Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an EOGRTS study including Cohorts 1, 2 and 3, parental toxicity was observed from the medium dose level (1500 ppm) based on decreased body weights and decreased food consumption. Reproduction was affected at the high dose level (5000 ppm) as shown by on a decreased number of implantation sites. At the highest dose level, spleen weights of pups were also reduced. No effects were reported regarding developmental neurotoxicity and developmental immunotoxicity.

In two preceding OECD 422 studies with both the source and the target chemical (and higher dose levels), effects on body weight gain and food consumption were reported as well. In addition, lower implantation sites were reported in high-dose females, in line with the results of the EOGRTS. The maternal toxicity at the highest dose level was more severe, and as a secondary effect the estrous cycle was disturbed.

Link to relevant study records

Reference

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

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.)

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.

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.

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

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.

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 (%)

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: 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.

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

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 mean25 (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.

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

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.

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:

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.

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

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.

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

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

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 means^a	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 means^a	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 means^a	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 paired^1)	28	28	28	28
Males mated^1)	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

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).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

109 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An Extended One Generation Reproductive Toxicity Study was performed with the registered substance including Cohorts 1A, 1B (extended to a second generation), 2 (developmental neurotoxicity) and 3 (developmental immunotoxicity). Wistar Han rats were administered the test item by dietary administration (powder diet) with diet concentrations of 500, 1500 and 5000 ppm. During the lactation period, animal received 250, 750 and 2500 ppm. The rats of the control group received standard powder rodent diet.

F0-males were administered the test diet for at least 11 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. F0-females were administered the test diet for a minimum of 16 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for at least 13 weeks.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, spilled diet from the food hopper or when they commence eating for themselves.

From weaning onwards (PND 21), F1-animals of Cohorts 1A received diet containing test item up to and including the day before scheduled necropsy (9-10 weeks). The F1-animals of Cohorts 2A, and 3 were treated for at least 7 weeks and 5 weeks until the day of necropsy. Animals of Cohorts 2A and 3 respectively. The F1-animals of Cohorts 2B and Surplus, as well as Spares (not assigned to one of the cohorts) were not separated from their dams before necropsy and had therefore access to the test diet present in the cage intended for the dam.

The F1-animals of Cohort 1B were treated from weaning onwards, including at least 11 weeks prior to the second mating period, the duration of pregnancy, and at least 21 days after delivery up to and including the day of scheduled necropsy. The F2-animals were not separated from their dams before necropsy and had therefore access to the test diet present in the cage intended for the dam. Animals of Cohort 1B were exposed for at least 13 weeks (males) or 16 weeks (females). F1-Females which failed to deliver were treated for at least 14 weeks.

Diet analysis

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 the acceptable concentration range of 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. Relative 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 implantation 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 counts in F0-males at 1500 and 5000 ppm were 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 considered decreased by treatment with the test item 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, 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. 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 of Cohort Surplus, a treatment related decrease in spleen weights was observed 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 0.84-0.87 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 estrous 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 and as no effects on mating and fertility were observed for the F1-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 non-adverse.

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.

Conclusion

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) 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).

Effects on developmental toxicity

Description of key information

The target substance and the source substance did not produce toxicity to development in both rats and rabbits in guideline-compliant OECD 414 studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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 4 hours at room temperature under normal laboratory light conditions has been confirmed over the concentration range 1 to 250 mg/mL (suspensions). Homogeneity of formulations was considered acceptable for the purpose of this study.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France
- Age at study initiation: 17-19 weeks
- Weight at study initiation: between 3064 and 4252 g
- Fasting period before study:
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet: Pelleted diet for rabbits (Global Diet 2030 from Envigo Teklad®, Mucedola, Milanese, Italy) was provided ad libitum t
- Water: ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C
- Humidity (%): 48 to 97%. The values that were outside the targeted range (40-70%) occurred for 15 days with a minimum or maximum of 97% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 4 hours after adding the vehicle to the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80
- Concentration in vehicle: 1.5, 5, 15 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis during week 1. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. For the formulation of Group 2, the mean accuracy was 125%, i.e. outside the acceptance criterion of 85% to 115% of nominal concentration. However, it should be noted that the mean accuracy of the 1 mg/mL QC samples was also relatively high at 117%. The concentrations analyzed in the formulations of Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

Details on mating procedure:

Animales were delivered time-mated

Duration of treatment / exposure:

Day 6 to 28 post-coitum

Frequency of treatment:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive.

Duration of test:

gestation day 29

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder, and in an attempt to produce graded responses to the test item.

Maternal examinations:

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 was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

DETAILED CLINICAL OBSERVATIONS:
Clinical observations were performed at least once daily, beginning on Day 6 post-coitum and lasting 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 1, 2, 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 WEIGHT:
Animals were individually weighed on Days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In addition, data on body weight Day 0 post-coitum (i.e. the day of mating) was provided by the Supplier (non-GLP) and included in the report. In order to monitor the health status, animal No. 44 (6 mg/kg/day) was also weighed on Day 26 post-coitum (documented in study raw data).

FOOD CONSUMPTION
Food consumption was quantitatively measured for Days 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 29

Unscheduled Deaths – F0-Generation
If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%), underwent
necropsy, and specified tissues were retained.

Scheduled Euthanasia – F0-Generation
Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%) on Day 29 post-coitum. No body weight was recorded at necropsy.

Necropsy – F0-Generation
All animals (including animals sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed.

Ovaries and uterine content:

Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.

Fetal examinations:

Method of Euthanasia – F1-Generation
Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube1.

Fetal Examinations (unscheduled) – F1-Generation
For normal implantations in development of females sacrificed before planned necropsy, a gross external examination was performed (if possible).

Fetal Examinations (scheduled) – F1-Generation
Litters of females surviving to scheduled necropsy, or that delivered on the day of scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described in the following sections.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined. Late resorptions were not examined or fixed.

Visceral Examinations – F1-Generation
All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice. All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations – F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from all groups. All specimens were archived in glycerin with bronopol as preservative.

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 as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) 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.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.

Indices:

