11

11 - 14

14 - 17

17 - 20

0 (Control)

mean

21.3

21.7

23.5

24.3

23.5

25.9

sd

2.8

2.8

2.8

2.5

3.0

3.2

n

24

24

24

24

24

24

30

mean

22.1

22.1

24.1

24.6

23.1

26.4

sd

3.7

1.8

2.0

2.3

2.5

2.8

n

24

24

24

24

24

24

175

mean

22.1

21.8

23.4

24.7

23.6

26.9

sd

2.9

2.6

3.5

2.8

2.9

3.2

n

24

24

24

24

24

24

1000

mean

22.5

13.1***

19.8***

21.7**

18.8***

25.1

sd

2.6

2.7

3.5

3.2

3.0

4.5

n

24

24

24

24

24

24

 


 

Table 7     Summary Incidence of Necropsy Findings

 

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

TERMINAL DEATH

 

 

 

 

Number of animals examined

24

24

24

24

Uterus: filled with yellow color fluid

0

0

0

1

No abnormalities detected

24

24

24

23

 

 

 


Table 8     Group Mean Litter DataValues

Dose Level (mg/kg bw/day)

 

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Implantation Loss
%

Number of Live Implants

%
Male
Fetuses

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

Mean Fetal Weight (g)

MeanPlacentalWeight
(g)

Litter Weight (g)

TotalPlacentalWeight
(g)

Early

Late

Total

Pre

Post

Male

Female

Total

0 (Control)

mean

13.8

13.5

0.0

0.1

0.2

2.1

1.1

6.9

6.5

13.4

52.0

4.062

3.881

3.982

0.581

53.128

7.717

sd

2.1

2.0

0.2

0.3

0.4

3.8

2.5

2.2

2.4

1.9

15.0

0.224

0.212

0.208

0.064

7.286

1.128

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

30

mean

14.1

13.8

0.3

0.0

0.3

2.8

2.4

6.8

6.6

13.4

50.7

4.187

4.008

4.095

0.582

54.687

7.798

sd

1.4

1.8

0.6

0.2

0.6

6.7

4.1

2.1

2.1

1.8

14.7

0.316

0.344

0.314

0.071

6.547

1.341

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

175

mean

14.0

13.7

0.2

0.1

0.3

1.7

2.1

6.6

6.8

13.5

49.7

4.113

3.899

4.014

0.554

53.839

7.415

sd

1.5

1.3

0.5

0.4

0.6

3.9

4.2

1.6

2.1

1.1

13.7

0.212

0.222

0.196

0.069

5.135

0.964

n

23

23

24

24

24

23

23

24

24

24

24

24

24

24

24

24

24

1000

mean

13.6

13.3

0.1

0.4

0.5

2.1

4.1

7.0

5.7

12.8

55.5

3.744***

3.602**

3.683***

0.544

46.796**

6.887*

sd

2.2

2.1

0.3

0.9

0.9

4.2

7.1

2.1

2.1

2.2

13.7

0.289

0.321

0.294

0.065

8.099

1.207

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

 


 

Table 9     Summary Incidence of Fetal External Findings

External Findings

Dose level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of fetuses (litters) examined

321 (24)

322 (24)

323 (24)

306 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Total Number Affected

3

3

1.1

4

4

1.2

3

2

0.9

6

6

1.9

Additional Tail

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Additional Forelimb

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Eyes malpositioned

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Full Body Odema

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Open Eye Macroglossia

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Encephalocoele

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Malpositioned Pinnae

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Bulging Eyes

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Small

1

1

0.3

1

1

0.3

2

1

0.6

5

5

1.6

Hematoma - Left Hind Limb

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Hematoma - Above Tail

0

0

0.0

1

1

0.3

0

0

0.0

0

0

0.0

Thread Like Tail

0

0

0.0

1

1

0.3

0

0

0.0

0

0

0.0

Hematoma - Right Side of Head

0

0

0.0

1

1

0.3

0

0

0.0

0

0

0.0

Hematoma - Back

0

0

0.0

0

0

0.0

1

1

0.3

1

1

0.3

Hematoma - Top of Head

0

0

0.0

0

0

0.0

1

1

0.3

0

0

0.0

Pale

0

0

0.0

0

0

0.0

1

1

0.3

0

0

0.0

 


