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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals : Ten weeks premating exposure duration was requested due to lack of data supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment R.7a, chapter R.7.6 (version 6.0, July 2017).
- Basis for dose level selection : The dose levels were selected based on the findings of a previously conducted study (OECD 422) with administration in feed.
- Inclusion/exclusion of extension of Cohort 1B: Cohort 1B was extended to produce an F2 based on findings on implantation sites during first pairing.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : developmental neurotoxicity Cohorts 2A and 2B were requested based on the concern on (developmental) neurotoxicity triggered by the findings observed in previously conducted in vivo studies (ptosis of eye lids, muscular hypotonia, stiff movements and tremor)
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : the previously conducted OECD 422 study showed effects on the thymus, therefore cohort 3 was included.
- Route of administration : the oral route is the most appropriate route of administration for substances to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 6.0, July 2017) R.7a, chapter R.7.6.2.3.2.)

Test material

1
Chemical structure
Reference substance name:
Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate
EC Number:
258-207-9
EC Name:
Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate
Cas Number:
52829-07-9
Molecular formula:
C28H52N2O4
IUPAC Name:
bis(2,2,6,6-tetramethylpiperidin-4-yl) sebacate
Details on test material:
Test item storage: At room temperature
Stability at higher temperatures: Stable, maximum temperature: 40°C
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 5 days at room temperature under normal laboratory light conditions (open container) was confirmed over the concentration range 500 to 15000 ppm. Stability for at least 10 days at room temperature under normal laboratory light conditions (open container) was confirmed over the concentration range 250 to 5000 ppm.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6 weeks
- Weight at study initiation: (P) between 138 and 174 g (males) and between 106 and 141 g (females)
- Fasting period before study: no
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm) with a maximum of 5 males/cage). Females were individually housed in Macrolon plastic cages (type III, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21). During locomotor activity monitoring, F1-Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- Diet:ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 45 to 74%. The values that were outside the targeted range occurred for 19 days with a maximum of 74% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
For preparation of the diets for Groups 2 to 4, the test item was mixed without the use of a vehicle, directly with the required amount of powder feed, i.e. standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Animals of Group 1 (control) received this standard powder rodent diet unprocessed and as received from the supplier. The diets prepared for animals of Groups 2 to 4 were stored in the freezer (≤-15°C) until use. On the day of use, it was provided to the animals in food hoppers, where it was kept at room temperature and under normal light conditions for a maximum of 5 days. Any remaining food left after filling the food hoppers might have been stored in the animal-room in a closed bag also for a maximum of 5 days, for supplementing food during the respective food consumption measurement interval. From study Week 8 onwards and based on the stability data obtained in this study, the diets provided in the food hoppers or stored in the animal room in closed bags were kept for a maximum of 10 days. The standard powder rodent diet for Group 1 animals was taken from the stock of the animal facility, which was stored as prescribed by the supplier.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: evidence of sperm in the vaginal lavage
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the diets of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). Small responses at the retention time of the test item were observed in the chromatograms of the Group 1 diet prepared for use in Week 1, Week 4, Week 13, Week 24 and Week 32. It was considered not to derive from the diet since a similar response was obtained in the
analytical blanks.
Duration of treatment / exposure:
F0 Males: 11-13 weeks (including 10 weeks pre-mating)
F0 Females: 16-18 weeks (including 10 weeks pre-mating)
F0 Females which failed to deliver or had total litter loss: 13-14 weeks
F1 Cohort 1A : 10 weeks
F1 Cohort 1B Males: 11-13 weeks (including 11-12 weeks pre-mating)
F1 Cohort 1B Females: 16-18 weeks (including 11-12 weeks pre-mating)
F1 Cohort 2A: 7-8 weeks
F1 Cohort 2B: n/a
F1 Cohort 3: 5 weeks
F1 Females which failed to deliver: 14-16 weeks (including 11 weeks pre-mating)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm
Remarks:
As food intake is considerably higher in lactating females, dietary concentrations were lowered by 50% during the lactation period
Dose / conc.:
1 500 ppm
Remarks:
As food intake is considerably higher in lactating females, dietary concentrations were lowered by 50% during the lactation period
Dose / conc.:
5 000 ppm
Remarks:
As food intake is considerably higher in lactating females, dietary concentrations were lowered by 50% during the lactation period
No. of animals per sex per dose:
see below
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The dose levels in this study were selected based the results of a preliminary reproductive toxicity study with dietary exposure of the test item in rats and in an attempt to produce graded responses to the test item. In this study, disruption of the estrous cycle regularity and lower implantation sites were observed at 5000 ppm and higher (highest dose level used was 15000 ppm) and reduced body weight gain of pups of pups during lactation at 15000 ppm. In the preliminary study, parental toxicity was observed as dose-related increase in red blood cells in males (maximum increase of approximately 10% at 15000 ppm) and decreased white blood cell counts in treated females (approximately 30-40% decrease at all dose levels of 1500, 5000 and 15000 ppm, with no clear dose response). Furthermore, inflammatory changes in the heart were observed in a few males among all test item-treated groups. In a single male at 1500 and 15000 ppm this finding was accompanied by myofiber necrosis. As this finding is uncommon in Wistar (Han) rats at this age, further investigations was made in the current study to determine whether this finding was of toxicological significance for this compound. Reproduction and developmental toxicity observed in the preliminary study comprised lower the regularity of the estrous cycle was disrupted and slightly lower implantation sites in female rats at dose levels of 5000 ppm and higher. At 15000 ppm, lower litter sizes were observed, the pup growth was reduced during the lactation period and T4 levels in male and female pups (at age of approximately 2 weeks) were increased.

Examinations

Parental animals: Observations and examinations:
IN-LIFE OBSERVATIONS

F0 Parental Animals:

CAGE SIDE OBSERVATIONS
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily, beginning prior to first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables. Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

BODY WEIGHT
Animals were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 postcoitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was quantitatively measured twice weekly during the first 8 weeks of premating and weekly afterwards, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. Food spillage was estimated in all cages over the first five weeks of the study and over Week 10 by means of sieving the bedding material, including the enrichments, with a metal sieve (mesh-size 1 mm) each time the cage was cleaned. The sieved amount, assumed to be mainly powder diet remains, was weighed and recorded. In addition, food spillage was estimated at discretion of the Study Director, e.g. in case of food hopper incidents.

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

GENERAL REPRODUCTION DATA
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were 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 PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.


F1B Parental animals:

Body Weights – Cohort 1B
Mated females were weighed individually on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

Food Consumption – Cohort 1B
Food consumption was not determined for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was quantitatively measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14, 17 and 21.

Estrous Cycle Determination – Cohort 1B
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed from start of the mating period until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

Cohabitation/Mating Procedure – Cohort 1B
Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating after at least 10 weeks of treatment. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males. For two couples (Male No. 321 and Female No. 666 - Male No. 596 and Female No. 916), detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, these couples were separated 2 and 3 days after the actual mating date, respectively. The actual mating date was designated Day 0 post-coitum.

General Reproduction Data – Cohort 1B
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were 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 PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.


CLINICAL PATHOLOGY

SAMPLE COLLECTION

F0-animals and Cohort 1A animals
Blood of 10 selected animals/sex/group5 of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anaesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals5 housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.

HEMATOLOGY F0 and F1A
Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the following parameters:
White blood cells (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC), (absolute) Red blood cells, Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets
A blood smear was prepared from each hematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated when required to confirm analyser results.

COAGULATION F0 and F1A
Blood samples at a target volume of 0.45 mL were collected into tubes containing citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY F0 and F1A
Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-heparin as anticoagulant. Samples at a target volume of 1.0 mL were collected in tubes without anticoagulant (same sample as for thyroid hormone measurement). Blood samples were processed for plasma or serum (bile acids), which was analyzed for the following parameters: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos)

THYROIDE HORMONE F0 and F1A
Blood samples at a target volume of 1.0 mL (same sample as for bile acid measurement) were collected into tubes without anticoagulant. Blood samples were processed for serum. Serum was used for measurement of both T4 and TSH.

URINALYSIS F0 and F1A
Urine samples were analyzed for the following parameters: Volume Specific gravity, Clarity, Colour, pH, Blood, White blood cells (WBC), Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite; Sediment: White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria, Other
Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. On the day of scheduled necropsy, a vaginal lavage was also taken.
Sperm parameters (parental animals):
For all surviving males, the following assessments were performed: Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples. One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
Litter observations:
IN-LIFE OBSERVATIONS

F1-Generation and F2-Generation until Weaning (PND 21)

Mortality/Moribundity Checks – F1 and F2-Generation
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F1 and F2-Generation
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check are given in the respective report tables.

Body Weights – F1 and F2-Generation
Live pups were weighed individually on PND 1, 4, 7, 13 and 21. For animals of Cohort 1A, 1B, 2A 2B and Surplus and F2-animals of Cohort 1B (10 selected litters/group; one male and one female), a terminal weight was recorded on the day of scheduled necropsy.

Sex – F1 and F2-Generation
Sex was externally determined for all pups on PND 1, 4 and 13.

Anogenital Distance – F1 and F2-Generation
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention – F1 and F2-Generation
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Culling – F1 and F2-Generation
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.


F1-Generation and F2-Generation from Weaning (PND 21) onwards

The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort 2B, Cohort Surplus and spare F1-animals as these were terminated on PND 22-24.

Mortality/Moribundity Checks – Cohorts 1A, 1B, 2A and 3
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

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

Arena Observations – Cohorts 1A, 1B, 2A and 3
Clinical observations were conducted in a standard arena once on day of weaning and at weekly intervals thereafter.

Body Weights – Cohorts 1A, 1B, 2A and 3
Animals were weekly weighed individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. In order to monitor the health status, Male Nos. 615, 618 and Female Nos. 930 and 958 (Group 4) were also weighed on Days 9, 10 and 11 of the treatment period. For animals of Cohorts 1A, 1B, and 2A, a terminal weight was recorded on the day of scheduled necropsy.

Food Consumption– Cohorts 1A, 1B, 2A and 3
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy. Food spillage was estimated in Cohort 1A animals over Week 5 of treatment of the F1-animals by means of sieving the bedding material, including the enrichments, with a metal sieve (mesh-size 1 mm) each time the cage is cleaned. The sieved amount, assumed to be mainly powder diet remains, was weighed and recorded. In addition, food spillage may be estimated at discretion of the study director, e.g. in case of food hopper incidents.

Water Consumption – Cohorts 1A, 1B, 2A and 3
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Vaginal Patency – Cohorts 1A, 1B, 2A and 3
Vaginal patency (vaginal opening) was monitored daily for all females from PND 24-25 onwards Ref. 1 until vaginal patency was present, by visual inspection of the vaginal area. Body weight was recorded on the day of acquisition of vaginal patency.

Balanopreputial Separation – Cohorts 1A, 1B, 2A and 3
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 34,35 or 36 onwards until balanopreputial separation was present, by visual inspection of the genital area.
Body weight was recorded on the day of acquisition of balanopreputial separation.

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

Estrous Cycle Determination – Cohort 1A Females
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods. During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day after onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88. The estrous cycle data of the first period is not reported.


CLINICAL PATHOLOGY

Sample Collection:

Culled pups of the F1-generation and F2-generation (PND 4) pups
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.

F1-animals of Cohort Surplus on PND 22-24
On PND 22-24, blood was collected from all Cohort Surplus animals (10/sex/group). Blood was drawn, between 8.00 and 11.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Samples were collected according to the following table. After collection, samples were transferred to the appropriate laboratory for processing.

Thyroid Hormone
Blood samples at a target volume of 0.5 mL (pooled PND 4 pups) and 1.0 mL (Cohort Surplus PND 22 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum.
Serum of Cohort Surplus animals (i.e. PND 22 pups) was used for measurement of both T4 and TSH. Pooled serum of culled PND 4 pups was used for measurement of T4 only. Any remaining sample was discarded.



ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

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

Functional Observation Battery – Cohort 2A
The Functional Observation Battery (FOB) tests were conducted once between PND 63-75 in the order of sequence indicated below and were divided between several days. The detailed clinical observations and locomotor activity were conducted in separate room(s) specially equipped for these purposes. The other FOB tests were conducted in the study room.
1. Detailed clinical observations: Detailed clinical observations consisted of a number of tests conducted in- and out-side the home cage as specified in Appendix 17. To the extent possible, testing of animals was counterbalanced across dose groups.
2. Rectal temperature: Rectal temperature was measured immediately after the detailed clinical observations.
3. Locomotor activity was tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions. To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
4. Hearing ability: Score 0 = normal/present, score 1 = abnormal/absent.
5. Pupillary reflex (both eyes).
6. Fore- and hindlimb grip strength. This was recorded per animal as the mean of three measurements, using a grip strength meter (Series M4-10, Mark-10 Corporation).
7. Landing (hind) foot splay. This was recorded per animal as the mean of three measurements


ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:

Immunization with SRBC – Cohort 3 and Positive Control Animals
All F1-animals of Cohort 3 and all positive control animals were immunized once, 5 days before scheduled necropsy (i.e. once between PND 52-55) via intravenous injection into the tail vein (approximately 1 mL/min) with 0.5 mL of the 4*108 SRBC/mL in sterile PBS formulation. Due to difficulties with the intravenous injection in the tail vein, Male No. 982 (positive control) received only 0.35 mL of the 4*108 SRBC/mL in sterile PBS formulation. The immunizations were given on approximately the same time of the day (within a time frame of 6 hours between the earliest and latest treated animal). After injection, the injection site was not marked with indelible ink). The positive control animals received the immunization 2-4 hours after having received the first cyclophosphamide treatment (see above).

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

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

Body Weights – Positive Control Animals
Animals were individually weighed once, on the first day of treatment.

Food and Water Consumption – Positive Control Animals
Food consumption was quantitatively measured weekly, from weaning onwards up to start treatment.

T-Cell Dependent Antibody Response (TDAR) Assay:

Sample Collection:
Blood of all Cohort 3 animals (10 animals/sex/group) and positive control animals (10 animals/sex), was collected on pre-immunization (PND 51-54) and 5 days after immunization (PND 57-60).
Animals were not deprived of food prior to sampling. Samples were collected, between 7.00 and 10.30 a.m., from the jugular vein in the animal facility. After collection, samples were transferred to the appropriate laboratory for processing. Time points for samples were as follows:
All Cohort 3 animals (10/sex/group): Preimmunization (PND 51-54), Day 5 after immunization (PND 57-60)
All positive control animals (10/sex): Preimmunization (PND 51-54), Day 5 after immunization (PND 57-60)

TDAR Assay Evaluation
Blood samples at a target volume of 0.3 mL were collected into tubes without anticoagulant. Blood samples were allowed to clot for at least 30 minutes and centrifuged within 2 hours after collection. Within 1 hour after centrifugation, serum of these samples were divided into 2 aliquots and subsequently stored in labeled polypropylene tubes at ≤-75C until analysis. The antibody response to the immunization with SRBC was determined by measuring the anti-SRBC IgM levels in serum using Rat Anti-SRBC IgM ELISA Kits (Life Diagnostics, Inc., West Chester, PA, USA) and an EL808™ Absorbance Microplate reader including Gen5 Secure software version 1.11.5 (BioTek Instruments, Inc., Winooski Vermont, USA) according to a validated method.
Postmortem examinations (parental animals):
TERMINAL PROCEDURES

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

Scheduled Euthanasia – F0-Generation
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies are summarized below:
- Males (which sired or failed to sire): After successful mating and a minimum of 10 weeks of treatment.
- Females which delivered: LD 23-25.
- Females which failed to deliver (Nos. 149, 151, 202, 218): With evidence of mating: Post-coitum Day 26-28.
- Female with total litter loss (No. 119): Within 24 hours after the last pup was found dead or missing.
Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.