Preimplantation loss (%), Postimplantation loss (%), Viable fetuses affected/litter (%)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs were noted during the observation period that were considered to be related to treatment with the test item.
One animal at 6 mg/kg/day (No. 44) showed hunched posture, swelling of the genital region, piloerection, and lean appearance between post-coitum Days 24 and 28 (necropsy did not reveal any correlating findings). In absence of similar findings among other animals of this dose group or other dose groups, these findings were considered incidental and not related to treatment with the test item. Two animals at 60 mg/kg/day showed piloerection on post-coitum Day 28 (No. 67) or Days 25 and 26 (No. 85). Given the very incidental occurrence, this symptom was considered not to be related to treatment with the test item. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
Two animals (one animal at 6 mg/kg/day (No. 30) and one animal at 20 mg/kg/day (No. 48)) were sacrificed for ethical reasons on Days 1 and 11 of treatment, respectively. Both mortalities were attributed to a gavage incident, since blood was noted in or on the dosing tube immediately after dosing, followed by laboured respiration and pallor (Nos. 30 and 48) and gasping and breathing rales (Nos. 30 and 48, respectively). Necropsy showed red foci or perforations in the lungs (Nos. 30 and 48, respectively). Both females were gravid.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg/day, a minor but statistically significantly lower mean body weight gain was recorded throughout the post-coitum period. At the end of the treatment period, mean body weights were the same as the control mean. Mean body weight gain corrected for gravid uterus at this dose level appeared lower than the control mean (not statistically significant and within the historical control range; -226.5 grams (-6.2%) vs. -120.6 grams (-3.3%) in the control group, and -195.4 grams (-5.1%) and -188.4 grams (-5.0%) at 6 and 20 mg/kg/day, respectively). Body weights and body weight gain at 6 and 20 mg/kg/day remained in the same range as controls over the study period, except for a statistically significantly lower body weight gain at 6 mg/kg/day on post-coitum Day 15. Since this occurred on a single occasion and in the absence of a dose-related response, and since this change was not consistently noted as treatment progressed, this was considered not to be related to treatment with the test item. Body weights on the day of necropsy corrected for gravid uterus weight were considered not affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg/day, food consumption before and after correction for body weight was statistically significantly lower on several occasions between post-coitum Days 6 and 21. Mean over mean absolute food intake at this dose level over the post coitum period was 0.88x of the control mean. Food consumption before and after correction for body weight at 6 and 20 mg/kg/day was considered not affected by treatment with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy showed no lesions that were considered to be related to treatment with the test item. Necropsy findings of the two animals that were sacrificed due to a gavage incident consisted of red foci or perforations in the lungs. All necropsy findings recorded for animals surviving until scheduled necropsy are occasionally seen among rabbits used in this type of study, and their incidence did not indicate a relationship to treatment with the test item.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites, pre- and postimplantation loss and early and late resorptions in the control and test groups were similar and in the range of normal biological variation. For one animal at 60 mg/kg/day (A084), a relatively high number of early resorptions was recorded (30.8%, exceeding the historical data range). Since the same number of early resorptions was also recorded for one control animal (A006), this was considered not to be related to treatment with the test item.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of two females in the control group, three females at 6 mg/kg/day, two females at 20 mg/kg/day and one female at 60 mg/kg/day were non-gravid. The number of non-gravid females remained within the historical control range.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no adverse effects reported

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights (both sexes) were not affected by treatment with the test item. Mean combined (male and female) fetal body weights were 37.1, 38.3, 37.2 and 37.0 gram for the control, 6, 20 and 60 mg/kg/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered not affected by treatment with the test item. Mean sex ratios (males:females) were 47.8:52.2, 49.5:50.5, 50.3:49.7 and 52.4:47.6 for the control, 6, 20 and 60 mg/kg/day groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was not affected by treatment with the test item. Mean litter sizes were 9.1, 9.8, 10.0 and 9.0 fetuses/litter for the control, 6, 20 and 60 mg/kg/day groups, respectively.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on external morphology following treatment up to 60 mg/kg/day.
External malformations occurred in one fetus each in the control, 6 and 20 mg/kg/day groups and five fetuses from two litters at 60 mg/kg/day. Four of the affected fetuses at 60 mg/kg/day (A078-01, A078-02, A078-08 and A084-09) had carpal flexures that not occurred in the other groups. These malformations were not confirmed during skeletal examinations. Additionally, one of these fetuses, A078-02, had cleft palate that also occurred in littermate A078-04, and which was confirmed during skeletal examinations. Two of the aforementioned fetuses in litter A078 were also viscerally affected, i.e. fetus A078-01 had multiple cardiovascular defects and A078-08 had diaphragmatic hernia and malpositioned kidneys. Although external malformations occurred in five fetuses at 60 mg/kg/day, this was considered not related to treatment with the test item, as the finding “carpal and/or tarsal flexures” are the most common external malformation among historical control fetuses and none were skeletally confirmed.
Moreover, the occurrence of flexed carpal(s) was essentially confined to one litter (A078) which contained three fetuses showing this malformation (A078-01, -02 and -08). Also, since all these fetuses also had other external and/or visceral malformations as described above, it could be considered that the malformations had a maternal or hereditary origin and hence were considered not related to treatment with the test item. Other malformations in this study consisted of distended abdomen (fetus A029-06 and A056-01 at 6 and 20 mg/kg/day, respectively) and brachydactyly (control fetus A003-12). As these were recorded as single occurrences and/or in a control fetus, they were considered chance findings.
External variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on skeletal morphology following treatment with 60 mg/kg/day.
Skeletal malformations (vertebral anomalies) occurred in one control (A015-08) and four fetuses of two litters at 20 mg/kg (A045-03, -09, -10 and A059-04). These malformations occurred in absence of a dose-related incidence (i.e. absence of this finding at 60 mg/kg/day) and were essentially confined to one litter. Also, vertebral anomaly is the most common skeletal malformation among historical control foetuses. Therefore, it was considered that these malformations were not related to treatment with the test item. A caudal shift of the pelvic girdle was observed at a statistically significantly higher incidence at 60 mg/kg/day. This variation occurred at 10.2%, 20.8%, 18.3% and 38.8% in the control, 6, 20 and 60 mg/kg/day group, respectively. The incidence at the high dose marginally exceeded the historical control maximum value (38.5% per litter) and was not accompanied by a significant increase of 13th full ribs, which are frequently encountered in parallel. Therefore, the occurrence of caudal shift of pelvic girdle was considered not to be related to treatment.
A statistically significantly higher incidence of unossified hyoid bodies was recorded at 60 mg/kg/day. Mean incidences per litter of this variation were 0.0%, 0.4% (one fetus, i.e. A026-07), 0.0% and 1.9% (four fetuses, i.e. A075-03, A076-06, A078-02 and A088-06) in the control, 6, 20 and 60 mg/kg/day group, respectively. Three of these fetuses at 60 mg/kg/day (A075-03, A076-06 and A088-06) had a body weight that was notably lower than the mean litter weight and mean group litter weight. However, mean fetal weights were considered not affected by treatment with the test item. Also, the mean incidence of unossified metacarpals and/or metatarsals and unossified sternebra nos. 5 and/or 6 (the two main ossification parameters) appeared higher or lower than the control mean, respectively, instead of a consistent increase that would be expected in case of skeletal ossification delay. Therefore, it was considered that unossified hyoid bodies were not related to treatment with the test item.
All other skeletal variations that occurred were not considered treatment related as they occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 60 mg/kg/day.
Visceral malformations occurred in one fetus of the control and 20 mg/kg/day group, five fetuses of three litters at 6 mg/kg/day and in three fetuses of two litters at 60 mg/kg/day. At 60 mg/kg/day, one fetus (A084-05) had one large lung cyst, one fetus (A078-01) had
multiple cardiovascular defects and one fetus (A078-08) had diaphragmatic hernia and malpositioned kidneys. One fetus at 20 mg/kg/day (A059-07) had two cysts associated with the colon. At 6 mg/kg/day, three fetuses (A026-01, -07 and A033-12) had one or more
cardiovascular abnormalities and two fetuses (A037-02 and -12) had internal hydrocephaly. One control fetus (A003-10) had a malpositioned testis. Based on the group distribution, single occurrence and/or occurrence in a control fetus, these malformations were considered not to be related to treatment with the test item.
All variations noted were considered unrelated to treatment with the test item as they occurred in the absence of a dose-related trend, occurred infrequently and/or at frequencies that were within the range of historical control data.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects reported