 

Table 10   Summary Incidence of Fetal Visceral Findings

Visceral Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

167 (24)

167 (24)

168 (24)

158 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

External/General

 

 

 

 

 

 

 

 

 

 

 

 

Hemorrhage

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Head

 

 

 

 

 

 

 

 

 

 

 

 

Tongue - short

0

0

0.0

2

2

1.1

0

0

0.0

0

0

0.0

Rugae - non-uniform patterning

15

13

8.9

15

9

10.0

9

7

5.2

6

6

3.8

Micropthalmia

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Anopthalmia

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Brain - lateral ventricle - enlarged

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Brain - third ventricle - enlarged

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Abdomen

 

 

 

 

 

 

 

 

 

 

 

 

Liver - additional lobe between right and left median

0

0

0.0

0

0

0.0

2

2

1.0

0

0

0.0

Liver - papillary process - reduced in size

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Spleen - reduced in size

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Umbilical artery - left-sided

1

1

0.7

1

1

0.6

0

0

0.0

2

2

1.2

Testis - partially undescended

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

Ureter - kinked

34

14

20.7

29

18

18.2

15

8

9.4

19

9

11.5

Ureter - dilated

30

12

18.4

20

13

12.9

15

7

9.2

12

8

7.2

Renal pelvis cavitation - increased

13

8

7.9

20

10

12.1

8

6

5.0

15

6

9.2

Renal papilla - absent

1

1

0.6

3

3

1.8

2

2

1.1

1

1

0.6

l


 

Table10(continued)           Summary Incidence of Fetal Visceral Findings

Visceral Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

167 (24)

167 (24)

168 (24)

158 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Thorax

 

 

 

 

 

 

 

 

 

 

 

 

Thymus - lobe partially undescended

4

4

2.3

2

2

1.1

10

8

6.1

11

9

7.1

Lungs - irregular surface throughout

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Right subclavian artery - retro-oesophageal

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Atrium - enlarged

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Ventricular septal defect

1

1

0.7

0

0

0.0

1

1

0.6

0

0

0.0

Total

54

22

32.6

50

22

31.0

37

19

22.3

41

17

25.5

 


 

Table 11   Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

154 (24)

155 (24)

155 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

 

 

 

 

 

 

 

 

 

 

 

 

Nasal - incomplete ossification

14

8

9.2

7

6

4.6

12

6

7.6

21

11

13.8

Frontal - incomplete ossification

3

3

1.7

3

3

2.0

1

1

0.7

3

3

1.8

Frontal - unossified area

4

2

2.9

0

0

0.0

3

3

1.9

0

0

0.0

Frontal - misshapen

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Intraorbital process of frontal - incomplete ossification

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Parietal - incomplete ossification

0

0

0.0

1

1

0.7

0

0

0.0

1

1

0.6

Parietal - unossified area(s)

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

Parietal - absent

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Interparietal - incomplete ossification

7

6

4.2

18

9

11.7

12

8

7.9

2

2

1.6

Interparietal - unossified area(s)

5

5

3.6

0

0

0.0*

0

0

0.0*

1

1

0.5

Interparietal - absent

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Occipital (Supra-occipital) - incomplete ossification

7

5

4.1

17

10

10.5

6

6

4.1

35

14

23.3**

Occipital (Supra-occipital) - unossified area(s)

7

5

4.3

15

10

9.7

6

5

3.6

13

10

8.1

Occipital - absent

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Squamosal - incomplete ossification

6

5

3.5

14

7

8.6

7

7

4.5

8

5

5.2

a


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

154 (24)

155 (24)

155 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Squamosal - unossified area(s)