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

Organ Weights and Tissue Collection/Preservation – F0-Generation
The organs specified (see table below) were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for Male No. 1 euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated. Representative samples of the tissues identified (see table below) were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

Necropsy – F1B-Generation
Scheduled necropsy of the F1-Generation of Cohort 1B was conducted on the following days:
- F1-Males (which sired or failed to sire): Following completion of the mating period.
- F1-Females which delivered: LD 21-23
- F1-Females which failed to deliver with evidence of mating (No. 940): Post-coitum Day 27.
- F1-Females which failed to deliver without evidence of mating (No. 854): 24 days after the last day of the mating period.
Due to the preterm death of one Cohort 1A female in Groups 2 and 4 each, only 24 females (instead of 25) were available for mating in these two groups. Consequently, Male Nos. 409 (Group 2) and 585 (Group 4) were
not used for mating. The F1-animals of Cohort 1B were not deprived of food overnight before necropsy. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.


HISTOLOGY
F0- and F1-Generation
Tissues mentioned in the table below from a selection of the F0- and F1-animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).


HISTOPATHOLOGY
F0- and F1-Generation
All tissues mentioned in the table below from a selection of the F0- and F1-animals were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):
TERMINAL PROCEDURES – F1- and F2-Generation until Weaning

Unscheduled Deaths– F1- and F2-Generation
Pups that were sacrificed in extremis, younger than 7 days, were euthanized by decapitation. Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally). Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Culled Pups (PND 4) – F1- and F2-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood was collected, if possible. Sex was determined both externally and internally (if possible). Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Scheduled Euthanasia – F2-Generation
Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23. The animals were not deprived of food overnight. From 10 selected litters/group, terminal body weight was determined for one male and one
female pup. Subsequently, these pups were deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table in Appendix 16 were weighed and collected. The selected litters were documented in the study files by the study director in advance. All remaining pups were sacrificed using Euthasol®20% by intraperitoneal (ip) injection.
Also these pups were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.


TERMINAL PROCEDURES – F1-Generation from Weaning Onwards

If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed. Spare F1-animals which were not assigned to one of the cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded. For all animals, necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Organ weights were not recorded for Female Nos. 754 and 930 euthanized in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated. The organs identified for weighing and representative samples of the tissues (see table below) were weighed and collected.

Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 89-93. Cohort 1A animals were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

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

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

Cohort 2A and 2B
Scheduled necropsy of Cohort 2A was conducted on PND 76-82. Scheduled necropsy of Cohort 2B was conducted on PND 21-22. The animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. The animals were first anaesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.). All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 6-7 days prior to removal from the skull (see deviation in Appendix 19). The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology7. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.

Cohort 3 and Positive Control Animals
Scheduled necropsy of Cohort 3 was conducted on PND 58-62. Positive control animals were euthanized on the same date(s). These animals were not deprived of food overnight before necropsy. The animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. On PND 22-24, blood samples (1.0 mL) were collected between 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 were collected into serum tubes for measurement of thyroidstimulating hormone (TSH) and thyroxine (T4).


HISTOLOGY
Tissues mentioned in the table below from a selection of the F1-animals identified in the Terminal Procedures table were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).

Cohort 1A
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Groups 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.

Cohort 2A and 2B
In addition to the procedures described above, the entire brain from all groups was processed to the block stage up front at the same time to avoid effects of fixation duration on morphometry. Furthermore, the time period between start of fixation in 10% buffered formalin and embedding in paraffin for brains of Cohort 2A and for brains of 2B animals was kept similar also to avoid effects of duration of fixation on morphometry. Sections of the brains of all Cohort 2A and 2B animals (all groups) were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet. In addition, an unstained section was also prepared from the homologous areas used for morphometric analysis, i.e. neocortical, hippocampal and cerebellar area, for the event that other stains might be needed to be applied in the future. For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections are obtained.


HISTOPATHOLOGY
F0- and F1-Generation
All tissues mentioned in the table below from a selection of the F1-animals were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist.

Cohort 2A and 2B animals
Morphometric (quantitative) analyses of CNS tissues was performed for Cohort 2A and 2B animals of Groups 1 and 4.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group 5 vs. Group 1

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating index males (%), Mating index females (%), Precoital time, fertility index males (%), fertility index females (%), gestation index (%), duration of gestation, Post-implantation survival index (%), Live birth index (%), Percentage live males at First Litter Check (%), Percentage live females at First Litter Check (%), Viability index (%), Weaning index (%), Percentage live males at weaning (%), Percentage live females at weaning (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations up to 5000 ppm. Incidental findings that were noted included scabs, scales, wounds, alopecia, black discoloration and discharge of the eye, exophthalmos, and piloerection. 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. At the incidence observed, these were considered not to be signs of toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No test item-related mortality occurred during the study period up to 5000 ppm. One control male was (No. 01) sacrificed moribund for humane reasons (exophthalmos of the right eye).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated animals were affected by treatment with the test item at all tested diet concentrations.
Males:
In males at 500 ppm, body weights progressively decreased from 0.95x (Week 5) to 0.92x (Week 13) of control and body weight gain was decreased to 0.76-0.85x of control from Week 2 until 13, resulting in a terminal body weight of 0.92x of control. At 1500 ppm, body weights progressively decreased from 0.96x (Week 4) to 0.92x (Week 13) of control and body weight gain was decreased to 0.86-0.91x of control from Week 2 until 13, resulting in a terminal body weight of 0.92x of control. At 5000 ppm, body weights progressively decreased from 0.93x (Week 2) to 0.88x (Week 13) of control and body weight gain was decreased to 0.71-0.81x of control from Week 2 until 13 of treatment, resulting in a terminal body weight of 0.87x of control.

Females:
In females at 500 ppm, body weights and body weight gain were decreased compared with control to 0.95x and to 0.85-0.92x of control, respectively, during Week 3, 4 and 7 of the premating period. At the end of the post-coitum period, body weights were 0.95x of control. At 1500 ppm, body weights and body weight gain were lower than control from Week 2 of the pre-mating period onwards. Mean body weights were 0.93-0.96x of control during Week 2-9 and 11 and body weight gain 0.76-0.92x of control during Week 2-11. During the post-coitum and lactation period, body weights were decreased to 0.92-0.93x and 0.94-0.96x of control, respectively, resulting in a terminal body weight of 0.94x of control. No statistically significantly differences were observed in body weight gains. At 5000 ppm, body weights and body weight gain were lower than controls from Week 2 and 3 of the pre-mating period onwards, respectively. Mean body weights were decreased to 0.92-0.96x of control during Week 2-9 and 11 and body weight gain 0.83-0.90x of control during Week 3-9 and 11. The decreased body weights and body weight gains persisted throughout the post-coitum and the lactation period. Body weights were decreased up to 0.88x of control at the end of the post-coitum period and were decreased to 0.90x of control at the start of the lactation period up to 0.96x of control at the end of the lactation period. Body weight gains were lower than control (0.56-0.86x) during the post-coitum period and were increased during the lactation period reaching 1.70x of control at lactation Day 21, resulting in a terminal body weight of 0.93x of control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight were affected by treatment with the test item from 1500 ppm.

Males:
In males at 1500 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.89x of control). Absolute food consumption recovered to normal levels in the following weeks, but relative food consumption was increased on several occasions (Week 2, 7-8 and 9-11, 1.07-1.09x of control). At 5000 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.78x of control). While absolute food consumption levels were normal in the following weeks, relative food consumption was increased until Week 11 of treatment (1.05- 1.15x of control).

Females:
In females at 1500 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.86x and 0.89x of control, respectively). While absolute food consumption recovered to normal levels in the following weeks, relative food consumption was increased on several occasions during the pre-mating period (Week 2, 3-4 and 8-10, 1.11-1.14x). During the post-coitum period, absolute food consumption was decreased during Days 0-11 to 0.84-0.88x of control while relative food consumption was only decreased during Days 7-11 to 0.88x of control. At 5000 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.86x and 0.88x of control, respectively). During the following weeks, absolute food consumption recovered to normal levels until the end of the pre-mating period (0.93x of control), while relative food consumption was increased during most weeks of the pre-mating period (Week 2, 3-6 and 7-10, 1.09-1.15x of control) but was decreased at the end of the premating period (0.91x of control). During the post-coitum period, absolute (Days 0-11 and 17- 20) and relative (Days 0-11) food consumption levels were decreased to 0.79-0.86x and 0.87- 0.89x of control, respectively. Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment. In addition, changes observed in males during Week 4 and in females during lactation Days 1-4 were considered to be the result from slightly higher control.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were observed in hematological parameters up to 5000 ppm. The statistically significant increase in mean corpuscular haemoglobin concentration (MCHC) in females at 500 ppm was considered unrelated to treatment as there was no dose related trend. Note: For the female control group, only 4 out of 10 samples were available due to clotting of the remaining samples. This somewhat lower number of samples did not affect the evaluation of the results since all data were within the historical control range.
Coagulation parameters of treated rats were unaffected by treatment with the test item up to 5000 ppm.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were not affected by treatment with the test item up to 1500 ppm.
In males at 5000 ppm, a statistically significant increase in total bilirubin (1.19x of control) and bile acid (2.14x) concentrations were observed. Mean values remained within the historical control range. In females at 5000 ppm, a statistically significant increase in alanine aminotransferase activity (ALAT; 1.75x of control) was observed. Mean values exceeded the historical control range. In females, potassium concentrations were increased at 500, 1500 and 5000 ppm. As all values were within the historical control range and in absence of a dose response, these changes were considered unrelated to treatment. The statistically significant decrease in albumin concentration was considered to be unrelated to treatment with the test item as it occurred in the absence of a dose-related trend.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone analyses:
Serum T4 levels were lower in males (0.82x of control) and females (0.66x) at 5000 ppm. Mean values at 5000 ppm remained within the historical control range for both males and
females. In males, this difference could be contributed to the relatively high serum T4 levels in concurrent controls compared to the historical control mean. Mean T4 levels at 5000 ppm were similar to historical control mean and therefore, the observed difference was considered unrelated to treatment. For females, similarly, a relatively high control mean compared to historical control mean was observed, partly contributing to the observed lower T4 values at 5000 ppm. However, as the mean T4 levels at 5000 ppm were also slightly decreased compared to historical control mean (0.84x), a treatment related effect could not be excluded. However, as values remained within the historical control range12, this potential effect was considered non-adverse. The statistically significantly decreased TSH serum levels in males at 1500 ppm, were considered unrelated to treatment with the test item as no dose response was observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters of treated rats were unaffected by treatment with the test item up to 5000 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment with the test item up to 5000 ppm. Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for Female No. 201 and for Female No. 206 no cycle classification could be determined (both with normal litter at 5000 ppm). 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, non-treatment-related
Description (incidence and severity):
Sperm motility and morphology were unaffected by treatment up to 5000 ppm. Sperm count of the epididymides was statistically significantly lower at 1500 and 5000 ppm, 0.86x of controls in both groups. No dose response was observed and values remained within historical control data. Sperm counts at 500 ppm were within the same range as concurrent controls.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- Mating Index - F0-Generation
Mating index was unaffected by treatment with the test item up to 5000 ppm. All females showed evidence of mating.

- Precoital Time
Precoital time was unaffected by treatment up to 5000 ppm. Most females were mated within the first 4 days of the mating period. One female was mated after 5 days (500 ppm), one after 12 days (5000 ppm) and four after 13 days (one control female, one female at 500 ppm and two females at 1500 ppm).

- Number of Implantation Sites - F0-Generation
The mean number of implantation sites in all treatment groups was lower compared to concurrent controls, reaching statistical significance at 5000 ppm only. Number of implantation sites were 12.6, 11.5, 11.5 and 11.1 for the control, 500, 1500 and 5000 ppm groups, respectively. Notably, control mean was similar to the historical control mean (12.6 vs. 12.1, respectively) and 2, 4 and 3 females in the 500, 1500 and 5000 ppm groups, respectively, had less than 10 implantations, the lowest number of implantations in the concurrent controls.

- Fertility Index - F0-Generation
Fertility index was not affected by treatment with the test item up to 5000 ppm. The fertility indices were 100, 96, 100 and 93% for the control, 500, 1500 and 5000 ppm groups, respectively. One female at 500 ppm and two females at 5000 ppm were not pregnant. This incidence was within the normal range.

- Histopathological evaluation of reproductive performance - F0-Generation
There were 1/28 couples of the control group with total litter loss on postnatal Day 2, 1/28 couples at 500 ppm with implantation sites only, and 1/28 couples at 500 ppm with a female that was not pregnant and 2/28 couples at 5000 ppm that were not pregnant. No abnormalities were seen in the reproductive organs or mammary gland, which could explain the absence of pregnancy, or account for their lack of healthy offspring. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

- Gestation Index - F0-Generation
Gestation index and duration of gestation were unaffected by treatment with the test item up to 5000 ppm. Except for one female at 500 ppm, all pregnant females had live offspring. The gestation indices were 100, 96, 100 and 100% for the control, 500, 1500 and 5000 ppm groups, respectively.

- Parturition/Maternal Care - F0-Generation
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- Post-Implantation Survival Index - F0-Generation
The total number of offspring born compared to the total number of uterine implantations was unaffected by treatment with the test item up to 5000 ppm. Post-implantation survival index was 97, 92, 93 and 94% for the control, 500, 1500 and 5000 ppm groups, respectively. For one female of the control (No. 127), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 25 days of lactation. No toxicological relevance was attached to this finding in the current study.

- Litter Size - F0-Generation
The mean live litter sizes in all treatment groups was lower compared to concurrent controls, reaching statistical significance at 1500 and 5000 ppm. Mean litter size was 12.1, 11.0, 10.7 and 10.4 living pups per litter for the control, 500, 1500 and 5000 ppm groups, respectively). Historical control mean is 11.3 for this parameter15 and 3 females in each treatment groups had less than 9 live pups, the lowest number of pups in the concurrent controls.

- Sex Ratio – F1-pups
Sex ratio was not affected by treatment with the test item up to 5000 ppm.