Developmental effects observed:

no

BODY WEIGHTS (GRAM) SUMMARY FEMALES

		Group 1	Group 2	Group 3	Group 4
		Control	6 mg/kg bw	20 mg/kg bw	60 mg/kg bw
DAY 0	MEAN	3525	3599	3640	3587
	ST.DEV.	303.3	316.2	298.5	236.8
	N	20	19	20	21
DAY 3	MEAN	3463	3589	3574	3539
	ST.DEV.	322.7	308.4	279.4	235.1
	N	20	19	20	21
DAY 6	MEAN	3514	3673	3649	3623
	ST.DEV.	336	320.7	272.7	237.6
	N	20	19	20	21
DAY 9	MEAN	3602	3724	3712	3618
	ST.DEV.	357.2	329	286.4	238
	N	20	18	20	21
DAY 12	MEAN	3655	3782	3762	3658
	ST.DEV.	345.2	309.6	292.5	239.5
	N	20	18	20	21
DAY 15	MEAN	3726	3819	3819	3703
	ST.DEV.	343.3	297.2	287.6	222.4
	N	20	18	20	21
DAY 18	MEAN	3755	3868	3853	3747
	ST.DEV.	364.3	299	293.8	240.3
	N	20	18	19	21
DAY 21	MEAN	3791	3900	3887	3763
	ST.DEV.	358.9	308.2	275.5	238.8
	N	20	18	19	21
DAY 24	MEAN	3815	3910	3916	3801
	ST.DEV.	352.8	357.9	263.2	259.8
	N	20	18	19	21
DAY 27	MEAN	3834	3942	3942	3845
	ST.DEV.	343	337.3	258.6	240.1
	N	20	18	19	21
DAY 29	MEAN	3867	3987	3980	3867
	ST.DEV.	364.7	320.9	259	252.3
	N	20	18	19	21

BODY WEIGHT GAIN (%) SUMMARY FEMALES

		Group 1	Group 2	Group 3	Group 4
		Control	6 mg/kg bw	20 mg/kg bw	60 mg/kg bw
DAY 6	MEAN	0	0	0	0
	ST.DEV.	0	0	0	0
	N	20	19	20	21
DAY 9	MEAN	2	1	2	0 **
	ST.DEV.	2.2	1.5	1.6	1.7
	N	20	18	20	21
DAY 12	MEAN	4	3	3	1 **
	ST.DEV.	2.6	1.8	2.2	1.5
	N	20	18	20	21
DAY 15	MEAN	6	4 *	5	2 **
	ST.DEV.	2.8	2.8	3	2.5
	N	20	18	20	21
DAY 18	MEAN	7	5	6	3 **
	ST.DEV.	3	2.4	3.8	2.8
	N	20	18	19	21
DAY 21	MEAN	8	6	7	4 **
	ST.DEV.	3.7	2.7	3.8	3
	N	20	18	19	21
DAY 24	MEAN	9	6	8	5 **
	ST.DEV.	3.4	3.5	3.9	2.9
	N	20	18	19	21
DAY 27	MEAN	9	7	8	6 *
	ST.DEV.	3.6	3.3	4.3	3.4
	N	20	18	19	21
DAY 29	MEAN	10	8	9	7 *
	ST.DEV.	4.1	3.7	4.3	3.7
	N	20	18	19	21

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

SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

DOSE GROUP	1		2		3		4
	NO.	%	NO.	%	NO.	%	NO.	%
FEMALES ON STUDY	22		22		22		22
FEMALES THAT ABORTED OR DELIVERED	0	0	0	0	0	0	0	0
FEMALES THAT DIED	0	0	0	0	0	0	0	0
FEMALES THAT ABORTED	0	0	0	0	0	0	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	0	0	0	0	0	0
FEMALES THAT WERE EUTHANIZED	0	0	1	4.5	1	4.5	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	1	100	1	100	0	0
FEMALES EXAMINED AT SCHEDULED NECROPSY	22	100	21	95.5	21	95.5	22	100
NONGRAVID	2	9.1	3	14.3	2	9.5	1	4.5
GRAVID	20	90.9	18	85.7	19	90.5	21	95.5
WITH RESORPTIONS ONLY	0	0	0	0	0	0	0	0
WITH VIABLE FETUSES	20	100	18	100	19	100	21	100
TOTAL FEMALES GRAVID	20	90.9	19	86.4	20	90.9	21	95.5

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

Group		Males	Females	Viable Fetuses	Dead Fetuses	Early Resorptions	Late resorptions
1	TOTAL	87	94	181	0	8	4
	MEAN	4.4	4.7	9.1	0	0.4	0.2
	S.D.	1.87	1.84	1.96	0	0.94	0.52
2	TOTAL	91	86	177	0	4	2
	MEAN	5.1	4.8	9.8	0	0.2	0.1
	S.D.	2.29	1.26	2.96	0	0.43	0.32
3	TOTAL	95	95	190	0	7	3
	MEAN	5	5	10	0	0.4	0.2
	S.D.	1.6	1.76	1.7	0	0.68	0.37
4	TOTAL	98	92	190	0	9	3
	MEAN	4.7	4.4	9	0	0.4	0.1
	S.D.	1.43	2.01	1.99	0	0.93	0.36

None significantly different from control group

Group		Post Implantation Loss	Implantation Sites	Corpora Lutera	Pre-Implantation Loss	Fetal weights (g)	No. of gravid females
1	TOTAL	12	193	206	13	NA	20
	MEAN	0.6	9.7	10.3	0.7	37.1
	S.D.	1.39	2.28	2.58	0.75	4.52
2	TOTAL	6	183	192	9	NA	18
	MEAN	0.3	10.2	10.7	0.5	38.3
	S.D.	0.49	3.15	3.25	0.71	4.25
3	TOTAL	10	200	205	5	NA	19
	MEAN	0.5	10.5	10.8	0.3	37.2
	S.D.	0.84	1.84	2.02	0.56	3.68
4	TOTAL	12	202	219	17	NA	21
	MEAN	0.6	9.6	10.4	0.8	37
	S.D.	0.93	2.11	1.83	1.36	5.66

None significantly different from control group

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY (% PER LITTER)

Group	1	2	3	4
CORPORA LUTEA
MEAN	10.3	10.7	10.8	10.4
S.D.	2.58	3.25	2.02	1.83
N	20	18	19	21
IMPLANTATION SITES
MEAN	9.7	10.2	10.5	9.6
S.D.	2.28	3.15	1.84	2.11
N	20	18	19	21
VIABLE FETUSES (%)
MEAN	95	97.1	95.4	94.6
S.D.	10.97	4.46	7.21	7.76
N	20	18	19	21
DEAD FETUSES (%)
MEAN	0	0	0	0
S.D.	0	0	0	0
N	20	18	19	21
EARLY RESORPTIONS (%)
MEAN	3.5	2	3.2	4
S.D.	7.62	4.13	5.86	7.71
N	20	18	19	21
LATE RESORPTIONS (%)
MEAN	1.5	0.9	1.4	1.4
S.D.	4.03	2.59	3.37	3.59
N	20	18	19	21
TOTAL RESORPTIONS (%)
MEAN	5	2.9	4.6	5.4
S.D.	10.97	4.46	7.21	7.76
N	20	18	19	21
PRE-IMPLANTATION LOSS (%)
MEAN	5.7	4.7	2.1	7.6
S.D.	6.52	6.36	4.44	12.04
N	20	18	19	21
POST-IMPLANTATION LOSS (%)
MEAN	5	2.9	4.6	5.4
S.D.	10.97	4.46	7.21	7.76
N	20	18	19	21
MALES (%)
MEAN	47.8	49.5	50.3	52.4
S.D.	15.09	13.1	15.38	16.13
N	20	18	19	21
FEMALES (%)
MEAN	52.2	50.5	49.7	47.6
S.D.	15.09	13.1	15.38	16.13
N	20	18	19	21
MALE FETAL WEIGHTS (g)
MEAN	37.6	39.5	38.3	37.6
S.D.	4.87	4.61	4.65	5.31
N	20	18	19	21
FEMALE FETAL WEIGHTS (g)
MEAN	37	37.2	35.4	36.9
S.D.	4.66	4.65	3.89	5.98
N	20	18	19	21
COMBINED FETAL WEIGHTS (g)
MEAN	37.1	38.3	37.2	37
S.D.	4.52	4.25	3.68	5.66
N	20	18	19	21