7

5

5.6

3

3

2.0

1

1

0.7

5

5

3.5

Squamosal - misshapen

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Jugal - incomplete ossification

1

1

0.5

4

3

2.6

2

2

1.4

0

0

0.0

Zygomatic process of maxilla - incomplete ossification

6

4

3.8

6

4

3.9

4

2

2.6

0

0

0.0

Zygomatic process of squamosal - incomplete ossification

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Premaxilla - incomplete ossification

2

2

1.1

2

1

1.4

0

0

0.0

0

0

0.0

Hyoid - incomplete ossification

18

12

11.1

14

8

9.3

6

5

3.8

1

1

0.6**

Hyoid - not ossified

7

6

4.6

3

3

2.1

4

4

2.5

0

0

0.0

Presphenoid - incomplete ossification

0

0

0.0

0

0

0.0

2

2

1.4

0

0

0.0

Presphenoid - misshapen

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Basisphenoid - incomplete ossification

1

1

0.6

3

3

1.8

0

0

0.0

1

1

0.7

Basisphenoid - misshapen

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

a


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

154 (24)

155 (24)

155 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Vertebral Column

 

 

 

 

 

 

 

 

 

 

 

 

Odontoid - ossification present

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Ventral arch of vertebra 1 - ossification present

27

17

18.0

44

12

28.9

34

16

22.0

11

5

6.7**

Cervical (neural) arch - incomplete ossification

5

4

2.9

7

5

4.4

1

1

0.7

2

2

1.3

Thoracic centrum - incomplete ossification

0

0

0.0

2

2

1.2

4

4

2.6*

12

7

7.8**

Thoracic centrum - not ossified

1

1

0.6

1

1

0.6

0

0

0.0

1

1

0.7

Thoracic centrum - bipartite ossification

0

0

0.0

0

0

0.0

2

2

1.4

2

1

1.0

Thoracic centrum - dumb-bell-shaped

5

4

2.9

11

10

6.7

17

13

11.1**

22

12

14.3**

Thoracic centrum - asymmetrically ossified

1

1

0.6

0

0

0.0

3

3

1.9

4

4

2.5

Lumbar centrum - absent

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Lumbar centrum - bipartite ossification

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Lumbar centrum - dumb-bell-shaped

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

Lumbar vertebra - absent (arches and centrum)

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Sacral (neural) arch - incomplete ossification

6

6

3.7

12

7

7.5

7

5

4.5

4

3

2.4

Sacral (neural) arch - not ossified

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Sacral vertebrae - absent (arches and centrum)

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

a


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

154 (24)

155 (24)

155 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Vertebral Column (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Number of pre-sacral vertebrae = 25/27

0

0

0.0

2

2

1.3

0

0

0.0

2

2

1.2

Caudal vertebrae - less than 4 ossified

25

12

15.8

31

14

19.1

34

17

21.8

79

21

51.4***

Caudal vertebrae - all absent

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Conjoined twin fetuses

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Ribs

 

 

 

 

 

 

 

 

 

 

 

 

Ossification centre - associated with 7th cervical vertebra

1

1

0.7

0

0

0.0

0

0

0.0

1

1

0.7

Ossification centre - associated with 1st lumbar vertebra

4

1

2.4

3

2

1.8

1

1

0.8

7

5

4.7

One or more ribs - thickened

1

1

1.0

0

0

0.0

0

0

0.0

0

0

0.0

Rib - short

6

2

3.8

0

0

0.0

1

1

0.6

2

2

1.3

Rib - rudimentary

1

1

0.7

0

0

0.0

1

1

0.7

0

0

0.0

Costal cartilage - misaligned

4

4

2.9

3

3

1.7

2

2

1.3

4

4

3.4

Costal cartilage - not fused to sternebra

18

10

12.0

21

12

13.1

16

10

10.2

33

16

23.6

a


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

154 (24)

155 (24)

155 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Sternebrae

 

 

 

 

 

 

 

 

 

 

 

 