- Live Birth Index - F0-Generation
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item up to 5000 ppm. The live birth indices were 99, 100, 100 and 99% for the control, 500, 1500 and 5000 ppm groups, respectively. Three pups (Litter Nos. 119, 128 and 133) of the control group, one pup (Litter No. 193) at 1500 ppm and two pups (Litter Nos. 197 and 204) at 5000 ppm were found dead at first litter check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

- Viability Index - F0-Generation
The number of live offspring on Day 4 before culling compared with the number of offspring on Day 1 (viability index) was unaffected by treatment with the test item up to 5000 ppm. Viability indices were 96, 99, 100 and 100% for the control, 500, 1500 and 5000 ppm groups, respectively. Eleven pups (nine pups of Litter No. 119 and one of Litter Nos. 118 and 131 each) were missing and one pup was killed in extremis (i.e the last remaining pup of Litter No. 119 resulting in a total litter loss) of the control group on PND 1 or 2. At 500 ppm, two pups (Litter Nos. 142 and 150) were missing on PND 2 and one pup (Litter No. 159) was killed in extremis on PND 3. At 1500 ppm, one pup (Litter No. 192) was missing on PND 3. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. Statistical significance observed at 1500 and 5000 ppm for postnatal loss was the result of the relatively high postnatal loss in the controls, mainly Litter No. 119).

- Weaning Index - F0-Generation
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was unaffected by treatment with the test item up to 5000 ppm. The weaning indices were 100% for the control, 500, 1500 ppm groups, respectively, and 99% at 5000 ppm. One pup (Litter No. 135) of the control group and three pups (Litter Nos. 204, 219 and 220) at 5000 ppm were found dead or missing on PND 10, 12 or 19. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General Toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: corresponding to 36 mg/kg bw in males and 41 mg/kg bw in females
Dose descriptor:
NOAEL
Remarks:
Reproduction Toxicity
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased number of implantation sites
Remarks on result:
other: corresponding to 109 mg/kg bw in males and 126 mg/kg bw in females

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
F1-Generation (Cohort 1A)
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 5000 ppm. Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for three females of the control group (Nos. 643, 645 and 652), six females at 500 ppm (Nos. 727, 728, 729, 731, 737 and 741), seven females at 1500 ppm (Nos. 811, 814, 815, 818, 820, 823 and 830) and for four females at 5000 ppm (Nos. 896, 902, 907 and 915). One female at 500 ppm (No. 745) had an extended estrus and for two females at 500 ppm (Nos. 740 and 742), one female at 1500 ppm (No. 829) and one female at 5000 ppm (No. 913) the cycle classification could not be determined. In absence of a dose-related incidence and of any apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
F1-Generation (Cohort 1A)
Sperm motility, concentration and morphology were considered not affected by treatment with the test item. The statistically significantly higher spermcount in the epididymides at 5000 ppm was considered not to be of toxicological relevance as the opposite effect (i.e. a decrease) would be expected in case of target organ toxicity.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reproduction Data – F1-Generation (Cohort 1B)

- Mating Index – F1-Generation
Mating index was unaffected by treatment with the test item up to 5000 ppm. The mating indices were 100, 100, 96 and 100% for both males and females in the control, 500, 1500 and 5000 ppm groups, respectively. At 1500 ppm, one male and one female were
not mated.

- Precoital Time – F1-Generation
Precoital time was unaffected by treatment up to 5000 ppm. All females were mated within the first 4 days of the mating period.

- Number of Implantation Sites – F1-Generation
The mean number of implantation sites at 1500 and 5000 ppm was lower compared to concurrent controls, reaching statistical significance at 5000 ppm only. Number of implantation sites were 12.4, 12.5, 11.9 and 10.1 for the control, 500, 1500 and 5000 ppm groups, respectively. Control mean was similar to the historical control mean (12.4 vs. 12.5, respectively) and 1 and 3 females in the 500, 1500 and 5000 ppm groups, respectively, had less than 9 implantations, the lowest number of implantations in the concurrent controls.

- Fertility Index – F1-Generation
Fertility index was not affected by treatment with the test item up to 5000 ppm. The fertility indices were 100, 100, 100 and 96% for both males and females in the control, 500, 1500 and 5000 ppm groups, respectively. One female at 5000 ppm was not pregnant and as this was a single incidence of non-pregnancy, this was considered not to be related to treatment with the test item.

- Histopathological evaluation of reproductive performance – F1-Generation
There was one male at 500 ppm and one male at 5000 ppm that were not used for mating. There were 1/25 couples at 1500 ppm that had not mated and 1/24 females at 5000 ppm that was not pregnant. No abnormalities were seen in the reproductive organs, which could explain the absence of pregnancy, or account for their lack of offspring.


Developmental Data – F1-Generation

- Gestation Index and Duration – F1-Generation
Gestation index and duration of gestation were unaffected by treatment with the test item up to 5000 ppm. All pregant females had live pups.

- Parturation/Maternal Care – F1-Generation
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- Post-Implantation Survival Index – F1-Generation
The total number of offspring born compared to the total number of uterine implantations was unaffected by treatment with the test item up to 5000 ppm. Post-implantation survival index was 94, 93, 93 and 90% for the control, 500, 1500 and 5000 ppm groups, respectively. For one control female (No. 675) and one female at 500 ppm (No. 750), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 21 days of lactation. No toxicological relevance was attributed to this finding in the current study.

- Litter Size – F1-Generation
The mean live litter sizes at 1500 and 5000 ppm was lower compared to concurrent controls, reaching statistical significant at 5000 ppm only . Mean litter size was 11.7, 11.5, 10.8 and 9.0 living pups per litter for the control, 500, 1500 and 5000 ppm groups, respectively. Historical control mean is similar to control mean for this parameter26 and 1 and 7 females at 1500 and 5000 ppm, respectively, had less than 8 live pups, the lowest number of pups in the concurrent controls.

- Live Birth Index – F1-Generation
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item up to 5000 ppm. The live birth indices were 100, 100, 98 and 99% for the control, 500, 1500 and 5000 ppm groups, respectively. One pup (Litter No. 759) at 500 ppm, five pups (Litter Nos. 840 and 852) at 1500 ppm and two pups (Litter Nos. 916 and 923) at 5000 ppm were found dead at first litter check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

- Viability Index – F1-Generation
The number of live offspring on Day 4 before culling compared with the number of offspring on Day 1 (viability index) was unaffected by treatment with the test item up to 5000 ppm. Viability indices were 99% for the control, 500, 1500 and 5000 ppm groups. One pup in the control group (Litter No. 684) was found dead on PND 2 and had beginning autolysis and one pup at 1500 ppm (Litter No. 842) was sacrificed in extremis on PND 1 after it was fallen accidentally during animal handling. After the fall, the animal had a bump on its head and difficulties with breathing. Moreover, three control pups (Litter Nos. 662, 675 and 680), two pups at 500 ppm (Litter Nos. 751 and 760), one pup at 1500 ppm (Litter No. 852) and two pups at 5000 ppm (Litter Nos. 916 and 931) were found missing between PND 2 and 4. These pups were most likely cannibalized.
No toxicological relevance was attributed to these dead/missing pups as the mortality incidence did not show a dose-related trend and remained within the normal range of biological variation.

- Weaning Index – F1-Generation
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item up to 5000 ppm. Weaning indices were 100, 99, 100 and 99% for the control, 500, 1500 and 5000 ppm groups, respectively. One pup at 500 ppm (Litter No. 748) was found dead on PND 16 and one pup at 5000 ppm (Litter No. 919) went missing on PND 13 (most likely cannibalized). No toxicological relevance was attributed to the dead/missing pups as the mortality incidence did not show a dose-related trend and remained within the normal range of biological variation.

- Sex Ratio – F2-Pups
Sex ratio was not affected by treatment with the test item up to 5000 ppm. Sex ratios were 59:41, 51:49, 54:46 and 49:51 for the control, 500, 1500 and 5000 ppm groups, respectively. The statistically significant difference observed at 5000 ppm, was considered to be the result of a somewhat deviating control value and unrelated to treatment with the test item.

Effect levels (P1)

Dose descriptor:
NOAEL
Remarks:
Reproduction Toxicity
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased number of implantation sites
Remarks on result:
other: corresponding to 121-163 mg/kg bw in males and 130-165 mg/kg bw in females

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- Clinical Signs during Lactation – F1-Pups
No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 5000 ppm. For the pup of female No. 119 (control) that was killed in extremis, a wound on the back and on the left hindleg was noted on Day 1. The pup of female No. 150 (500 ppm) that was killed in extremis, was cold and dehydrated. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

- Clinical Observations – F1-Generation from Weaning onwards
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations up to 5000 ppm. Incidental findings that were noted included scabs, scales, alopecia, bent tail, erythema focal of the skin, dehydrated, ptosis, exophthalmos, lethargy, hunched posture, lean and piloerection. 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. At the incidence observed, these were considered not to be signs of toxicological relevance.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- Mortality – F1-Generation
No test item-related mortality occurred during the study period up to 5000 ppm. Two Cohort 1B females (Nos. 754 at 500 ppm and 930 at 5000 ppm) were sacrificed moribund for humane reasons. Female No. 754 was injured when closing the cage, all macroscopic and microscopic findings were in line with this accident. Female No. 930 was sacrificed on PND 29 as she had a hunched posture, piloerection, ptosis and was dehydrated and lean. Within one day, she had lost 9% of her body weight and at macroscopic examination she was emaciated. There were no microscopic findings in these animals that indicated a relation to the test item. Therefore, these deaths were considered incidental and not related to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Body Weights during Lactation – F1-Pups
Body weights of pups were affected by treatment with the test item from 500 ppm. At 5000 ppm, statistically significantly reduced pup body weights were observed on PND 14, which progressively decreased until PND 21. For males, weights decreased from 0.94x to 0.86x of control and for females, weights decreased from 0.93x to 0.85x. Male and female body weights combined decreased from 0.94x to 0.86x of control. At 500 and 1500 ppm, pup body weights also progressively decreased between PND 14 and PND 21 to 0.97 and 0.92x of control respectively (statistically significant at 1500 ppm only).

- Body Weights and Body Weight Gains – F1-Generation from weaning onwards
Note: “Day 1” of treatment is the day that the first animals were weaned. Body weights of animals that were weaned during the subsequent 7 days were all entered under that day. Due to the effect on pup body weight gain during the last week of the lactation period, body weights at weaning were reduced compared to concurrent controls in all treatment groups, all cohorts and both sexes (0.94-0.98, 0.90-0.94 and 0.84-0.87x of control for the 500, 1500 and 5000 ppm groups; not always statistically significant at 500 and 1500 ppm). One week after weaning, body weight gain was significantly reduced in all treatment groups, resulting in further reduced body weights in all groups (body weights were 0.83-0.90, 0.83-0.94 and 0.62-0.76x of control for the 500, 1500 and 5000 ppm groups). Notably, this effect was slightly more pronounced in males compared to females. From Day 15 onwards, body weight gain started to recover. For males at 5000 ppm, body weight gain did not fully recover to similar levels of control, and was statistically significantly lower for most cohorts in Week 4 and 5 of the treatment period. Consequently, terminal body weights in Cohort 1A and 1B were statistically significantly lower (0.80-0.84x of control). At 1500 ppm, the body weight gain recovered to levels similar to or slightly below concurrent control. Any differences were not statistically significant, however at the end of the treatment period in Cohort 1A and 1B, terminal body weights remained statistically significantly lower (0.88-0.91x of control). For females at 5000 ppm, body weight gain recovered to normal levels from Day 22 onwards. However, terminal body weights remained significantly lower 0.87-0.89x of control). At 1500 ppm, body weight gain returned to similar or slightly increased levels compared to concurrent controls. Statistical significance for increased body weight gains was reached on several occasions. After a treatment period of at least 10 weeks, only slightly lower terminal body weights were noted in Cohort 1A and 1B (0.95-0.96x of control; not statistically siginicant). Overall, body weight gain remained within the same range as concurrent controls throughout the post-coitum and lactation period in all treatment groups. Any statistical differences were considered unrelated to treatment in the absence of a dose-related response. The slightly reduced body weight gain during pre-mating in both males and females at 500 ppm was considered unrelated to treatment as no dose related response was observed. Slightly lower terminal body weights were observed in Cohort 1A and 1B (0.92-0.94 and 0.96-0.97x of controls for males and females, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Food Consumption – F1-Generation from Weaning Onwards
Food consumption before or after correction for body weight were affected by treatment with the test item at 5000 ppm. In males at 5000 ppm, food consumption was decreased for all cohorts. For Cohort 1A, absolute food consumption was decreased to 0.56-0.96x of control (in Weeks 1-2, 3-5, 6-8 and 9-10) and relative food consumption was decreased to 0.80x in Week 1-2 and increased to 1.07-1.25x of control in Week 2-11. For Cohort 1B, absolute food consumption was decreased to 0.56-0.91x of control (in Weeks 1-2, 3-7 and 13-14) and relative food consumption was decreased to 0.86x in Weeks 1-2 and increased to 1.08-1.33x of control in Week 2-14. For Cohorts 2A and 3 similar patterns were observed and absolute food consumption was decreased to 0.63-0.84x of control for Cohort 2A (in Weeks 1-2 and 3-4) and to 0.44-0.86x of control for Cohort 3 (in Weeks 1-2 and 3-5). Relative food consumption was increased to 1.06-1.21x of control for Cohort 2A (Week 2-4 and 6-8) and 1.09-1.22x of control for Cohort 3 (Weeks 2-5). In females at 5000 ppm, absolute food consumption was decreased for Cohorts 1A and 1B to 0.75-0.93x of control (Weeks 1-2, 4-5 and 7-9) and to 0.63-1.00x of control (Weeks 1-2, 7-8, 10-11 and 12), respectively. Relative food consumption was increased for all cohorts (with the exception of Week 1-2 of Cohort 1B (0.85x of control)) and was increased to 1.07-1.22x (Weeks 2-8), 1.07-1.20x (Weeks 2-12), 1.11-1.19x (Weeks 2-4 and 6-7) and 1.15-1.21x (Weeks 2-3 and 4-5) for Cohorts 1A, 1B, 2A and 3, respectively. Changes in absolute and relative food consumption observed for males and females at 500 and 1500 ppm were considered unrelated to treatment with the test item at the incidence and magnitude observed, and in the absence of a dose related response.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1-Generation (Cohort 1A)
In males and females up to 5000 ppm, hematology parameters were considered unaffected by treatment with the test item. In females at 5000 ppm, lymphocytes were statistically significantly decreased to 0.78x of control. This could be partly contributed to a relatively high control mean compared to historical control mean and values remained within historical control range. The statistically significant increase in mean corpuscular volume (MCV) in females at 5000 ppm was minimal (1.03x of control) and therefore considered not toxicologically relevant. Statistically significant changes observed in haemoglobin (males), red blood cell distribution width and mean corpuscular haemoblobin (females) were considered unrelated to treatment with the test item in absence of a dose response. Coagulation parameters of treated rats were unaffected by treatment with the test item up to 5000 ppm.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Clinical Biochemistry (T4 and TSH levels) on PND 4 and 22 – F1-Pups
Serum T4 levels in male and female pups (pooled samples) culled at PND 4, and serum T4 and TSH levels in male and female pups of Cohort Surplus at PND 22-24 were unaffected by treatment with the test item up to 5000 ppm. The higher serum T4 levels in female PND 22-24 pups at 5000 ppm was likely attributed to a high value of female No. 976 (after exclusion: mean T4 value was 5.77 μg/dL) and was considered unrelated to treatment with the test item in absence of a statistically significant change and as levels remained within the historical control range. At 1500 ppm, mean serum T4 levels in male PND22-24 pups were slightly increased (not statistically significant). In the absence of a dose related response and as the mean value remained within historical control range, this is considered unrelated to treatment with the test item.