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

CORPORA LUTEA AND IMPLANTATION SITES COMPARED USING DUNNETT'S TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

	FETUSES				LITTERS
Dose Group	1	2	3	4	1	2	3	4
NUMBER EXAMINED EXTERNALLY	181	177	190	190	20	18	19	21
ABDOMEN- DISTENDED	0	1	1	0	0	1	1	0
BRACHYDACTYLY	1	0	0	0	1	0	0	0
CARPAL AND/OR TARSAL FLEXURE	0	0	0	4	0	0	0	2
CLEFT PALATE	0	0	0	2	0	0	0	1
NUMBER EXAMINED VISCERALLY	181	177	190	190	20	18	19	21
AORTIC ARCH- DILATED	0	2	0	0	0	1	0	0
PULMONARY TRUNK- NARROW	0	1	0	0	0	1	0	0
ATRIAL SEPTUM DEFECT	0	1	0	0	0	1	0	0
ATRIOVENTRICULAR VALVE- ABSENT	0	1	0	1	0	1	0	1
TESTIS- MALPOSITIONED	1	0	0	0	1	0	0	0
TETRALOGY OF FALLOT	0	1	0	0	0	1	0	0
AORTIC ARCH- NARROW	0	0	0	1	0	0	0	1
PULMONARY TRUNK- DILATED	0	0	0	1	0	0	0	1
VENTRICULAR SEPTUM DEFECT	0	0	0	1	0	0	0	1
ATRIUM- SMALL	0	0	0	1	0	0	0	1
DIAPHRAGMATIC HERNIA	0	0	0	1	0	0	0	1
KIDNEY(S)- MALPOSITIONED	0	0	0	1	0	0	0	1
HYDROCEPHALY- INTERNAL	0	2	0	0	0	1	0	0
VISCERA- CYST(S)	0	0	1	0	0	0	1	0
LUNG- CYST	0	0	0	1	0	0	0	1
NUMBER EXAMINED SKELETALLY	181	177	190	190	20	18	19	21
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	1	0	4	0	1	0	2	0
TOTAL NUMBER WITH MALFORMATIONS
EXTERNAL	1	1	1	5	1	1	1	2
SOFT TISSUE	1	5	1	3	1	3	1	2
SKELETAL	1	0	4	0	1	0	2	0
COMBINED	3	6	6	6	2	4	3	2

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS % PER LITTER

Dose Group		1	2	3	4
NUMBER OF LITTERS EXAMINED EXTERNALLY		20	18	19	21
ABDOMEN- DISTENDED	MEAN	0	0.5	0.4	0
	S.D.	0	1.96	1.91	0
BRACHYDACTYLY	MEAN	0.5	0	0	0
	S.D.	2.24	0	0	0
CARPAL AND/OR TARSAL FLEXURE	MEAN	0	0	0	2.3
	S.D.	0	0	0	8.42
CLEFT PALATE	MEAN	0	0	0	1.2
	S.D.	0	0	0	5.46

None significantly different from control group

NUMBER OF LITTERS EXAMINED VISCERALLY		20	18	19	21
AORTIC ARCH- DILATED	MEAN	0	0.8	0	0
	S.D.	0	3.37	0	0
PULMONARY TRUNK- NARROW	MEAN	0	0.4	0	0
	S.D.	0	1.68	0	0
ATRIAL SEPTUM DEFECT	MEAN	0	0.4	0	0
	S.D.	0	1.68	0	0
ATRIOVENTRICULAR VALVE- ABSENT	MEAN	0	0.4	0	0.6
	S.D.	0	1.68	0	2.73
TESTIS- MALPOSITIONED	MEAN	0.5	0	0	0
	S.D.	2.24	0	0	0
TETRALOGY OF FALLOT	MEAN	0	0.4	0	0
	S.D.	0	1.81	0	0
AORTIC ARCH- NARROW	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
PULMONARY TRUNK- DILATED	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
VENTRICULAR SEPTUM DEFECT	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
ATRIUM- SMALL	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
DIAPHRAGMATIC HERNIA	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
KIDNEY(S)- MALPOSITIONED	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
HYDROCEPHALY- INTERNAL	MEAN	0	1	0	0
	S.D.	0	4.29	0	0
VISCERA- CYST(S)	MEAN	0	0	0.6	0
	S.D.	0	0	2.55	0
LUNG- CYST	MEAN	0	0	0	0.5
	S.D.	0	0	2.42

None significantly different from control group

NUMBER OF LITTERS EXAMINED SKELETALLY		20	1819	19	21
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	MEAN	0.6	0	1.9	0
	S.D.	2.48	0	6.15	0

None significantly different from control group

NUMBER OF LITTERS EXAMINED		20	18	19	21
PERCENT PER LITTER WITH EXTERNAL MALFORMATIONS	MEAN	0.5	0.5	0.4	2.9
	S.D.	2.24	1.96	1.91	11.06
PERCENT PER LITTER WITH SOFT TISSUE MALFORMATIONS	MEAN	0.5	2.2	0.6	1.7
	S.D.	2.24	5.45	2.55	5.86
PERCENT PER LITTER WITH SKELETAL MALFORMATIONS	MEAN	0.6	0	1.9	0
	S.D.	2.48	0	6.15	0

None significantly different from control group

Executive summary:

The objectives of this study were to determine the potential of the test item to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. Based on the results of the dose range finder, groups of 22 females per dose group were exposed to the test article at dose levels of 0, 6, 20, and 60 mg/kg body weight per day. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, organ weights, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80 were prepared accurately. Homogeneity of

formulations was considered acceptable for the purpose of this study. No adverse maternal effects were recorded up to a dose level of 60 mg/kg/day. There were no test item-related effects in fetal external, visceral or skeletal morphology or other developmental parameters. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test item was established as being at least 60 mg/kg/day. Based on the Dose Range Finding Study, a slightly higher dose level of 100 mg/kg/day was not tolerated.

Reference 2

Endpoint:

developmental toxicity

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 185 and 284 g
- Fasting period before study:
- Housing: in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 54 to 70%.
- Air changes (per hr): ten or more
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared as a solution for maximally 8 days, filled out in daily portions and stored in the refrigerator at 2-8ºC. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity/composition of the test item.
Details of the preparation and dispensing of the test item have been retained in the Study Records. Any residual volumes were discarded.