Sternebra - incomplete ossification

2

2

1.2

1

1

0.6

5

5

3.1

8

8

5.3*

Sternebra - not ossified

1

1

0.6

3

1

1.8

1

1

0.7

2

2

1.2

Sternebra - bipartite ossification

1

1

0.6

1

1

0.6

0

0

0.0

2

1

1.4

Sternebra - misaligned

2

2

1.1

0

0

0.0

4

4

2.6

9

6

6.2

Xiphoid cartilage - partially split

2

2

1.4

1

1

0.8

6

5

3.7

9

8

5.9

Pectoral Girdle

 

 

 

 

 

 

 

 

 

 

 

 

Scapula - misshapen

2

2

1.2

2

1

1.0

9

6

5.8

12

7

7.4

Pelvic Girdle

 

 

 

 

 

 

 

 

 

 

 

 

Ischium - incomplete ossification

0

0

0.0

1

1

0.6

1

1

0.7

0

0

0.0

Pubis - not ossified

0

0

0.0

0

0

0.0

1

1

0.7

0

0

0.0

Pubis - incomplete ossification

2

2

1.1

5

4

3.2

3

3

2.0

5

5

3.3

a


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

30

175

1000

Number of Fetuses (litters) Examined

154 (24)

155 (24)

155 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Forelimbs

 

 

 

 

 

 

 

 

 

 

 

 

Metacarpal - not ossified

23

11

14.2

42

14

26.2

20

12

13.1

52

18

34.7*

Metacarpal - incomplete ossification

0

0

0.0

2

2

1.4

2

2

1.4

0

0

0.0

Forepaw phalanges - 1 or more - ossified

26

9

17.3

33

11

21.0

36

11

22.0

30

12

20.7

Humerus - incomplete ossification

0

0

0.0

4

3

2.5

0

0

0.0

0

0

0.0

Humerus - hole

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Humerus - short

2

1

1.2

0

0

0.0

0

0

0.0

0

0

0.0

Hindlimbs

 

 

 

 

 

 

 

 

 

 

 

 

Metatarsal - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.6

5

2

3.0

Femur - incomplete ossification

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Total

117

24

75.9

126

24

80.5

122

24

78.5

136

24

91.2**

NOTE: a fetus may appear in more than one category

 

Number of dams with abortions, early deliveries, stillbirths, resorptions, and/or dead foetuses

 

Dose level

0 mg/kg (control)

30 mg/kg

175 mg/kg

1000 mg/kg

Abortions

0

0

0

0

Early deliveries

0

0

0

0

Still births

0

0

0

0

Resorptions/ dead fetuses

4/24

7/24

5/24

9/24

 

 

Pre-and post-implantation (fetus) loss, number and percent

 

Dose level

Number pre-implantation

Number post implantation

Percentage live offspring#

0 mg/kg (control)

332-325=7

325-321=4

13.4/13.5*100=99.26%

30 mg/kg

339-330=9

330-322=8

13.4/13.8*100=97.1%

175 mg/kg

322-316=6

316-309=7

13.5/13.7*100=98.54%

1000 mg/kg

326-319=7

319-306=13

12.8/13.3*100=96.24%

 

 

 

Conclusions:
The oral administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to pregnant rats by gavage during gestation Days 5 to 19, at dose levels of 30, 175 or 1000 mg/kg bw/day resulted in an adverse effect on body weight gain and food consumption at the high dose level. Although some improvement was evident, cumulative body weight gains for females treated with 1000 mg/kg bw/day remained lower than controls throughout the dosing period with body weight gain adjusted for the contribution of gravid uterus also being markedly lower than controls. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 175 mg/kg bw/day within the confines of this type of study.
At 1000 mg/kg bw/day, fetal and litter weights were marginally lower than control with skeletal examination identifying a number of treatment-related variants. These observations were, however, considered to be due to maternal toxicity which results in slight reduction in fetal growth rather than an indication of a direct effect of the test item on fetal growth and development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day whilst the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was deemed to be 175 mg/kg bw/day within the confines of this type of study.
Executive summary:

Introduction

The study was performed according to the study plan and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 30, 175 or 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil BP) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results…….