- Clinical Biochemistry F1-Generation (Cohort 1A) including T4 and TSH
Urea levels were increased in males at 500, 1500 and 5000 ppm, at 1.13x (500 and 1500 ppm) and 1.24x (5000 ppm) of control. As this change was minimal and all mean and individual values remained within the historical control range, this was considered not toxicologically relevant. In females, chloride levels were increased at 1500 and 5000 ppm (both 1.02x of control). As the magnitude of the effect was minimal and in the absence of a clear dose-response, these increases were considered not toxicologically relevant. In females at 500 ppm, clinical chemistry parameters were unaffected by treatment with the test item.
Thyroid hormones were unaffected by treatment with the test item up to 5000 ppm. In males, serum levels of total T4 were higher (1.24x of control values) at 1500 ppm. As this difference occurred in the absence of a dose response and the mean level of total T4 remained within the historical control range, the higher total T4 levels were considered unrelated to treatment with the test item.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1-Generation (Cohort 1A)
Urinalysis parameters of treated rats were unaffected by treatment with the test item up to 5000 ppm. In males at 5000 ppm, the pH value was statistically significantly lower compared with the control. As the difference was minimal (7.2 vs 6.8) and mean pH and all individual values remained within the historical control range24, the lower pH level was considered not toxicologically relevant. In females, urine volume was statistically lower at 5000 ppm. This was considered unrelated to treatment with the test item in absence of a clear dose-response and effects on other urinalysis parameters.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Sexual maturation was delayed in both sexes at 1500 and 5000 ppm. At 1500 and 5000 ppm , the age at attainment of balonopreputial separation or vaginal opening was statistically significantly increased in the majority of the cohorts and in remaining cohorts a similar trend was observed. Body weight at attainment was similar (females 1500 and 5000 ppm, males at 1500 ppm) or reduced (males at 5000 ppm) compared to controls, indicating that the observed delay in sexual maturation for both males and females was due to the test item-related growth retardation observed at these dose levels. The statistically significant decrease in body weight at balanopreputial separation for Cohort 1B at 500 ppm and increase of body weight at vaginal opening for Cohort 1B at 1500 ppm, were considered unrelated to treatment with the test item as these changes were present for this cohort only and occurred in absence of a dose related trend. The age at first estrus was statistically significantly increased at 1500 and 5000 ppm. This was directly related to the increased age at attainment of vaginal opening and considered due to growth retardation. Time between attainment of vaginal opening and first estrus was similar in all groups.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance was unaffected by treatment with the test item up to 5000 ppm. Normalized anogenital distance in male pups were statistically significantly lower at 5000 ppm compared to controls. As the effect was minimal (1.41 vs 1.45 mm, respectively) and as all individual values were within the historical control range, this was considered unrelated to treatment with the test item. The statistically significantly higher mean normalized anogenital distance of female pups at 1500 ppm occurred without a dose related-trend and was considered unrelated to treatment with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment with the test item up to 5000 ppm had no effect on areola/nipple retention. For none of the examined male pups, nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Organ weights until Weaning – F1-Pups (Cohort Surplus)
Organ weights were considered affected by treatment with the test item from 1500 ppm. Spleen weights were considered decreased by treatment with the test item at 500 (females) and 1500 and 5000 ppm (males and females), as both absolute organ weight and organ/relative to body weight were decreased. Absolute and relative mean values at 5000 ppm and absolute weights at 1500 ppm were below the historical control range for both sexes, whereas, organ/relative to body weight at 1500 ppm and all mean values at 500 ppm were (just) within this range. Both absolute and organ/relative to body weight of the thymus for males and females at 5000 ppm were decreased. All mean values remained within the historical control range For the brain, an increased organ/relative to body weight was noted, with minor or no changes in absolute weights, which was considered the result to be the result of lower terminal body weights.

- Organ weights F1-Generation from weaning onward
Cohort 1A (PND 89-93)
There were organ weight changes as depicted in the table below. The organ weight changes were all considered to be the result of a lower terminal body weight. For some organs (brain, spleen, testes, epididymides) an increased organ/relative to body weight was noted, with minor or no changes in absolute weights. In other organs (heart, pituitary gland, liver, thymus, mesenteric lymph node, kidneys, adrenal glands, prostate gland, seminal vesicles, axillary lymph nodes, ovaries) the decrease in body weight resulted in lower absolute organ weights, with minor or no changes in the organ/relative to body weight. There were no macroscopic of microscopic findings in any of these organs, further indicating that these organ weight changes were a consequence of the decrease in terminal body weight and not directly test item-related.

Cohort 1B (≥ PND 90)
The same reasoning applies as used in the cohort 1A organ weight section, the decrease in terminal body weight in all dose groups resulted in many organ weights that are statistically significant different from control animals, both absolute and relative to body weight. The absence of any macroscopic of microscopic test item-related finding seem to suggest these organ weight changes are primarily due to the decrease in terminal body weight and not a direct test item-related effect.

Cohort 2B (PND 21-22)
There was a significant increase in brain weight in males treated at 1500 (absolute and relative to body weight) and 5000 ppm (relative to body weight only). The absolute brain weight was not affected at the high dose (5000 ppm) and there was a significant decrease in final body weight in males at 5000 ppm. This indicates this was not a direct test item-related effect on brain weight but rather a test item-related effect on final body weight.

Cohort 2A (PND 76-82)
There was a significant increase in brain weight (relative to body weight only) in males treated at 1500 and 5000 ppm and in females at 5000 ppm. The absolute brain weights were not affected and there was a significant decrease in terminal body weight in males and females at 5000 ppm. This indicates this was not a direct test item-related effect on brain weight but rather a test item-related effect on terminal body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Macroscopy until Weaning – F1-Pups
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test item up to 5000 ppm.

- F1-Generation from weaning onward
There were no test item-related gross observations in Cohorts 1A, 1B, 2A, 2B and Surplus). All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in Cohort 1A. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Ovarian Follicle Counts (Cohort 1A)
There were no test item-related effects on the ovarian follicle counts or corpora lutea counts in the F1-females of the 5000 ppm group when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.

Spermatogenesis-staging (Cohorts 1A and 1B)
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional Tests – F1-Generation (Cohort 2A)

- Acoustic Startle Response
Acoustic startle response was unaffected by treatment with the test item up to 5000 ppm.

- Detailed Clinical Observations
Detailed clinical observations revealed no symptoms that were considered to be related to treatment with the test item. The clinical symptoms that were observed were within the normal range of behavioural findings for this type of study, and were generally also observed in control animals. These findings were therefore considered not to be related to treatment.

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

- Functional Observations
Grip strength of the fore leg was decreased to 0.88x of control in males by treatment with the test item at 5000 ppm. Mean grip strength of the fore leg remained within the historical control range. Significant changes observed in foot splay in males at 500 ppm and in grip strength of the fore leg in females at 500 and 1500 ppm were considered unrelated to treatment in absence of a dose related trend. Grip strength of the hind leg was unaffected by treatment and hearing ability and pupillary reflex were normal in all examined animals.


Neuropathology and Morphometry– F1-Generation (Cohorts 2A and 2B)

- Fixed Brain Weights
There were changes to the fixed brain weights as shown in the table below. These fixed brain weight changes were considered to be related to the lower terminal body weight and were not directly test item-related.

- Brain Dimensions
Brain dimensions (length and width of brain) at PND 21-22 (Cohort 2B) and PND 76-82 (Cohort 2A) were not affected by treatment up to 5000 ppm.

- Brain Histopathology
In the F1-animals (Cohort 2A), there were no test item-related effects on the H&E or Luxol Fast Blue/Cresyl Violet stained sections of brain in the control or high-dose group males or females.

- Brain Morphometry
- Morphometric analysis of the brain at PND 21-22 revealed no statistically significant differences in animals potentially exposed to bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate compared to control animals. Morphometric analysis at PND 76-82 revealed no statistically significant differences in animals administered the test item via dietary administration up to 5000 ppm compared to control animals. In conclusion, there were no differences using morphometric analysis in brains of treated animals at either PND 21-22 or PND 76-82.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
- T-Cell Dependent Antibody Response (TDAR) Evaluation (Cohort 3 and Positive control animals)
Background anti-SRBC IgM levels in pre-immunization serum samples were similar for all Cohort 3 groups. All control animals (Group 1) showed a clear increase in anti-SRBC IgM levels 5 days after immunization with SRBC which confirmed the TDAR assay was performed successfully. As expected, positive control animals treated with cyclophosphamide (Group 5) showed no increase in anti-SRBC IgM levels 5 days after immunization with SRBC. This clear sign of immunosuppression caused by treatment with cyclophosphamide was in accordance with the reduced size of thymus observed for 6 out of 10 positive control males. All animals treated with the test item up to 5000 ppm showed a clear increase in anti-SRBC IgM levels 5 days after immunization with SRBC, except for one male at 500 ppm which showed a mild increase in anti-SRBC IgM levels. Mean anti-SRBC IgM levels appeared lower for males at 500, 1500, 5000 ppm when compared to control males. Although thisdifference was statistically significant, it was considered to be caused by biological variability and to be of no immunotoxicological relevance because of the following reasons: anti-SRBC IgM levels of most of the treated males remained within range of control males, no doserelated trend in anti-SRBC IgM levels was observed for treated males, anti-SRBC IgM values in treated males were similar to those in treated females and control females, and all but one of the treated males showed a clear induction of antibody response. Mean anti-SRBC IgM levels in treated females and control females appeared similar. Therefore, it is considered that treatment with the test item up to 5000 ppm did not result in immunotoxicologically relevant effects on anti-SRBC IgM levels 5 days after immunization. This conclusion is in accordance with the absence of test item-related changes in splenocyte subpopulations and with the absence of microscopic observations of Cohort 1A animals.

- Splenic Lymphocyte Subpopulation – F1-Generation (Cohort 1A)
There were no test item-related effects on splenic lymphocyte subpopulations observed up to 5000 ppm.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General Toxicity
Generation:
F1
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: corresponding to 121-163 mg/kg/day in males and 130-165 mg/kg/day in females
Dose descriptor:
NOAEL
Remarks:
Developmental General Toxicity
Generation:
F1
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 121-163 mg/kg/day in males and 130-165 mg/kg/day in females
Dose descriptor:
NOAEL
Remarks:
Developmental Neurotoxicity
Generation:
other: F1 cohort 2A and 2B
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: corresponding to 428-552 mg/kg/day in males and 451-573 mg/kg/day in females
Dose descriptor:
NOAEL
Remarks:
Developmental Immunotoxicity
Generation:
F1 (cohort 3)
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: corresponding to 428-552 mg/kg/day in males and 451-573 mg/kg/day in females

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical Signs during Lactation – F2-Pups
No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 5000 ppm. For the pup at 5000 ppm missing on Day 2 (Litter No. 916), a pale appearance and a blue spot on the head was observed at first litter check. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body Weights during Lactation – F2-Pups
Body weights of pups were affected by treatment with the test item from 1500 ppm onwards. At 1500 and 5000 ppm, body weights of male and female pups were decreased at PND 21. At 5000 ppm, weights decreased to 0.88x of control in males, 0.86x in females and to 0.89x in males and females combined. At 1500 ppm, weights decreased to 0.94x in males, 0.93x in females and to 0.93x in males and females combined.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was unaffected by treatment with the test item up to 5000 ppm.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment with the test item up to 5000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weights were considered affected by treatment with the test item at 5000 ppm. The lower mean spleen weights in at 1500 and 5000 ppm when compared with concurrent controls was regarded to be related to treatment with the test item as both absolute organ weight and organ/relative to body weight were decreased. With the exception of mean absolute spleen weight in females at 5000 ppm, all mean values remained within the historical control range. Other organ weight changes as depicted above were all considered to be the result of a lower terminal body weight. For the brain and thymus, an increased organ/relative to body weight was noted, with minor or no changes in absolute weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test item up to 5000 ppm. At 5000 ppm, for three pups of one litter (Litter No. 931) an enlarged thyroid was observed and for four pups of this litter, reddish foci were observed on the thymus. Reddish foci were also observed for three pups of Litter No. 920. At 1500 ppm, for four pups (Litter No. 833) the lungs were not collapsed. The nature and incidence of these and other macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
Developmental General Toxicity
Generation:
F2
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 121-163 mg/kg/day in males and 130-165 mg/kg/day in females

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
5 000 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Any other information on results incl. tables

Test Item Intake - F0 Generation

Group No. Mean over means intake
[mg test item/kg body weight] (mean range indicated within brackets)
2 3 4
Nominal dietary inclusion level (ppm) during (pre-/post-) mating and post-coitum 500 1500 5000
Nominal dietary inclusion level (ppm) during lactation 250 1250 2500
Sex Study period            
Males Pre-mating 38 (28-53) 115 (88-155) 400 (302-543)
Post-mating 26 (25-27) 81 (80-82) 266 (264-268)
Mean of meansa 36 109 378
Females Pre-mating 42 (33-54) 130 (100-158) 440 (317-539)
Post-coitum 33 (31-36) 100 (96-107) 342 (310-447)
Lactation 45 (29-59) 136 (86-175) 467 (291-609)
Mean of meansa 41 126

427

aMean of means of all periods, weighed for number of measurement days per period:

Males: ((70x mean premating) + (14x mean post-mating)) / 84

Females: ((70x mean premating) + (20x mean post-coitum) + (20x mean lactation)) / 110

Test Item Intake – F1-Generation

Group No. Mean over means intake
[mg test item/kg body weight] (mean range indicated within brackets)
2 3 4
Nominal dietary inclusion level (ppm) during (pre-/post-) mating and post-coitum 500 1500 5000
Nominal dietary inclusion level (ppm) during lactation 250 1250 2500
Sex Study period            
Males Cohort 1A Treatment 43 (30-58) 132 (92-178) 463 (353-645)
Cohort 2A Treatment 48 (35-58) 142 (105-179) 497 (387-644)
Cohort 3 Treatment 56 (51-59) 163 (149-176) 552 (407-647)
Cohort 1B Treatment 40 (28-58) 121 (88-179) 428 (302-643)
Females Cohort 1A Treatment 46 (36-60) 136 (107-183) 480 (386-659)
Cohort 2A Treatment 49 (37-60) 150 (111-202) 504 (393-664)
Cohort 3 Treatment 54 (48-59) 165 (144-199) 573 (478-674)
Cohort 1B:            
Treatment (Pre-mating) 44 (34-59) 132 (103-186) 458 (333-653)
Post-coitum  38 (34-52) 114 (103-141) 372 (316-466)
Lactation 47 (32-56) 143 (100-177) 504 (337-621)
Mean of meansa 43 130 451

a Mean of means of all periods, weighed for number of measurement days per period:

Cohort 1B females: ((76x mean premating) + (20x mean post-coitum) + (20x mean lactation)) / 116

BODY WEIGHTS (GRAM) SUMMARY - F0

MALES GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
PRE MATING          
DAY 1 MEAN 153 158 153 154
WEEK 1 ST.DEV 8.3 7.5 9.5 7.4
  N 28 28 28 28
DAY 9 MEAN 205 199 200 190 **
WEEK 2 ST.DEV 11.4 12.3 13.6 10.7
  N 28 28 28 28
DAY 15 MEAN 238 230 230 219 **
WEEK 3 ST.DEV 14.6 13.9 15 14.5
  N 28 28 28 28
DAY 22 MEAN 255 248 244 * 229 **
WEEK 4 ST.DEV 15.9 13 15.3 16.5
  N 28 28 28 28
DAY 29 MEAN 298 282 ** 284 * 271 **
WEEK 5 ST.DEV 21.3 17.8 20.2 21
  N 28 28 28 28
DAY 36 MEAN 315 297 ** 297 ** 283 **
WEEK 6 ST.DEV 22.5 19.1 22.9 24
  N 28 28 28 28
DAY 43 MEAN 334 312 ** 312 ** 298 **
WEEK 7 ST.DEV 26.1 21 25.8 26.6
  N 27 28 28 28
DAY 50 MEAN 346 323 ** 324 ** 307 **
WEEK 8 ST.DEV 29.1 22 28.5 28.8
  N 27 28 28 28
DAY 57 MEAN 361 334 ** 335 ** 319 **
WEEK 9 ST.DEV 30.8 23.8 30.3 30.4
  N 27 28 28 28
DAY 64 MEAN 370 341 ** 342 ** 326 **
WEEK 10 ST.DEV 31.3 25.1 32 33.4
  N 27 28 28 28
           
MATING PERIOD MEAN 379 351 ** 351 ** 335 **
DAY 1          
WEEK 1 ST.DEV 33.2 25.8 33.8 34.2
  N 27 28 28 28
DAY 8 MEAN 383 355 ** 354 ** 339 **
WEEK 2 ST.DEV 33.8 25.5 32.9 34.2
  N 27 28 28 28
DAY 15 MEAN 391 360 ** 361 * 346 **
WEEK 3 ST.DEV 34.3 28.6 32.1 33.5
  N 19 20 20 20
DAY 22 MEAN 401 349 397 352
WEEK 4 ST.DEV 25.5 10.9 44.4 16.6
  N 3 4 4 4

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
PRE MATING          
DAY 1 MEAN 122 121 122 120
WEEK 1 ST.DEV 6.1 6.6 8.1 5.6
  N 28 28 28 28
DAY 9 MEAN 148 143 142 * 142 *
WEEK 2 ST.DEV 8.2 7.2 10.5 9.2
  N 28 28 28 28
DAY 15 MEAN 165 157 * 156 ** 155 **
WEEK 3 ST.DEV 9.2 10.2 11.9 10.5
  N 28 28 28 28
DAY 22 MEAN 180 171 * 168 ** 167 **
WEEK 4 ST.DEV 9.9 12.4 13.8 10
  N 28 28 28 28
DAY 29 MEAN 192 187 179 ** 180 **
WEEK 5 ST.DEV 10.5 12.9 14.8 12.6
  N 28 28 28 28
DAY 36 MEAN 198 193 187 ** 187 **
WEEK 6 ST.DEV 11.4 14.1 15.5 12.2
  N 28 28 28 28
DAY 43 MEAN 210 200 * 196 ** 194 **
WEEK 7 ST.DEV 11.2 15.1 14.9 12.8
  N 28 28 28 28
DAY 50 MEAN 217 210 205 ** 201 **
WEEK 8 ST.DEV 11.7 15.3 14.3 12
  N 28 28 28 28
DAY 57 MEAN 221 213 205 ** 204 **
WEEK 9 ST.DEV 12.2 15.7 15.3 13.7
  N 28 28 28 28
DAY 64 MEAN 224 221 215 216
WEEK 10 ST.DEV 12 15.9 15.8 13.8
  N 28 28 28 28
           
MATING PERIOD          
DAY 1 MEAN 226 219 214 ** 212 **
WEEK 1 ST.DEV 11.8 16.7 16.2 14.4
  N 28 28 28 28
DAY 8 MEAN 264 233 232 224
WEEK 2 ST.DEV --- --- 5.7 ---
  N 1 1 2 1
DAY 15 MEAN 260      
WEEK 3 ST.DEV ---      
  N 1      
           
POST COITUM          
DAY 0 MEAN 228 222 212 ** 211 **
  ST.DEV. 13.4 16.1 16 14.2
  N 28 27 28 26
DAY 4 MEAN 242 236 226 ** 220 **
  ST.DEV. 15 15.8 15.1 16
  N 28 27 28 26
DAY 7 MEAN 248 242 228 ** 222 **
  ST.DEV. 14.1 15.4 16.4 16
  N 28 27 28 26
DAY 11 MEAN 262 255 244 ** 237 **
  ST.DEV. 14.8 13.8 15.2 17.4
  N 28 27 28 26
DAY 14 MEAN 272 263 250 ** 242 **
  ST.DEV. 14.8 16.2 15.7 18.6
  N 28 27 28 26
DAY 17 MEAN 294 283 274 ** 264 **
  ST.DEV. 17.5 18.3 17 19.1
  N 28 27 28 26
DAY 20 MEAN 330 314 * 304 ** 289 **
  ST.DEV. 18.2 20 21.9 22.4
  N 28 27 28 26
           
LACTATION          
DAY 1 MEAN 251 244 237 ** 227 **
  ST.DEV. 13.9 15.3 17 17.2
  N 28 26 28 26
DAY 4 MEAN 264 260 250 ** 243 **
  ST.DEV. 15.1 16.1 19.2 17.2
  N 27 26 28 26
DAY 7 MEAN 273 271 259 * 252 **
  ST.DEV. 15.9 16.4 18.8 18.5
  N 27 26 28 26
DAY 14 MEAN 274 275 264 256 **
  ST.DEV. 22.6 15.3 18.2 19.5
  N 27 26 28 26
DAY 21 MEAN 276 276 266 * 264 *
  ST.DEV. 15.6 14.6 15.5 17.9
  N 27 26 28 26

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

BODY WEIGHT GAIN (%) SUMMARY - F0

MALES GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
PRE MATING          
DAY 1 MEAN 0 0 0 0
WEEK 1 ST.DEV 0 0 0 0
  N 28 28 28 28
DAY 9 MEAN 34 26 ** 30 ** 24 **
WEEK 2 ST.DEV 4.3 3.9 4.8 3.6
  N 28 28 28 28
DAY 15 MEAN 55 46 ** 50 ** 43 **
WEEK 3 ST.DEV 6.8 5.4 5.8 6.6
  N 28 28 28 28
DAY 22 MEAN 67 57 ** 59 ** 49 **
WEEK 4 ST.DEV 8 6 7.8 8.3
  N 28 28 28 28
DAY 29 MEAN 95 79 ** 85 ** 77 **
WEEK 5 ST.DEV 12.4 8.9 10.2 11
  N 28 28 28 28
DAY 36 MEAN 106 88 ** 93 ** 84 **
WEEK 6 ST.DEV 13.6 9.4 11.9 12.7
  N 28 28 28 28
DAY 43 MEAN 119 98 ** 103 ** 94 **
WEEK 7 ST.DEV 14.7 11.7 12.4 14.2
  N 27 28 28 28
DAY 50 MEAN 127 105 ** 111 ** 100 **
WEEK 8 ST.DEV 16.8 12.7 14.6 15.6
  N 27 28 28 28
DAY 57 MEAN 136 112 ** 118 ** 108 **
WEEK 9 ST.DEV 17.9 14 15.5 16.6
  N 27 28 28 28
DAY 64 MEAN 143 116 ** 123 ** 112 **
WEEK 10 ST.DEV 19 15.1 16.3 18.6
  N 27 28 28 28
           
MATING PERIOD          
DAY 1 MEAN 148 123 ** 129 ** 118 **
WEEK 1 ST.DEV 20.5 16 17.5 18.9
  N 27 28 28 28
DAY 8 MEAN 151 125 ** 131 ** 121 **
WEEK 2 ST.DEV 20.4 15.6 16.4 19.2
  N 27 28 28 28
DAY 15 MEAN 155 129 ** 135 ** 126 **
WEEK 3 ST.DEV 22.5 17.1 14.1 18.8
  N 19 20 20 20
DAY 22 MEAN 161 123 ** 147 131 *
WEEK 4 ST.DEV 16.7 15.3 11.1 7.3
  N 3 4 4 4

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
PRE MATING          
DAY 1 MEAN 0 0 0 0
WEEK 1 ST.DEV 0 0 0 0
  N 28 28 28 28
DAY 9 MEAN 21 19 16 ** 19
WEEK 2 ST.DEV 4.1 5.1 4.6 5.4
  N 28 28 28 28
DAY 15 MEAN 35 30 ** 28 ** 29 **
WEEK 3 ST.DEV 4.7 6.8 5.8 7.1
  N 28 28 28 28
DAY 22 MEAN 48 41 ** 37 ** 40 **
WEEK 4 ST.DEV 6.1 6.9 8 7.2
  N 28 28 28 28
DAY 29 MEAN 58 55 46 ** 51 **
WEEK 5 ST.DEV 7.6 7.9 8.6 8.6
  N 28 28 28 28
DAY 36 MEAN 63 60 53 ** 56 *
WEEK 6 ST.DEV 8.5 8.8 8.9 8.1
  N 28 28 28 28
DAY 43 MEAN 72 66 * 61 ** 62 **
WEEK 7 ST.DEV 8.5 10.8 8.7 9.8
  N 28 28 28 28
DAY 50 MEAN 79 74 68 ** 68 **
WEEK 8 ST.DEV 8.3 9.8 8.7 9.3
  N 28 28 28 28
DAY 57 MEAN 82 77 68 ** 71 **
WEEK 9 ST.DEV 9 10.1 9.5 10.5
  N 28 28 28 28
DAY 64 MEAN 84 83 77 * 81
WEEK 10 ST.DEV 9.1 10.8 9.4 9
  N 28 28 28 28
           
MATING PERIOD          
DAY 1 MEAN 86 82 75 ** 77 **
WEEK 1 ST.DEV 9.5 12.2 9.8 10.4
  N 28 28 28 28
DAY 8 MEAN 115 99 84 96
WEEK 2 ST.DEV --- --- 15.1 ---
  N 1 1 2 1
DAY 15 MEAN  111      
WEEK 3 ST.DEV ---
  N 1      
           
POST COITUM          
DAY 0 MEAN 0 0 0 0
  ST.DEV. 0 0 0 0
  N 28 27 28 26
DAY 4 MEAN 6 6 6 4 *
  ST.DEV. 2.2 2.4 3 2.9
  N 28 27 28 26
DAY 7 MEAN 9 9 8 5 **
  ST.DEV. 2.4 2.7 3.2 3.4
  N 28 27 28 26
DAY 11 MEAN 15 15 15 12 **
  ST.DEV. 3.1 4.2 3.7 3.9
  N 28 27 28 26
DAY 14 MEAN 19 19 18 15 **
  ST.DEV. 3.5 3.8 3.9 4.1
  N 28 27 28 26
DAY 17 MEAN 29 28 29 25 *
  ST.DEV. 3.8 5.3 5.2 4.4
  N 28 27 28 26
DAY 20 MEAN 45 42 43 37 **
  ST.DEV. 4.6 8.1 6.2 6
  N 28 27 28 26
           
LACTATION          
DAY 1 MEAN 0 0 0 0
  ST.DEV. 0 0 0 0
  N 28 26 28 26
DAY 4 MEAN 5 6 6 7
  ST.DEV. 4.4 3.5 4 3.5
  N 27 26 28 26
DAY 7 MEAN 9 11 9 11
  ST.DEV. 4.1 4.3 4.3 4.4
  N 27 26 28 26
DAY 14 MEAN 9 13 12 13
  ST.DEV. 7.1 4.1 7.9 5.8
  N 27 26 28 26
DAY 21 MEAN 10 13 12 17 **
  ST.DEV. 4.8 5 6.3 5
  N 27 26 28 26

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

BODY WEIGHTS OF F1 PUPS (GRAM)

DAY SEX   GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
1 M MEAN 6.3 6.6 6.4 6.4
    ST.DEV. 0.5 0.6 0.6 0.5
    N 28 26 28 26
  F MEAN 6 6.2 6.1 6
    ST.DEV. 0.5 0.5 0.5 0.5
    N 28 26 28 26
  M+F MEAN 6.2 6.4 6.2 6.2
    ST.DEV. 0.5 0.5 0.5 0.5
    N 28 26 28 26
4 M MEAN 9.6 10.1 9.7 9.6
    ST.DEV. 0.7 1 1 1
    N 27 26 28 26
  F MEAN 9.3 9.6 9.4 9.2
    ST.DEV. 0.7 1 1 0.9
    N 27 26 28 26
  M+F MEAN 9.4 9.8 9.5 9.4
    ST.DEV. 0.7 1 0.9 1
    N 27 26 28 26
7 M MEAN 16.2 16.7 16 15.7
    ST.DEV. 1.2 1.3 1.2 1.3
    N 27 26 28 26
  F MEAN 15.8 15.9 15.5 15
    ST.DEV. 1.1 1.3 1.3 1.4
    N 27 26 28 25
  M+F MEAN 16 16.3 15.8 15.4
    ST.DEV. 1.1 1.3 1.2 1.3
    N 27 26 28 26
14 M MEAN 33.4 34.2 32.6 31.4 **
    ST.DEV. 1.5 1.9 1.8 2.5
    N 27 26 28 26
  F MEAN 32.6 33.2 32 30.3 **
    ST.DEV. 1.7 1.9 1.8 2.5
    N 27 26 28 25
  M+F MEAN 33 33.6 32.3 30.9 **
    ST.DEV. 1.5 1.8 1.7 2.5
    N 27 26 28 26
21 M MEAN 51.1 49.6 46.8 ** 44.2 **
    ST.DEV. 3.8 2.6 3 3.7
    N 27 26 28 26
  F MEAN 49.5 47.9 45.5 ** 42.1 **
    ST.DEV. 3.1 3.4 2.8 3.3
    N 27 26 28 25
  M+F MEAN 50.3 48.7 46.2 ** 43.4 **
    ST.DEV. 3.4 2.9 2.7 3.9
    N 27 26 28 26