VEHICLE
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis during week 1 from all groups (concentration analysis) and from groups 2 and 4 (homogeneity). Analyses were performed using a validated analytical procedure For concentration analysis duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. For homogeneity analysis duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

Animals were received time-mated

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum

Frequency of treatment:

daily

Duration of test:

until day 21 post-coitum (necropsy)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of the dose range finder, and in an attempt to produce graded responses to the test item.

Maternal examinations:

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.

DETAILED CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting 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 WEIGHT: Yes
Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS
Animals were euthanized by an oxygen/carbon dioxide procedure on Day 21 post-coitum. No terminal body weight was recorded.
All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus.
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites. 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.
The thyroid gland and uterus were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated. The thyroid gland was collected from all animals and preserved in 10% neutral buffered formalin. Additional tissue samples were collected to elucidate abnormal findings. Thyroid glands of all animals of Groups 1 and 4 were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin and were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected randomized at the discretion of the Study Director, between 7:00 and 09:00 a.m., from the jugular vein in the animal facility. After collection, samples were transferred to the appropriate laboratory for processing. Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for the following parameters: Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)

Fetal examinations:

Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube.
Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations, as described in the following sections. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight. For late resorptions a gross external examination was performed. Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral Examinations– F1-Generation
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. As visceral malformations were suspected for fetus (A088-09), selected for skeletal examination, this fetus was also subjected to visceral examination. Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded. The carcasses were processed and stained with Alizarin Red S (as described below), but not examined.

Skeletal Examinations– F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

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 as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) 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.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Pre-implantation loss (%), Post-implantation loss (%), Viable fetuses affected/litter (%)

Historical control data:

Corrected body weight gain (gram): mean: 29, P5-P95:14.8-44.4, n=1077
Corrected body weight gain (%): mean: 13, P5-P95:7.0-20.7, n=1077
Absolute food consumption (gram), post-coitum Days 2-6: mean: 21, P5-P95: 16.3-25.8, n=1049
Total T3 (ng/dL): mean: 59.7, P5-P95:43.20-79.50, n=197
Total T4 (μg/dL): mean: 2.24, P5-P95:1.470-3.300, n=223

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was recorded for 4/22 animals at 500 mg/kg/day (Nos. 71, 74, 82 and 83) between post-coitum Days 7 and 20. Piloerection was also recorded for 1/22 animals (No. 56) at 150 mg/kg/day between postcoitum Days 6 and 10, but given its incidental and temporary occurrence, and absence of corroborative changes in body weight or food intake, this was considered unrelated to treatment with the test item.
Other clinical signs were considered not to represent toxicity, and mainly consisted of salivation which was recorded after dosing among all animals at 150 and 500 mg/kg/day from post-coitum Day 7 onwards and among most animals at 50 mg/kg/day from post-coitum Day 8 onwards. This was considered to be a physiological response rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, a statistically significantly reduced mean body weight gain was recorded throughout the treatment period, with slight mean weight loss (2%) between post-coitum Days 6 and 9 (individual weight loss values ranged between 1 and 8%). Absolute mean body weights were also slightly lower than control means throughout the treatment period at this dose, but were only statistically significant on post-coitum Day 21. Mean absolute body weight at the end of the treatment period was at 0.93x of the control mean. When corrected for gravid uterus weight, mean body weight and body weight showed a more pronounced (statistically significant) change at this dose level, with absolute weight gain being 0.42x of the control mean. Both absolute and relative body weight gain corrected for gravid uterus were below the historical control data range.
At 50 and 150 mg/kg/day, mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, a statistically significantly lower mean absolute and relative food consumption was recorded over post-coitum Days 6 to 9 and Days 9 to 12 post-coitum (0.59x and 0.84x control mean for absolute food intake, respectively). Absolute food consumption was also statistically significantly lower than the control mean on post-coitum Days 18-21 (0.87x).
At 50 and 150 mg/kg/day, absolute and relative food consumption of treated animals remained in the same range as controls over the treatment period.
Note: Several individual food consumption values across the dose groups over post-coitum Days 2-6 were considered unrealistically low, as these well exceeded the historical control data range and since these low values were recorded before treatment was initiated. In absence of corroborative changes in clinical appearance or body weight gain, these low values were attributed to incorrect technical conduct of these measurements.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Note: Values for non-gravid animals (No. 13 (Control group) and No. 49 (150 mg/kg/day) were excluded from the data tables and reported in a separate table.
At 500 mg/kg/day, mean total Triiodothyronine (T3) was statistically significantly lower than control mean (0.70x). Mean T3 values remained within the historical control data range. At 150 mg/kg/day, mean total Thyroxine (T4) appeared lower than the control mean. Since this mean was less than 20% different from the control mean (0.88x), was not statistically significant and remained within the historical control data range, this variation was considered not to be related to treatment. Serum levels of thyroid stimulating hormone (TSH) were unaffected by treatment with the test item up to 500 mg/kg/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related changes in thyroid gland weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item. All necropsy findings among control and treated animals were incidental, did not show a dose-related incidence trend and are occasionally seen among rats used in this type of study. Therefore, these necropsy findings were considered not to be related to treatment with the test item.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The recorded microscopic findings of the thyroid 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.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights (both sexes) were not affected by treatment with the test item up to 500 mg/kg/day. Mean combined (male and female) fetal body weights were 5.2, 5.4, 5.2 and 5.1 gram for the control, 50, 150 and 500 mg/kg/day groups, respectively. The statistically significantly higher mean fetal body weights of males (and combined for both sexes) at 50 mg/kg/day occurred in the absence of a dose-related trend and only marginally exceeded the historical control data range. As such, this variation was considered not to be related to treatment with the test item.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group. Mean litter sizes were 11.6 fetuses/litter in the control group and 11.0 fetuses/litter at 50, 150 and 500 mg/kg/day