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no clinical signs for any of the animals throughout the study.

Body Weight

At all dose levels, females showed a dose-related reduction in group mean body weight gains over Days 5 to 6 of gestation with most animals from the 1000 mg/kg bw/day showing actual body weight losses. Subsequent recovery was evident for females receiving 30 or 175 mg/kg bw/day with periodic body weight gains for these animals remaining comparable with controls from Day 6 of gestation. Females treated with 1000 mg/kg bw/day also showed improvement in body weight performance following the initial affect such that body weight gains over Days 7 to 14 of gestation were comparable with controls; however, further reduction in body weight gain was observed over Days 14 to 17 of gestation resulting in lower cumulative body weight gains for these animals throughout the treatment period. The overall body weight gain for these females was approximately 21% lower than controls with body weight gain adjusted for gravid uterus contribution also being markedly lower than controls. The effect on body weight development for the 1000 mg/kg bw/day females was considered to be of toxicological significance.

Food Consumption

There was marked reduction in food consumption at the start of dosing for females given 1000 mg/kg bw/day. Gradual improvement was apparent, however, dietary intake for these animals remained statistically significantly lower than controls up to Day 17 of gestation. Taken together with the effect on body weight development at this dose level, this observation was considered to be of toxicological significance.

There was no effect of treatment on food intake at 30 or 175 mg/kg bw/day.

Water Consumption

Daily visual inspection of water bottles did not reveal any treatment-related intergroup differences.

Post Mortem Studies

There were no treatment-related macroscopic findings at terminal necropsy.

Litter Data and Litter Placental and Fetal Weights

Treatment with the test item at 30, 175 or 1000 mg/kg bw/day did not result in any treatment-related effects onin uterosurvival. Sex ratios were also similar across all dose groups including controls. At 1000 mg/kg bw/day, group mean fetal weights were marginally lower than controls which resulted in slightly lower litter weight for this dose group. These findings were deemed likely due to maternal toxicity rather than a direct effect of treatment on fetal growth and development. Group mean total placental weight for the dams treated with 1000 mg/kg bw/day was also slightly lower than controls which was also considered likely to be due to maternal toxicity.

Fetal Examination

For all dose groups, there were no statistically significant treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities. At 1000 mg/kg bw/day, a number of skeletal anomalies achieved statistical significance. These included incomplete ossification of occipital (supra-occipital) region, thoracic centrum and sternbrae, no ossification of metacarpal, and caudal vertebrae - less than 4 ossified. These observations are minor variants and although group mean values were outside the historical background data ranges, they were deemed likely due to a slightly slower fetal growth rate resulting from maternal toxicity rather than an indication of a direct effect of the test item on fetal development. Any other skeletal findings at all dose levels were considered to be within normal biological variation.

Conclusion

The oral administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to pregnant rats by gavage during gestation Days 5 to 19, at dose levels of 30, 175 or 1000 mg/kg bw/day resulted in an adverse effect on body weight gain and food consumption at the high dose level. Although some improvement was evident, cumulative body weight gains for females treated with 1000 mg/kg bw/day remained lower than controls throughout the dosing period with body weight gain adjusted for the contribution of gravid uterus also being markedly lower than controls. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 175 mg/kg bw/day within the confines of this type of study.

At 1000 mg/kg bw/day, fetal and litter weights were marginally lower than control with skeletal examination identifying a number of treatment-related variants. These observations were, however, considered to be due to maternal toxicity which results in slight reduction in fetal growth rather than an indication of a direct effect of the test item on fetal growth and development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day whilst the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was deemed to be 175 mg/kg bw/day within the confines of this type of study.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

In rats reduced sperm concentration and motility which was associated with an increase in sperm morphological abnormalities following an OECD408. In an OECD443 these effects were also observed together with inability to reproduce at 450 mg/kg bw/d. Based on these results the substance is classified as toxic to reproduction category1b(f).