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

BODY WEIGHTS (GRAM) SUMMARY - F1B

MALES COHORT 1B GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
TREATMENT          
DAY 1 MEAN 52 50 47 ** 44 **
WEEK 1 ST.DEV 4.6 3.1 2.6 4.1
  N 25 24 25 25
DAY 8 MEAN 80 66 ** 66 ** 55 **
WEEK 2 ST.DEV 9.2 8.5 5.5 6.6
  N 25 25 25 25
DAY 15 MEAN 109 99 ** 96 ** 88 **
WEEK 3 ST.DEV 8.7 7.2 8.5 7
  N 25 25 25 25
DAY 22 MEAN 171 154 ** 153 ** 129 **
WEEK 4 ST.DEV 11.3 10.8 8.1 13.9
  N 25 25 25 25
DAY 29 MEAN 216 196 ** 193 ** 171 **
WEEK 5 ST.DEV 14.2 12.6 10.8 11.2
  N 25 25 25 25
DAY 36 MEAN 252 232 ** 231 ** 206 **
WEEK 6 ST.DEV 18.3 17 13.1 13.9
  N 25 25 25 25
DAY 43 MEAN 287 266 ** 264 ** 238 **
WEEK 7 ST.DEV 22.4 24.2 16 17.6
  N 25 25 25 25
DAY 50 MEAN 313 290 ** 285 ** 260 **
WEEK 8 ST.DEV 25.3 29.6 19.8 21.9
  N 25 25 25 25
DAY 57 MEAN 331 304 ** 298 ** 275 **
WEEK 9 ST.DEV 27.2 34.1 24.8 23.7
  N 25 25 25 25
DAY 64 MEAN 349 322 ** 313 ** 289 **
WEEK 10 ST.DEV 27.7 35.7 26.8 25.1
  N 25 25 25 25
DAY 71 MEAN 363 336 ** 327 ** 303 **
WEEK 11 ST.DEV 29.2 36.7 29.2 26.7
  N 25 25 25 25
DAY 78 MEAN 377 349 ** 339 ** 313 **
WEEK 12 ST.DEV 31.3 38.7 30.2 26.6
  N 25 25 25 25
DAY 84 MEAN 388 360 * 350 ** 323 **
WEEK 12 ST.DEV 32.9 39.5 33.4 27.3
  N 25 25 25 25
DAY 91 MEAN 393 366 * 358 ** 331 **
WEEK 13 ST.DEV 32.4 39.3 34.2 27.7
  N 25 25 25 25
DAY 98 MEAN 403 376 * 368 ** 339 **
WEEK 14 ST.DEV 34.4 42 35.3 29
  N 25 25 25 25

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES COHORT 1B GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
TREATMENT          
DAY 1 MEAN 49 48 46 * 42 **
WEEK 1 ST.DEV 4.4 4.4 3.6 3.4
  N 25 25 25 24
DAY 8 MEAN 71 63 ** 64 ** 53 **
WEEK 2 ST.DEV 7.2 9.2 6.4 7.3
  N 25 25 25 25
DAY 15 MEAN 104 93 ** 92 ** 81 **
WEEK 3 ST.DEV 6.5 9.6 6 11.2
  N 25 25 25 24
DAY 22 MEAN 135 123 ** 126 * 113 **
WEEK 4 ST.DEV 8.1 12.8 9.5 12.8
  N 25 25 25 24
DAY 29 MEAN 154 141 ** 143 ** 131 **
WEEK 5 ST.DEV 9.9 12.5 9.9 11.4
  N 25 25 25 24
DAY 36 MEAN 168 158 ** 160 148 **
WEEK 6 ST.DEV 11.1 15 10.9 10.6
  N 25 25 25 24
DAY 43 MEAN 181 169 ** 170 ** 159 **
WEEK 7 ST.DEV 12.5 13.9 11.2 12.6
  N 25 25 25 24
DAY 50 MEAN 189 179 * 179 * 166 **
WEEK 8 ST.DEV 14.1 13.1 12 12.8
  N 25 25 25 24
DAY 57 MEAN 198 188 * 182 ** 174 **
WEEK 9 ST.DEV 14.9 13.9 15.3 13.2
  N 25 24 25 24
DAY 64 MEAN 203 193 * 189 ** 180 **
WEEK 10 ST.DEV 14.8 15 11 12.8
  N 25 24 25 24
DAY 71 MEAN 210 197 ** 196 ** 184 **
WEEK 11 ST.DEV 15.4 14.9 11.8 13.3
  N 25 24 25 24
DAY 78 MEAN 213 203 * 200 ** 186 **
WEEK 12 ST.DEV 15.8 15.5 11.3 13
  N 25 24 25 24
DAY 84 MEAN 219 207 * 206 ** 193 **
WEEK 12 ST.DEV 16.4 15.3 11.7 13.9
  N 25 24 25 24
DAY 91 MEAN     190  
WEEK 13 ST.DEV     -  
  N     1  
DAY 98 MEAN     198  
WEEK 14 ST.DEV     -  
  N     1  
DAY 105 MEAN     200  
WEEK 15 ST.DEV -  
  N 1  
DAY 112 MEAN     195  
WEEK 16 ST.DEV     -  
  N 1  
DAY 119 MEAN     194  
WEEK 17 ST.DEV -  
  N     1  

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
POST COITUM          
DAY 0 MEAN 220 207 * 205 ** 194 **
  ST.DEV. 16.1 16.2 11.8 12.3
  N 24 24 24 22
DAY 4 MEAN 235 225 222 * 206 **
  ST.DEV. 17.1 17.9 13.3 14.8
  N 25 24 24 23
DAY 7 MEAN 239 229 228 * 209 **
  ST.DEV. 17.5 17.1 13.3 14.1
  N 25 24 24 23
DAY 11 MEAN 251 242 240 222 **
  ST.DEV. 17.9 18.8 14.2 14.2
  N 25 24 24 23
DAY 14 MEAN 259 248 248 227 **
  ST.DEV. 19.8 18.4 13.4 15.6
  N 25 24 24 23
DAY 17 MEAN 282 274 272 249 **
  ST.DEV. 21.2 21.3 14.6 19.8
  N 25 24 24 23
DAY 20 MEAN 317 307 304 273 **
  ST.DEV. 23.4 24.8 18.1 22.2
  N 25 24 24 23
           
LACTATION          
DAY 1 MEAN 242 229 ** 233 213 **
  ST.DEV. 17 16.4 13.5 14.9
  N 25 24 24 23
DAY 4 MEAN 260 250 249 231 **
  ST.DEV. 19.6 18 17.7 18.1
  N 25 24 24 23
DAY 7 MEAN 266 257 257 239 **
  ST.DEV. 18.2 18.9 17.6 17.7
  N 25 24 24 23
DAY 14 MEAN 274 273 273 247 **
  ST.DEV. 16.6 19.5 19 19.1
  N 25 24 24 23
DAY 21 MEAN 273 266 265 242 **
  ST.DEV. 18.8 19 18.5 17.6
  N 25 24 24 23

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

BODY WEIGHT GAIN (%) SUMMARY - F1B

MALES COHORT 1B GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
TREATMENT          
DAY 1 MEAN 0 0 0 0
WEEK 1 ST.DEV 0 0 0 0
  N 25 24 25 25
DAY 8 MEAN 54 33 ** 40 ** 26 **
WEEK 2 ST.DEV 13.6 16.4 10.6 15.9
  N 25 24 25 25
DAY 15 MEAN 109 100 104 101
WEEK 3 ST.DEV 12.5 15.8 15.6 20
  N 25 24 25 25
DAY 22 MEAN 231 212 * 224 193 **
WEEK 4 ST.DEV 21.6 24.2 19.3 31.2
  N 25 24 25 25
DAY 29 MEAN 317 296 * 311 290 **
WEEK 5 ST.DEV 29.8 28.9 25.5 30
  N 25 24 25 25
DAY 36 MEAN 387 371 391 369
WEEK 6 ST.DEV 39.7 35.6 31.5 32.6
  N 25 24 25 25
DAY 43 MEAN 456 439 460 441
WEEK 7 ST.DEV 52.9 48.5 41 41.2
  N 25 24 25 25
DAY 50 MEAN 506 487 506 492
WEEK 8 ST.DEV 54.9 58.7 48 49.2
  N 25 24 25 25
DAY 57 MEAN 540 518 533 527
WEEK 9 ST.DEV 56.6 66.8 58.3 54.6
  N 25 24 25 25
DAY 64 MEAN 575 554 564 558
WEEK 10 ST.DEV 57.8 70 62.5 55.5
  N 25 24 25 25
DAY 71 MEAN 601 582 596 591
WEEK 11 ST.DEV 62.4 72.2 68.2 59.4
  N 25 24 25 25
DAY 78 MEAN 629 608 620 612
WEEK 12 ST.DEV 67.6 76.1 70.7 61.4
  N 25 24 25 25
DAY 84 MEAN 649 631 645 637
WEEK 12 ST.DEV 71.7 76.8 79 63.6
  N 25 24 25 25
DAY 91 MEAN 660 643 660 653
WEEK 13 ST.DEV 72.3 79.8 80.2 65.1
  N 25 24 25 25
DAY 98 MEAN 679 664 682 671
WEEK 14 ST.DEV 79.1 83.5 83.6 67.1
  N 25 24 25 25

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES COHORT 1B GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
TREATMENT          
DAY 1 MEAN 0 0 0 0
WEEK 1 ST.DEV 0 0 0 0
  N 25 25 25 24
DAY 8 MEAN 47 30 ** 41 28 **
WEEK 2 ST.DEV 10.9 15.3 10.8 16.3
  N 25 25 25 24
DAY 15 MEAN 115 92 ** 102 ** 98 **
WEEK 3 ST.DEV 15.5 16.5 13.2 14.7
  N 25 25 25 23
DAY 22 MEAN 178 154 ** 175 174
WEEK 4 ST.DEV 21.4 23.5 17.6 21.9
  N 25 25 25 23
DAY 29 MEAN 218 191 ** 213 217
WEEK 5 ST.DEV 27 25.7 20.7 26
  N 25 25 25 23
DAY 36 MEAN 247 227 * 251 255
WEEK 6 ST.DEV 30.3 30.8 21.4 30.1
  N 25 25 25 23
DAY 43 MEAN 274 249 * 273 281
WEEK 7 ST.DEV 32.2 28.8 23.3 33.7
  N 25 25 25 23
DAY 50 MEAN 291 271 293 298
WEEK 8 ST.DEV 34.5 28.3 25.4 35.2
  N 25 25 25 23
DAY 57 MEAN 308 289 299 318
WEEK 9 ST.DEV 35.9 30.5 29.4 39
  N 25 24 25 23
DAY 64 MEAN 320 300 315 332
WEEK 10 ST.DEV 38.1 30.1 23.6 39
  N 25 24 25 23
DAY 71 MEAN 334 308 * 331 340
WEEK 11 ST.DEV 38.6 33.9 27.1 39.2
  N 25 24 25 23
DAY 78 MEAN 339 320 338 347
WEEK 12 ST.DEV 39 33.2 27.1 40
  N 25 24 25 23
DAY 84 MEAN 352 330 353 363
WEEK 12 ST.DEV 39.6 36.5 30.3 41.7
  N 25 24 25 23
DAY 91 MEAN     363  
WEEK 13 ST.DEV     -  
  N     1  
DAY 98 MEAN     383  
WEEK 14 ST.DEV     -  
  N     1  
DAY 105 MEAN     388  
WEEK 15 ST.DEV     -  
  N     1  
DAY 112 MEAN     376  
WEEK 16 ST.DEV     -  
  N     1  
DAY 119 MEAN     373  
WEEK 17 ST.DEV     -  
  N     1  

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
POST COITUM          
DAY 0 MEAN 0 0 0 0
  ST.DEV. 0 0 0 0
  N 24 24 24 22
DAY 4 MEAN 7 8 * 8 * 6
  ST.DEV. 2.2 2 2.2 2
  N 24 24 24 22
DAY 7 MEAN 9 10 11 8
  ST.DEV. 2.5 1.7 2.3 3.1
  N 24 24 24 22
DAY 11 MEAN 15 17 * 17 ** 15
  ST.DEV. 3.1 2.5 2.2 2.6
  N 24 24 24 22
DAY 14 MEAN 18 19 21 * 17
  ST.DEV. 2.5 2.6 3 2.7
  N 24 24 24 22
DAY 17 MEAN 29 32 * 32 ** 29
  ST.DEV. 3.5 3.3 4 4.7
  N 24 24 24 22
DAY 20 MEAN 45 48 48 41
  ST.DEV. 5.8 4.7 5.1 5.2
  N 24 24 24 22
           
LACTATION          
DAY 1 MEAN 0 0 0 0
  ST.DEV. 0 0 0 0
  N 25 24 24 23
DAY 4 MEAN 7 10 7 9
  ST.DEV. 4.4 3.4 4 4.7
  N 25 24 24 23
DAY 7 MEAN 10 12 10 12
  ST.DEV. 5.3 3.1 3.9 4.9
  N 25 24 24 23
DAY 14 MEAN 14 20 ** 17 16
  ST.DEV. 6.5 4.2 4.7 5.2
  N 25 24 24 23
DAY 21 MEAN 13 17 * 14 14
  ST.DEV. 5 4.6 4.8 5.1
  N 25 24 24 23

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

BODY WEIGHTS OF F2 PUPS (GRAM)

DAY SEX   GROUP 1 GROUP 2 GROUP 3 GROUP 4
CONTROL 500 PPM 1500 PPM 5000 PPM
1 M MEAN 6.4 6.3 6.3 6.5
    ST.DEV. 0.6 0.5 0.5 0.5
    N 25 24 24 23
  F MEAN 6.1 6 5.9 6.2
    ST.DEV. 0.6 0.5 0.5 0.6
    N 25 24 24 22
  M+F MEAN 6.3 6.2 6.1 6.3
    ST.DEV. 0.6 0.5 0.5 0.5
    N 25 24 24 23
4 M MEAN 9.9 9.7 9.8 10.1
    ST.DEV. 1 1 0.9 1.1
    N 25 24 24 23
  F MEAN 9.6 9.3 9.2 9.6
    ST.DEV. 1.1 1 0.8 1
    N 25 24 24 21
  M+F MEAN 9.8 9.5 9.5 9.9
    ST.DEV. 1 0.9 0.8 1.1
    N 25 24 24 23
7 M MEAN 16.3 16.2 16.1 16.1
    ST.DEV. 1.6 1.3 1.2 1.6
    N 25 24 24 23
  F MEAN 15.9 15.6 15.3 15.6
    ST.DEV. 1.7 1.2 1 1.5
    N 25 24 24 21
  M+F MEAN 16.1 15.9 15.7 16
    ST.DEV. 1.6 1.2 1 1.7
    N 25 24 24 23
14 M MEAN 30.8 31.1 31.1 30.2
    ST.DEV. 2.6 2 2 3.4
    N 25 24 24 23
  F MEAN 30 30.2 29.9 28.9
    ST.DEV. 2.8 1.9 1.6 2.9
    N 25 24 24 21
  M+F MEAN 30.4 30.6 30.4 29.9
    ST.DEV. 2.7 1.9 1.7 3.5
    N 25 24 24 23
21 M MEAN 49.8 47.8 46.7 * 44.0 **
    ST.DEV. 4.9 3.6 2.6 5.8
    N 25 24 24 23
  F MEAN 48.2 46 44.8 ** 41.5 **
    ST.DEV. 4.8 3.3 2.4 3.9
    N 25 24 24 21
  M+F MEAN 49.1 46.9 45.7 * 43.5 **
    ST.DEV. 4.7 3.3 2.5 5.8
    N 25 24 24 23