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment with the test item up to 500 mg/kg/day. Mean sex ratios (males:females) were 43.5:56.5, 52.4:47.6, 43.2:56.8 and 50.8:49.2 for the control, 50, 150 and 500 mg/kg/day groups, respectively. The statistically significant alteration in sex ratio at 50 mg/kg/day occurred in the absence of a dose-related trend and was slight in magnitude. As such, this variation was considered not to be related to treatment with the test item.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Fetal ano-genital distance of both sexes (before and after correction for body weight) was unaffected by treatment with the test item up to 500 mg/kg/day.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological examination were 243 (21), 243 (22), 231 (21) and 241 (22) in the control, 50, 150 and 500 mg/kg/day groups, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Moreover, as during eviscerating of the fetuses prior to skeletal staining, a prominent visceral malformation was noticed for one fetus (A088-09) at 500 mg/kg/day that was selected for skeletal examination, this fetus was also subjected to a visceral examination.
There were no treatment-related effects on external morphology following treatment up to 500 mg/kg/day.
The only two external malformations in this study were observed in the control group. Fetus A009-07 had an omphalocele and the late resorption in litter A020 had general anasarca. Since both malformations occurred in the control group, these were considered to be spontaneous in origin. There were no external variations in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on skeletal morphology following treatment up to 500 mg/kg/day.
Skeletal malformations in test item treated groups were confined to sternoschisis in one fetus at 500 mg/kg/day (A088-09) and bent scapula and humerus in one fetus at 50 mg/kg/day (A023-07). The group distribution and single occurrence of these two malformations did not suggest a relationship to treatment with the test item. Moreover, the control fetus with omphalocele externally (A009-07) also had sternoschis (in addition to a vertebral anomaly) and both malformations were previously observed in historical controls.
No treatment-related skeletal variations were recorded. Skeletal variations occurred at an incidence of 79.5%, 82.2%, 77.3% and 74.4% per litter in the control, 50, 150 and 500 mg/kg/day groups, respectively. The incidence of 14th full ribs in the control group (12.6%) was close to the historical control maximum value of 13.1% per litter. The statistically significantly lower incidence of this skeletal variation at 50 and 500 mg/kg/day (2.7% and 1.9% per litter at 50 and 500 mg/kg/day, respectively, and 6.0% at 150 mg/kg/day) was considered to have resulted from this relatively high control incidence. Moreover, since there was no apparent dose-related trend in incidence of 14th full ribs, the statistically significant changes in this skeletal variation were considered not to represent a test itemrelated effect. The statistically non-significant higher incidence of 7th cervical ossification sites at 500 mg/kg/day (11.3% per litter, i.e. 11 fetuses in 7 litters) was higher than the control mean (5 fetuses in 5 litters) and exceeded the historical control maximum value (mean: 3.8% per litter, and maximum value of 9.9% per litter). However, statistical significance was not achieved, the mean was only slightly above the upper historical control limit, and a more generalized/advanced ossification process is expected in case of a test item-related effect.
Therefore, the presence of 7th cervical ossification sites at 500 mg/kg/day was considered not to be related to treatment with the test item. Moreover, an extra cervical ossification site in the costal cartilage region will disappear postnatally by incorporation in the transverse process. All other variations occurred in the absence of a dose-related incidence trend, in control fetuses only and/or at frequencies that were within the range of available historical control data. Therefore, they were considered not related to treatment with the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 500 mg/kg/day.
Two viscerally malformed fetuses were observed. One fetus at 500 mg/kg/day (A088-09) had a narrow pulmonary trunk, and one fetus at 150 mg/kg/day (A050-08) had situs inversus. Due to the single occurrence and absence of any clear dose-related incidence trend, both were considered chance findings. Only one visceral variation (small supernumerary liver lobes) was observed in this study in two fetuses at 50 mg/kg/day and one fetus at 500 mg/kg/day. Given the low incidence and absence of any clear dose-related incidence trend, this was considered not to be related to treatment with the test item.

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects reported

Developmental effects observed:

no

BODY WEIGHTS (GRAM) SUMMARY FEMALES

		Group 1	Group 2	Group 3	Group 4
		Control	50 mg/kg bw	150 mg/kg bw	500 mg/kg bw
DAY 0	MEAN	213	213	215	214
	ST.DEV.	19.9	20.4	20.6	23.8
	N	21	22	21	22
DAY 3	MEAN	228	231	229	228
	ST.DEV.	18.2	20.2	19.6	24.9
	N	21	22	21	22
DAY 6	MEAN	235	238	236	223
	ST.DEV.	16.7	20.9	20.0	22.2
	N	21	22	21	22
DAY 9	MEAN	251	254	251	239
	ST.DEV.	18.0	21.7	20.3	25.3
	N	21	22	21	22
DAY 12	MEAN	267	269	264	253
	ST.DEV.	19.5	23.2	20.7	26.2
	N	21	22	21	22
DAY 15	MEAN	299	299	296	282
	ST.DEV.	22.6	26.6	23.9	29.4
	N	21	22	21	22
DAY 18	MEAN	338	337	332	316*
	ST.DEV.	27.5	29.5	26.5	37.3
	N	21	22	21	22
DAY 21	MEAN	3791	3900	3887	3763
	ST.DEV.	358.9	308.2	275.5	238.8
	N	20	18	19	21

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

BODY WEIGHT GAIN (%) SUMMARY FEMALES

		Group 1	Group 2	Group 3	Group 4
		Control	50 mg/kg bw	150 mg/kg bw	500 mg/kg bw
DAY 6	MEAN	0	0	0	0
	ST.DEV.	0.0	0.0	0.0	0.0
	N	21	22	21	22
DAY 9	MEAN	3	3	3	-2**
	ST.DEV.	2.3	1.5	1.8	2.8
	N	21	22	21	22
DAY 12	MEAN	10	10	9	5**
	ST.DEV.	2.1	1.8	2.4	3.3
	N	21	22	21	22
DAY 15	MEAN	17	17	15	11**
	ST.DEV.	2.7	2.4	2.5	4.1
	N	21	22	21	22
DAY 18	MEAN	31	30	29	24**
	ST.DEV.	4.0	2.5	4.1	5.3
	N	21	22	21	22
DAY 21	MEAN	49	46	45	38**
	ST.DEV.	6.1	5.1	6.9	7.8
	N	21	22	21	22

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

CLINICAL BIOCHEMISTRY SUMMARY FEMALES pregnant

		Group 1	Group 2	Group 3	Group 4
		Control	50 mg/kg bw	150 mg/kg bw	500 mg/kg bw
TSH	MEAN	0.300	0.287	0.286	0.247
uIU/mL	ST.DEV	0.176	0.178	0.251	0.172
	N	21	22	21	22
TotalT3	MEAN	62.0	55.3	48.5	43.2**
ng/dL	ST.DEV	19.7	17.0	18.1	18.4
	N	21	20	21	22
TotalT4	MEAN	2.08	2.25	2.25	1.83
ug/dL	ST.DEV	0.44	0.58	0.65	0.56
	N	21	22	21	22

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

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES pregnant

		Group 1	Group 2	Group 3	Group 4
		Control	50 mg/kg bw	150 mg/kg bw	500 mg/kg bw
BODYW.	MEAN	338	337	332	316*
(GRAM)	ST.DEV	28	30	26	37
	N	21	22	21	22
THYROIDS	MEAN	0.017	0.017	0.018	0.015
(GRAM)	ST.DEV	0.004	0.003	0.003	0.004
	N	21	22	21	22

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

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY FEMALES pregnant

		Group 1	Group 2	Group 3	Group 4
		Control	50 mg/kg bw	150 mg/kg bw	500 mg/kg bw
BODYW.	MEAN	338	337	332	316*
(GRAM)	ST.DEV	28	30	26	37
	N	21	22	21	22
THYROIDS	MEAN	0.005	0.005	0.005	0.005
(%)	ST.DEV	0.001	0.001	0.001	0.001
	N	21	22	21	22

SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

DOSE GROUP	1		2		3		4
	NO.	%	NO.	%	NO.	%	NO.	%
FEMALES ON STUDY	22		22		22		22
FEMALES THAT ABORTED OR DELIVERED	0	0	0	0	0	0	0	0
FEMALES THAT DIED	0	0	0	0	0	0	0	0
FEMALES THAT ABORTED	0	0	0	0	0	0	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	0	0	0	0	0	0
FEMALES THAT WERE EUTHANIZED	0	0	0	0	0	0	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	0	0	0	0	0	0
FEMALES EXAMINED AT SCHEDULED NECROPSY	22	100	22	100	22	100	22	100
NONGRAVID	1	4.5	0	0	1	4.5	0	0
GRAVID	21	95.5	22	100	21	95.5	22	100
WITH RESORPTIONS ONLY	0	0	0	0	0	0	0	0
WITH VIABLE FETUSES	21	100	22	100	21	100	22	100
TOTAL FEMALES GRAVID	21	95.5	22	100	21	95.5	22	100

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

Group		Males	Females	Viable Fetuses	Dead Fetuses	Early Resorptions	Late resorptions
1	TOTAL	108	135	243	0	6	3
	MEAN	5.1	6.4	11.6	0	0.3	0.1
	S.D.	2.06	1.86	2.38	0	0.56	0.36
2	TOTAL	127	116	243	0	15	0
	MEAN	5.8	5.3	11	0	0.7	0
	S.D.	2.27	2.33	3.03	0	0.84	0
3	TOTAL	101	130	231	0	9	1
	MEAN	4.8	6.2	11	0	0.4	0
	S.D.	1.86	1.72	2.3	0	0.6	0.22
4	TOTAL	123	118	241	0	13	0
	MEAN	5.6	5.4	11	0	0.6	0
	S.D.	2.17	1.89	2.5	0	0.85	0

Group		Post Implantation Loss	Implantation Sites	Corpora Lutera	Pre-Implantation Loss	Fetal weights (g)	No. of gravid females
1	TOTAL	9	252	262	10	NA	21
	MEAN	0.4	12	12.5	0.5	5.2
	S.D.	0.75	2	1.94	0.68	0.3
2	TOTAL	15	258	283	25	NA	22
	MEAN	0.7	11.7	12.9	1.1	5.4*
	S.D.	0.84	2.75	2.03	2.05	0.34
3	TOTAL	10	241	268	27	NA	21
	MEAN	0.5	11.5	12.8	1.3	5.2
	S.D.	0.6	2.16	1.84	1.71	0.23
4	TOTAL	13	254	284	30	NA	22
	MEAN	0.6	11.5	12.9	1.4	5.1
	S.D.	0.85	2.74	2.04	1.73	0.26

* = Significantly different from the control group at 0.05

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA, FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

Group	1	2	3	4
CORPORA LUTEA
MEAN	12.5	12.9	12.8	12.9
S.D.	1.94	2.03	1.84	2.04
N	21	22	21	22
IMPLANTATION SITES
MEAN
S.D.	12	11.7	11.5	11.5
N	2	2.75	2.16	2.74
VIABLE FETUSES (%)	21	22	21	22
MEAN
S.D.
N	95.7	92.8	95.6	95.4
DEAD FETUSES (%)	9.58	9.72	5.4	6.4
MEAN	21	22	21	22
S.D.
N	0	0	0	0
EARLY RESORPTIONS (%)	0	0	0	0
MEAN	21	22	21	22
S.D.
N
LATE RESORPTIONS (%)	2.8	7.2	3.8	4.6
MEAN	6.69	9.72	5.15	6.4
S.D.	21	22	21	22
N
TOTAL RESORPTIONS (%)
MEAN	4.3	7.2	4.4	4.6
S.D.	9.58	9.72	5.4	6.4
N	21	22	21	22
PRE-IMPLANTATION LOSS (%)
MEAN	3.8	8.7	9.7	10.9
S.D.	5.5	16.68	12.39	14.44
N	21	22	21	22
POST-IMPLANTATION LOSS (%)
MEAN	4.3	7.2	4.4	4.6
S.D.	9.58	9.72	5.4	6.4
N	21	22	21	22
MALES (%)
MEAN	43.5	52.4*	43.2	50.8
S.D.	14.2	15.31	12.35	15.73
N	21	22	21	22
FEMALES (%)
MEAN	56.5	47.6*	56.8	49.2
S.D.	14.2	15.31	12.35	15.73
N	21	22	21	22
MALE FETAL WEIGHTS (g)
MEAN	5.3	5.6*	5.3	5.2
S.D.	0.39	0.28	0.28	0.26
N	21	22	21	22
FEMALE FETAL WEIGHTS (g)
MEAN	5	5.3	5.1	4.9
S.D.	0.25	0.39	0.22	0.27
N	21	22	21	21
COMBINED FETAL WEIGHTS (g)
MEAN	5.2	5.4*	5.2	5.1
S.D.	0.3	0.34	0.23	0.26
N	21	22	21	22
MALE AGD (MM)
MEAN	3.03	2.98	3.08	3.09
S.D.	0.391	0.502	0.42	0.354
N	21	22	21	22
FEMALE AGD (MM)
MEAN	1.68	1.65	1.72	1.7
S.D.	0.156	0.205	0.2	0.207
N	21	22	21	21
MALE AGD corrected by Body Weight
MEAN	1.74	1.68	1.76	1.79
S.D.	0.211	0.288	0.238	0.207
N	21	22	21	22
FEMALE AGD corrected by Body Weight
MEAN	0.98	0.95	1	1
S.D.	0.087	0.118	0.121	0.13
N	21	22	21	21

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

CORPORA LUTEA AND IMPLANTATION SITES COMPARED USING DUNNETT'S TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

FETAL WEIGHTS AND (CORRECTED) AGD COMPARED USING DUNNETT'S TEST

* = Significantly different from the control group at 0.05

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

	FETUSES				LITTERS
Dose Group	1	2	3	4	1	2	3	4
NUMBER EXAMINED EXTERNALLY	243	243	231	241	21	22	21	22
TRUNK- OMPHALOCELE	1	0	0	0	1	0	0	0
NUMBER EXAMINED VISCERALLY	121	123	114	121	21	22	21	22
SITUS INVERSUS	0	0	1	0	0	0	1	0
PULMONARY TRUNK- NARROW	0	0	0	1	0	0	0	1
NUMBER EXAMINED SKELETALLY	122	120	117	121	21	22	21	22
BENT LIMB BONES	0	1	0	0	0	1	0	0
STERNOSCHISIS	1	0	0	1	1	0	0	1
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	1	0	0	0	1	0	0	0
TOTAL NUMBER WITH MALFORMATIONS
EXTERNAL :	1	0	0	0	1	0	0	0
SOFT TISSUE :	0	0	1	1	0	0	1	1
SKELETAL :	1	1	0	1	1	1	0	1
COMBINED :	1	1	1	1	1	1	1	1

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS % PER LITTER

Dose Group		1	2	3	4
NUMBER OF LITTERS EXAMINED EXTERNALLY		21	22	21	22
TRUNK- OMPHALOCELE MEAN	MEAN	0.5	0	0	0
	S.D.	2.18	0	0	0

None significantly different from control group

NUMBER OF LITTERS EXAMINED VISCERALLY		21	22	21	22
SITUS INVERSUS	MEAN	0	0	1	0
	S.D.	0	0	4.36	0
PULMONARY TRUNK- NARROW	MEAN	0	0	0	0.6
	S.D.	0	0	0	3.05

None significantly different from control group

NUMBER OF LITTERS EXAMINED SKELETALLY		20	18	19	21
BENT LIMB BONES	MEAN	0	0.6	0	0
	S.D.	0	3.05	0	0
STERNOSCHISIS	MEAN	1	0	0	0.9
	S.D.	4.36	0	0	4.26
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	MEAN	1	0	0	0
	S.D.	4.36	0	0	0

None significantly different from control group

NUMBER OF LITTERS EXAMINED		20	18	19	21
PERCENT PER LITTER WITH EXTERNAL MALFORMATIONS	MEAN	0.5	0	0	0
	S.D.	2.18	0	0	0
PERCENT PER LITTER WITH SOFT TISSUE MALFORMATIONS	MEAN	0	0	1	0.6
	S.D.	0	0	4.36	3.05
PERCENT PER LITTER WITH SKELETAL MALFORMATIONS	MEAN	1	0.6	0	0.9
	S.D.	4.36	3.05	0	4.26
TOTAL PERCENT PER LITTER WITH MALFORMATIONS	MEAN	0.5	0.6	1	0.6
	S.D.	2.18	3.05	4.36	3.05

None significantly different from control group

Conclusions:

Based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) were established:
- Maternal NOAEL: 150 mg/kg/day (based on reduced weight gain).
- Developmental NOAEL: at least 500 mg/kg/day.