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Organ Weights – F0-Generation

Mean Percent Organ Weight Differences from Control Groups – F0-Generation Males and Females

   Males       Females     
Dose level (ppm): 500 1500 5000 500 1500 5000
 Body Weight -8** -8** -13** -3 -6** -7**
Brain -8** -8** -13** -3 -6** -7**
Absolute -1 0 -2 1 -1 -1
Relative to body weight 5* 9** 12** 4 5* 6**
Pituitary gland        
Absolute -10 -10* -10** -8 -8 -8*
Relative to body weight 0 0 0 0 0 0
Heart    
Absolute -5 -6* -8** -3 -6* -6*
Relative to body weight 2 2 5* 1 0 2
Liver      
Absolute -8* -9* -11* -11** -11** -15**
Relative to body weight 0 0 2 -7* -6 -9**
Thyroid gland      
Absolute -5     -16*  -11 -6 -18** -12**
Relative to body weight 0 0 0 -13  -13*  -13
Kidneys    
Absolute -4 -7* -8** -3 -6** -6**
Relative to body weight 3 2 5** -1 -1 0
Adrenal glands    
Absolute 4 6 -2 -7 -6 -8**
Relative to body weight 7* 14** 7** -3 0 0
Spleen    
Absolute 0 0 1 n.s. n.s. n.s.
Relative to body weight 7 9* 15** n.s. n.s. n.s.
Testes    
Absolute 0 0 -3 n.a. n.a. n.a.
Relative to body weight 7* 9** 11** n.a. n.a. n.a.
Epididymides    
Absolute 1 -1 -3 n.a. n.a. n.a.
Relative to body weight 9** 8** 12** n.a. n.a. n.a.
Ovaries    
Absolute n.a. n.a. n.a. -3 -14** -15**
Relative to body weight n.a. n.a. n.a. -2 -9 -8
Seminal vesicles    
Absolute 4 -6 -7 n.a. n.a. n.a.
Relative to body weight 13* 3 7 n.a. n.a. n.a.
Thymus      
Absolute -12* -2 -11 n.s. n.s. n.s.
Relative to body weight -5 6 2 n.s. n.s. n.s.

*: P<0.05, **: P<0.01, n.s.: not significant, n.a.: not applicable

Organ weights until Weaning – F1-Pups (Cohort Surplus)

Mean Percent Organ Weight Differences from Control Groups – F1-Generation Cohort Surplus, Males and Females

   Males       Females     
Dose level (ppm): 500 1500 5000 500 1500 5000
Body Weight 0 -5 -9* -4 -7 -18**
Brain    
Absolute 0 -1 -1 4 1 1
Relative to body weight 0 6 9* 8 11* 23**
Thymus    
Absolute -3 -12 -19** 12 -8 -29**
Relative to body weight -1 -5 -10 -9 0 -13*
Spleen    
Absolute -9 -19* -26** -17* -31** -51**
Relative to body weight -9 -14 -20* -13 -24** -40**

*: P<0.05, **: P<0.01, n.s.: not significant, n.a.: not applicable

Organ Weights – F1-Generation - Cohort 1A

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

  Males Females
Dose level (ppm): 500 1500 5000 500 1500 5000
Body Weight -8* -12** -20** -4 -4 -13*
Brain    
Absolute -2 0 -3* -1 0 -2
Relative to body weight 5    11**  20** 3 3 13**
Pituitary gland    
Absolute -20** -10* -20** 0 -9 -9*
Relative to body weight 0 0 0 0 -17 0
Heart    
Absolute -4 -8* -12** 1 -2 -8*
Relative to body weight 4 4 10** 5 2 6
Liver      
Absolute -10* -12** -18** -3 -2 -7
Relative to body weight -2 0 3 0 1 8*
Thymus    
Absolute -15* -16* -27** n.s. n.s. n.s.
Relative to body weight -9 -5 -8 n.s. n.s. n.s.
Mesenteric Lymph node    
Absolute -31 -43* -50** n.s. n.s. n.s.
Relative to body weight -24 -33 -31 n.s. n.s. n.s.
Kidneys    
Absolute -9** -9** -16** -7* -3 -9**
Relative to body weight -1 3 6* -3 0 4
Adrenal glands    
Absolute -11 -10 -16** -2 -3 -14**
Relative to body weight 0 6 6 3 0 0
Spleen    
Absolute -5 -3 -8 1 8 2
Relative to body weight 4 10* 15** 6 12** 18**
Testes    
Absolute -5 -6* -9** n.a. n.a. n.a.
Relative to body weight 4 6 14** n.a. n.a. n.a.
Prostate gland    
Absolute -8 -13 -21** n.a. n.a. n.a.
Relative to body weight -1 -1 0 n.a. n.a. n.a.
Epididymides    
Absolute -4 -5 -12** n.a. n.a. n.a.
Relative to body weight 4 6 11** n.a. n.a. n.a.
Seminal vesicles    
Absolute -3 -2 -15* n.a. n.a. n.a.
Relative to body weight 6 11 8 n.a. n.a. n.a.
Ovaries    
Absolute n.a. n.a. n.a. -4 -8 -17**
Relative to body weight n.a. n.a. n.a. 0 -4 -3
Axillary lymph node    
Absolute -17 -33* -50** 0 0 25
Relative to body weight -12 -18 -24 -14 5 27*

*: P<0.05, **: P<0.01, n.s.: not significant, n.a.: not applicable

Organ Weights – F2-Pups

Mean Percent Organ Weight Differences from Control Groups – F1-Generation Cohort Surplus, Males and Females

  Males Females
Dose level (ppm): 500 1500 5000 500 1500 5000
Body Weight -6 -8* -14** -4 -6 -16**
Brain            
Absolute 0 -1 -1 1 2 -1
Relative to body weight 6 8 14** 4 7 17**
Thymus            
Absolute 0 0 -3 -2 -3 -8
Relative to body weight 6 9 12* 2 5 10*
Spleen            
Absolute -7 -18** -30** 1 -12 -33**
Relative to body weight -2 -11 -19** 3 -5 -20**

*: P<0.05, **: P<0.01, n.s.: not significant, n.a.: not applicable

Fixed Brain Weights

Cohort 2A:

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

  Males Females
Dose level (ppm): 500 1500 5000 500 1500 5000
Body weight 1 -8 -13** -1 -4 -10**
Brain            
Absolute 1 3 -1 2 2 -1
Relative to body weight 0  10*  14** 3 5 10*

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

Cohort 2B:

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

  Males Females
Dose level (ppm): 500 1500 5000 500 1500 5000
Body weight -4 -6 -10** -2 -4 -10
Brain            
Absolute 1 7** 1 0 1 -2
Relative to body weight 5 13** 12* 1 6 10

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

REPRODUCTION DATA SUMMARY - F0-generation

  GROUP 1 GROUP 2 GROUP 3 GROUP 4
  CONTROL 500 PPM 1500 PPM 5000 PPM
Males paired1) 28 28 28 28
Males mated1) 28 28 28 28
Females paired 28 28 28 28
Females mated 28 28 28 28
Pregnant females 28 27 28 26
Females with implantations only 0 1 0 0
Females with total litter loss on Day 2 1 0 0 0
Females with living pups on Day 1 28 26 28 26
Mating index males (%)
(Males mated / Males paired) * 100
100 100 100 100
Fertility index males (%)
(Pregnant females / Males mated) * 100
100 96 100 93
Mating index females (%)
(Females mated / Females paired) * 100
100 100 100 100
Fertility index females (%)
(Pregnant females / Females mated) * 100
100 96 100 93
Gestation index (%)
(Females with living pups on Day 1 / Pregnant females) * 100
100 96 100 100

1) Animal no. 1 was killed in extremis before start mating period, Animal no. 2 was used instead

IMPLANTATION SITES SUMMARY F0-generation

    GROUP 1 GROUP 2 GROUP 3 GROUP 4
    CONTROL 500 PPM 1500 PPM 5000 PPM
Implantations  MEAN 12.6 11.5 11.5 11.1 ++
ST.DEV 1.5 2.8 1.8 1.4
N 28 27 28 26

+/++ Steel-test significant at 5% (+) or 1% (++) level

Developmental Data - F0-generation

  GROUP 1 GROUP 2 GROUP 3 GROUP 4
 CONTROL 500 PPM 1500 PPM 5000 PPM
LITTERS        
TOTAL 28 26 28 26
         
DURATION OF GESTATION MEAN (+) 21.3 21.6 21.3 21.3
ST.DEV. 0.5 0.5 0.5 0.5
N 28 26 28 26
 
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#) 3 0 1 2
TOTAL 3 0 1 2
MEAN (+) 0.1 0 0 0.1
ST.DEV. 0.3 0 0.2 0.3
N 28 26 28 26
 
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#) 52 / 48 49 / 51 48 / 52 49 / 51
TOTAL 339 286 299 270
MEAN (+) 12.1 11 10.7 + 10.4 ++
ST.DEV. 1.5 2.3 1.8 1.8
N 28 26 28 26
         
POSTNATAL LOSS        
% OF LIVING PUPS 3.5 1 0.3 0
LITTERS AFFECTED (#) 3 3 1 0
TOTAL (#) 12 3 1 ## 0 ##
MEAN (+) 0.4 0.1 0 0
ST.DEV. 1.9 0.3 0.2 0
N 28 26 28 26
         
CULLED PUPS TOTAL 111 80 76 68
         
LIVING PUPS DAY 4 P.P. TOTAL 216 203 222 202
MEAN (+) 7.7 7.8 7.9 7.8
ST.DEV. 1.5 1 0.4 0.8
N 28 26 28 26
         
BREEDING LOSS DAYS 5 - 21 P.P.
% OF LIVING PUPS AT DAY 4 P.P.  0.5 0 0  1.5
LITTERS AFFECTED (#) 1 0 0 3
TOTAL (#) 1 0 0 3
MEAN (+) 0 0 0 0.1
ST.DEV. 0.2 0 0 0.3
N 28 26 28 26
         
LIVING PUPS DAY 21 P.P.
% OF MALES / FEMALES (#)  53 / 47  50 / 50  50 / 50  50 / 50
TOTAL 215 203 222 199
MEAN (+) 7.7 7.8 7.9 7.7
ST.DEV. 1.5 1 0.4 1
N 28 26 28 26

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

  GROUP 1 CONTROL GROUP 2
500 PPM
GROUP 3
1500 PPM
GROUP 4
5000 PPM
Total number of offspring born 342 286 300 272
Total number of uterine implantation sites 353 310 322 288
Number of live offspring on Day 1 after littering 339 286 299 270
Number of live offspring on Day 4 (before culling) 327 283 298 270
Number of live offspring on Day 4 (after culling) 216 203 222 202
Number of live offspring on Day 21 after littering 215 203 222 199
Post-implantation survival index (%)
(Total number of offspring born/Total number of uterine implantation sites) * 100
97 92 93 94
Live birth index (%)
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
99 100 100 99
Viability index (%)
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
96 99 100 100
Weaning index (%)
(Number of live offspring on Day 21 after littering/Number of live offspring on Day 4 (after culling)) * 100
100 100 100 99

REPRODUCTION DATA SUMMARY - F1-generation

  GROUP 1 GROUP 2 GROUP 3 GROUP 4
  CONTROL 500 PPM 1500 PPM 5000 PPM
Males paired 25 24 25 24
Males mated 25 24 24 24
Females paired 25 24 25 24
Females mated 25 24 24 24
Pregnant females 25 24 24 23
Females with living pups on Day 1 25 24 24 23
Mating index males (%)
(Males mated / Males paired) * 100
100 100 96 100
Fertility index males (%)
(Pregnant females / Males mated) * 100
100 100 100 96
Mating index females (%)
(Females mated / Females paired) * 100
100 100 96 100
Fertility index females (%)
(Pregnant females / Females mated) * 100
100 100 100 96
Gestation index (%)
(Females with living pups on Day 1 / Pregnant females) * 100
100 100 100 100

IMPLANTATION SITES SUMMARY F1-generation

    GROUP 1 GROUP 2 GROUP 3 GROUP 4
    CONTROL 500 PPM 1500 PPM 5000 PPM
Implantations  MEAN 12.4 12.5 11.9 10.1 ++
ST.DEV 1.8 1.7 2.2 2.3
N 25 24 24 23

+/++ Steel-test significant at 5% (+) or 1% (++) level

Developmental Data - F1-generation

  GROUP 1 GROUP 2 GROUP 3 GROUP 4
 CONTROL 500 PPM 1500 PPM 5000 PPM
LITTERS        
TOTAL 28 26 28 26
         
DURATION OF GESTATION        
MEAN (+) 21.4 21.3 21.2 21.4
ST.DEV. 0.5 0.5 0.4 0.7
N 25 24 24 23
 
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#) 0 1 2 2
TOTAL 0 1 5 2
MEAN (+) 0 0 0.2 0.1
ST.DEV. 0 0.2 0.8 0.3
N 25 24 24 23
 
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#) 59 / 41 51 / 49 54 / 46 49 / 51 #
TOTAL 292 277 259 207
MEAN (+) 11.7 11.5 10.8 9.0 ++
ST.DEV. 1.9 2.2 1.9 2.7
N 25 24 24 23
         
POSTNATAL LOSS
% OF LIVING PUPS 1.4 0.7 0.8 1
LITTERS AFFECTED (#) 4 2 2 2
TOTAL (#) 4 2 2 2
MEAN (+) 0.2 0.1 0.1 0.1
ST.DEV. 0.4 0.3 0.3 0.3
N 25 24 24 23
         
CULLED PUPS TOTAL 88 84 66 38
         
LIVING PUPS DAY 4 P.P. TOTAL 200 191 191 167
MEAN (+) 8 8 8 7.3
ST.DEV. 0 0.2 0.2 1.6
N 25 24 24 23
         
BREEDING LOSS DAYS 5 - 21 P.P.
% OF LIVING PUPS AT DAY 4 P.P. 0 0.5 0 0.6
LITTERS AFFECTED (#) 0 1 0 1
TOTAL (#) 0 1 0 1
MEAN (+) 0 0 0 0
ST.DEV. 0 0.2 0 0.2
N 25 24 24 23
         
LIVING PUPS DAY 21 P.P.
% OF MALES / FEMALES (#) 56 / 44 49 / 51 51 / 49 54 / 46
TOTAL 200 190 191 166
MEAN (+) 8 7.9 8 7.2 +
ST.DEV. 0 0.3 0.2 1.6
N 25 24 24 23

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

  GROUP 1 CONTROL GROUP 2
500 PPM
GROUP 3
1500 PPM
GROUP 4
5000 PPM
Total number of offspring born 292 278 264 209
Total number of uterine implantation sites 310 300 285 233
Number of live offspring on Day 1 after littering 292 277 259 207
Number of live offspring on Day 4 (before culling) 288 275 257 205
Number of live offspring on Day 4 (after culling) 200 191 191 167
Number of live offspring on Day 21 after littering 200 190 191 166
Post-implantation survival index (%)
(Total number of offspring born/Total number of uterine implantation sites) * 100
94 93 93 90
Live birth index (%)
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
100 100 98 99
Viability index (%)
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
99 99 99 99
Weaning index (%)
(Number of live offspring on Day 21 after littering/Number of live offspring on Day 4 (after culling)) * 100
100 99 100 99

Applicant's summary and conclusion

Executive summary:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of the test item on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the test article on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation. Furthermore, the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments characterized potential effects in those systems. The dose levels in this study were selected to be 0, 500, 1500, 5000 ppm, based on the results of a preliminary reproductive toxicity study with dietary exposure of the test substance in rats. As food intake is considerably higher in lactating females, dietary concentrations were lowered by 50% during the lactation period of the F0-females. Similar to the F0-generation, dietary concentrations for Cohort 1B females were lowered by 50% during the lactation period as indicated below.

Chemical analyses of dietary preparations were conducted on six occasions during the study to assess accuracy, homogeneity and/or stability over 10 days at room temperature under normal laboratory light conditions and 21 days in the freezer. For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrous, estrous cycle determination, functional observations including acoustic startle response, immunotoxicity assessments using TDAR assay, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations, neurohistopathological examinations and morphometric analysis of brain tissues. In addition, the following reproduction/developmental parameters were determined for the F0- and F1-generation: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).

Diet preparations were considered homogeneous at 5000 ppm and analysis of accuracy revealed acceptable levels at all concentrations. In 2 out of 5 occasions, the diets prepared for Group 2 (500 ppm) were slightly outside the target criteria for homogeneity. This deviation was considered minimal on both occasions (coefficient of variation was 12% and 18% instead of ≤10%) and accuracies of individual samples were within or above target criteria for accuracy (91-127% and 87-138% compared to 80-120%, respectively). Furthermore, for the remaining occasions, diets prepared at 500 ppm were considered homogeneous and accurate. Therefore, it was considered that all Group 2 animals were exposed to a diet concentration of 500 ppm or minimally above during the complete study. Although trial diets prepared at 250 ppm were inhomogeneous, diets prepared for use in Week 32 and administered to the animals were homogenous at this concentration. Therefore, the inhomogeneity during the trial preparation did not impact the study.

F0-Generation - Parental results

Parental effects were observed at all dose levels tested.

In males, body weight and body weight gain were lower in all treatment groups compared to concurrent controls on most days of treatment. Notably, there was no dose response between 500 and 1500 ppm. The lowered body weights and body weight gains occurred without a concurrent decrease in food consumption. In fact, increased relative food consumption was noted compared to concurrent controls at 1500 ppm on several occasions and at 5000 ppm throughout the treatment period. The delayed growth of 1500 and 5000 ppm males in the presence of higher relative food intake is indicative for a lower food efficiency, i.e. more food has to be ingested to reach the same growth in the animal’s mass. Given the magnitude of the effect at 1500 and 5000 ppm (approximately 8-13% lower terminal weight compared to controls) in combination with the decreased food efficiency, it was considered related to treatment with the test item and adverse from 1500 ppm. As food consumption was unaffected at 500 ppm, the effect on body weight was considered minimal and non-adverse at this dose level. For females at 5000 ppm, body weights and body weight gains were decreased compared to controls throughout the premating, mating and post-coitum period. Although body weight gains were increased during the last part of the lactation period, terminal body weights were lower compared to concurrent controls. Furthermore, increased relative food consumption was noted compared to concurrent controls at 5000 ppm throughout the treatment period, indicative for a lower food efficiency. Based on the magnitude of the effect that lasted throughout the largest part of the treatment period, together with the decreased food efficiency this was considered adverse. For females at 500 and 1500 ppm, a similar trend with lower body weights and body weight gains was observed during the premating and mating period. Body weight gains were not affected during the post-coitum and lactation period. No concurrent decrease in food consumption was observed. Food consumption was actually increased on several occasions during the treatment period at 1500 ppm. Based on the combination of lower body weights and a lower food efficiency, this was considered adverse at 1500 ppm. Whereas food consumption was unaffected at 500 ppm. In combination with the relatively small effect on body weight this dose level, this was considered non-adverse. The observed reduction in food consumption during Week 1 of the study in both males and females at 1500 and 5000 ppm, was most likely contributed a palatability issue with the diet containing test item, as it recovered to normal levels thereafter. Differences in clinical biochemistry parameters were observed at 5000 ppm. These consisted of higher total bilirubin and bile acid levels in males and higher alanine aminotransferase activity in females. These differences were considered non-adverse as they occurred in one sex only and in absence of a microscopic correlate at the organ level. Several changes were observed in organ weights (brain, spleen, adrenal glands, heart, liver, thyroid gland, kidneys, thymus, seminal vesicles, testes, epididymides and ovaries) at all dose levels. As changes in organ/relative to body weight occurred with minor or no changes in absolute weight or vice versa, these changes were regarded to be the result of a lower terminal body weight and not directly affected by treatment with the test item. There were no macroscopic or microscopic findings correlates for these organ weight changes. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/viability, clinical appearance, hematology and coagulation parameters, thyroid hormone analysis, urinalysis, macroscopic examination and microscopic examination).

F0- and F1-Generation - Reproductive results

In F0-females, a lower number of implantation sites was observed in all treatment groups. Overall, the effect was relatively small. Although statistical significance was reached at 5000 ppm only, the litter sizes were similar in all treatment groups. However, as the a control mean was comparable with historical control mean a treatment related effect could not be excluded. Futhermore, the decrease in number of implantation sites was accompanied by a lower live litter size in all groups compared to concurrent controls. Simlary to the implantation sites, differences in litter size between the treatment groups was relatively small. To elucidate this equivocal result and establish a NOAEL for the effect, the study was extended with a second generation (i.e. Cohort 1B animals were mated to produce an F2-generation). For the F1-females, the mean number of implantations were lower at 1500 and 5000 ppm. At both dose levels, this was accompanied by a lower live litter size compared to concurrent and historical control mean. Given the similar effects in the F0 and F1-generation at both dose levels, this was considered related to treatment with the test item. The number of implantation sites for F1-females at 500 ppm was similar to concurrent control and historical control mean. Furthermore, litter size was unaffected at 500 ppm. Therefore, the slightly lower number of implanation sites and live litter size in F0-females at 500 ppm was considered unrelated to treatment. Despite the overall lower number of implantation sites and smaller live litter size at 1500 ppm, the majority of the individual values remained within the same range as concurrent controls. Therefore, at this dose the effects were considered non-adverse. At 5000 ppm, however, the effects were more pronounced, especially in the F1-females with values at the lower end of historical controls and up to 7 females with values below the concurrent control range (live litter size). Given the magnitude of the effect and consistency between the F0 and F1-generation, these findings were considered adverse at 5000 ppm. The significantly lower sperm count of the epididymides at 1500 and 5000 ppm in F0-males remained within historical control range and there was no correlation between low sperm count and live litter size at the individual level. Furthermore, sperm counts in the Cohort 1A males were unaffected by treatment with the test item. Therefore, the lower sperm count in F0-males at 1500 and 5000 ppm was considered a spurious finding. No test item-related changes were noted in any of the remaining reproductive parameters investigated in this study for F0 and F1 animals (i.e. mating and fertility indices, precoital time, estrous cycle, and histopathological examination of reproductive organs including stagedependent qualitative evaluation of spermatogenesis in the testis).

F0-Generation / F1-Generation (pre-weaning) and F1-Generation / F2-Generation - Developmental results

Developmental toxicity was observed from 1500 ppm onwards.

The decreased litter size F0 and F1-females at 1500 and 5000 ppm were considered the result of the lower number of implantation sites (see above), and considered adverse at 5000 ppm. Body weights of F1-pups were progressively decreased during postnatal Day 14 until 21 from 500 ppm. The effect observed at 500 and 1500 ppm was minimal to slight, at PND 21 pup body weights were 3-8% lower compared to concurrent control, and were therefore considered non-adverse. As the decrease was progressive at an early stage in development and resulted in 14% lower pup body weights at 5000 ppm compared to concurrent controls on PND 21, this change was regarded adverse. At 1500 and 5000 ppm, body weights of pups of the F2-generation were decreased by treatment with the test item at PND 21. The progression and size of the effect were similar to F1-pups and considered adverse at 5000 ppm. In Cohort Surplus pups (PND 22-24), spleen weights were decreased at 500 (females) and 1500 and 5000 ppm (males and females). This could only be partly contributed to the lower body weights of the pups. The organ/body weight ratio at 1500 and 5000 ppm were >10% lower compare to concurrent controls. At 5000 ppm, mean values were 0.6x (females) and 0.8x (males) of control and outside the historical control range. The findings were corroborated by similar effects observed in F2-pups (see below). Given the magnitude of the effect, this decrease was considered adverse at 5000 ppm. As the mean values remained within historical control range at 500 and 1500 ppm this was considered non-adverse. The decreased thymus weights in Cohort Surplus pups at 5000 ppm remained within historical control range and were therefore considered not adverse. Similar to F1-pups, a treatment related decrease in spleen weights was observed in F2-pups at 1500 and 5000 ppm. All mean values remained within historical control range. Given the magnitude of the effect, this was considered adverse at 5000 ppm. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, live birth, viability and weaning indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, thyroid hormone levels in F1-animals (total T4 of PND 4 and 22 pups and TSH of PND 22 pups) and macroscopic examination).

F1-Generation (post-weaning) – Developmental results

(Developmental) toxicity was observed at 5000 ppm during the post-weaning phase. Due to the progressive body weight loss during the last week of lactation, body weights at weaning were reduced compared to controls in all treatment groups. Body weights were further reduced compared to controls in Week 2 after weaning, due to lower food intake in the first two weeks after weaning. This lowered food consumption was similar to the reduced food consumption observed in the F0-generation. Although pups already had access to the diet when housed with the dams, this reduction may still reflect a palatability issue as no alternative food source is available after weaning. Therefore, this effect was considered nonadverse. At 5000 ppm, body weight gain did not fully recover to normal levels for males. Consequently, differences in body weight between controls and 5000 ppm treated males increased from 0.85-0.87 at weaning to 0.80-0.84 at the end of the treatment period. Together with the increased relative food consumption indicative of decreased food efficiency similar to the F0-animals, this was considered adverse. For females at 5000 ppm, body weight gain recovered to normal levels from Day 15 onwards and consequently, differences between controls and 5000 ppm treated females remained similar at weaning and at the end of treatment. Body weight gain recovered (females) or almost recovered (males) to normal levels at 1500 ppm. At the end of the treatment period, differences in body weight compared to controls were similar compared to the differences in body weight at weaning. Therefore, the lower body weights at the end of the treatment period for females at 5000 and 1500 ppm and males at 1500 ppm were considered not a direct effect of treatment with the test item after weaning. At 1500 ppm and 5000 ppm, males and females reached balanopreputial separation and vaginal opening at a higher age than concurrent controls. As a consequence of the delayed time to vaginal opening, also the age that females reached first estrus was increased. The time between vaginal opening and first estrous was unaffected by treatment with the test item. As the delay in sexual maturation was attributed to the lower body weights of these animals, it was regarded non-adverse. Lymphocyte counts were lower in females at 5000 ppm. As values remained within the historical control range, were partly the result of relatively high controls (lymphocytes) and in the absence of macroscopic or microscopic correlates, these changes were considered nonadverse. Several statistically significant changes were observed in organ weights (brain and fixed brain weights, spleen, heart, pituitary gland, thyroids, liver, thymus, mesenteric and axillary lymph nodes, kidneys, adrenal glands, prostate gland, testes, epididymides, seminal vesicles and ovaries) at all dose levels. These changes were attributed to the lower terminal body weight and not directly caused by treatment with the test item. In absence of macroscopic or microscopic findings organ weight changes, they were considered non-adverse. No treatment related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/viability, clinical appearance, estrous cycle determination, sperm analysis, coagulation and clinical biochemistry parameters, thyroid hormones, urinalysis, gross necropsy findings, and histopathological examinations (including ovarian follicle and corpora lutea counts, stage-dependent qualitative evaluation of spermatogenesis).

F1-Generation (post-weaning) - Developmental Neurotoxicity

Lower grip strength of the foreleg was observed for males at 5000 ppm. As all values remained within the historical control range and there were no indications of neuro-muscular degeneration based on clinical signs and other parameters that were part of the detailed functional observation battery, the lower grip strength was regarded non-adverse. No treatment related changes were noted in any of the remaining parameters investigated in this study (i.e. acoustic startle response, detailed clinical observations, rectal temperature, motor activity, foot splay, grip strength of the hind leg, hearing ability, pupillary reflex, brain

dimensions, brain histopathology and morphometry).

F1-Generation (post-weaning) - Developmental immunotoxicity

No treatment related changes were noted in splenic lymphocyte subpopulation analysis and TDAR assay.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following no-observed-adverse-effect level (NOAEL) of the test item were established:

General Toxicity NOAEL:

F0-generation: 500 ppm (on average corresponding to 36 mg/kg/day in males and 41 mg/kg/day in females of the F0-generation; based on a decreased body weights and food consumption at 1500 ppm).

F1-generation: 1500 ppm (on average corresponding to 121-163 mg/kg/day in males and 130-165 mg/kg/day in females of the F1-generation; based on a decreased body weight and food consumption at 5000 ppm).

Reproduction Toxicity NOAEL

F0-generation and F1-generation: 1500 ppm (on average corresponding to 109 mg/kg/day in males and 126 mg/kg/day in females of the F0-generation, and 121-163 mg/kg/day in males and 130-165 mg/kg/day in females of the F1-generation; based on a decreased number of implantation sites at 5000 ppm).

Developmental General Toxicity NOAEL

F1-generation: 1500 ppm (on average corresponding to 121-163 mg/kg/day in males and 130-165 mg/kg/day in females of the F1-generation; based on decreased spleen and body weights at 5000 ppm).

F2-generation: 1500 ppm (on average corresponding to 121-163 mg/kg/day in males and 130-165 mg/kg/day in females of the F1-generation; based on decreased spleen and body weights at 5000 ppm).

Developmental Neurotoxicity NOAEL

F1-generation: At least 5000 ppm (on average corresponding to 428-552 mg/kg/day in males and 451-573 mg/kg/day in females).

Developmental Immunotoxicity NOAEL

F1-generation: At least 5000 ppm (on average corresponding to 428-552 mg/kg/day in males and 451-573 mg/kg/day in females).

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