Executive summary:

The objectives of this study were to determine the potential of the test item to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 50, 150 or 500 mg/kg/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4 and TSH), gross necropsy findings, number of corpora lutea, organ weights ((gravid) uterus and thyroid gland), uterine contents and histopathologic examination (thyroid gland). In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, ano-genital distance, external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

Maternal findings

Adverse effects on body weight were recorded at 500 mg/kg/day, at which absolute weight gain corrected for gravid uterus weight was less than half of the control mean (and below the historical control data range). This magnitude of change in weight gain was considered to represent an adverse effect. Over post-coitum Days 6-9, slight mean weight loss (2%) and lower food intake (0.59x control mean) was recorded, which showed a partial recovery as treatment progressed, although food intake was again reduced over post-coitum Days 18-21 (0.87x control mean). This was accompanied by piloerection for a few animals at 500 mg/kg/day between post-coitum Days 7 and 20. A non-adverse but statistically significant lower mean total T3 was recorded at 500 mg/kg/day (0.70x of control means, respectively). Since the mean remained within the historical control data range, and no treatment-related changes in thyroid histopathology or thyroid weight was recorded, this lower mean total T3 was considered not adverse.

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Fetal Examinations

There were no test item-related effects in external, visceral or skeletal morphology. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights and anogenital distance).

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) were established:

Maternal NOAEL: 150 mg/kg/day (based on reduced weight gain).

Developmental NOAEL: at least 500 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The test substance was investigated in a developmental toxicity study following OECD guideline 414. Time-mated female New Zealand White rabbits were treated from Day 6 to 28 post-coitum, inclusive by daily oral gavage at dose levels of 6, 20 and 60 mg/kg/day. The rabbits of the control group received the vehicle, 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80, alone.

Formulation analyses confirmed that formulations of test item in vehicle were prepared accurately. Homogeneity of formulations was considered acceptable for the purpose of this study.

No adverse maternal effects were recorded up to a dose level of 60 mg/kg/day. At 60 mg/kg/day, slightly lower mean body weight gain was recorded throughout the post-coitum period, also after correction for gravid uterus weight. This was accompanied by a slightly lower food intake during the first two weeks of treatment. Given the slight magnitude of these changes, they were considered not to be adverse. The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation. There were no test item-related effects in external, visceral or skeletal morphology. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio and fetal body weights).

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test article was established as being at least 60 mg/kg/day. Based on the Dose Range Finding Study, a slightly higher dose level of 100 mg/kg/day was not tolerated.

The structurally related source substance EC 915-687-0 was investigated in a developmental toxicity study (OECD 414). Time-mated female Wistar Han rats were treated from Day 6 to 20 post-coitum, inclusive by daily oral gavage at dose levels of 50, 150 and 500 mg/kg. The rats of the control group received the vehicle, corn oil, alone. Test formulations prepared were homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Adverse effects on body weight were recorded at 500 mg/kg/day, at which absolute weight gain corrected for gravid uterus weight was less than half of the control mean (and below the historical control data range). This magnitude of change in weight gain was considered to represent an adverse effect. Over post-coitum Days 6-9, slight mean weight loss (2%) and lower food intake (0.59x control mean) was recorded, which showed a partial recovery as treatment progressed, although food intake was again reduced over post-coitum Days 18-21 (0.87x control mean). This was accompanied by piloerection for a few animals at 500 mg/kg/day between post-coitum Days 7 and 20. A non-adverse but statistically significant lower mean total T3 was recorded at 500 mg/kg/day (0.70x of control means, respectively). Since the mean remained within the historical control data range, and no treatment-related changes in thyroid histopathology or thyroid weight was recorded, this lower mean total T3 was considered not adverse. The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation. There were no test item-related effects in external, visceral or skeletal morphology. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights and anogenital distance).

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) for TINUVIN® 292 were established:

- Maternal NOAEL: 150 mg/kg/day (based on reduced weight gain).

- Developmental NOAEL: at least 500 mg/kg/day.

Justification for classification or non-classification

There are conclusive but not sufficient data for classification of the test item with regard to developmental toxicity/teratogenicity. The test item is not classified for this endpoint in accordance to Directive 67/548/EEC or the CLP Regulation (EC) No 1272/2008 as well as GHS regulations.

The test article caused effects on fertility in an extended one-generation reproduction toxicity study (EOGRTS). Specifically, a lower mean number of implantation sites was observed. In F0-females the effect was relatively small. A statistical significance was reached at 5000 ppm only. The decrease in number of implantation sites was accompanied by a lower live litter size in all groups compared to concurrent controls. Similarly, to the implantation sites, differences in litter size between the treatment groups was relatively small However, a treatment related effect could not be excluded, the study was extended with a second 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.

  

	F0	F1
Implantations sites	12.6, 11.5, 11.5 and 11.1** (0, 500, 1500, and 5000 ppm)	12.4, 12.5, 11.9 and 10.1** (0, 500, 1500, and 5000 ppm)
Litter size	12.1, 11.0, 10.7*, 10.4** (0, 500, 1500, and 5000 ppm)	11.7, 11.5, 10.8, 9.0** (0, 500, 1500, and 5000 ppm)

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.

 

These findings occurred at dose levels that also caused maternal toxicity, manifesting as adverse effects on body weight and body weight gain. Reduced body weight gain, especially in combination with palatability issues can induce stress to the animals. Maternal stress exposure and related effects on the developing embryo during the preimplantation period has been described in literature. In a study with pregnant ICR mice, maternal restraint stress (3 times a day for 30 minutes from day 1 to day 4 of pregnancy) resulted in significantly increased serum corticosterone concentrations (Burkus et al., 2015). Further, a significant reduction in implantation sites in uteri was observed on gestational day 6 when compared with untreated controls. The blastocysts of stressed mothers showed reduced average cell numbers in the trophoectoderm and the inner cell mass lineages, which was indicated to contribute to the factors responsible for the decreased implantation rate.

 

Maternal preimplantation stress and its effects on implantation sites and pup numbers were investigated by comparing inhouse historical control data on Wistar rats, i.e. the strain used in the EOGRTS. The database consists of 80 studies (1787 litter) of time mated and subsequently transported rats and 35 studies (949 litter) with inhouse mated rats without further transportation (study dates 2013-2018). The time mated and transported rats (mean = 10.8; Range 9.4 – 11.9) showed a reduction of 1.4 in implantation sites when compared to inhouse mated animals (mean = 12.2; Range 9.4 – 13.9). Consequently, the difference in the mean number of delivered pups was 1.5 (transported: mean = 10.1; Range 8.5 – 11.2 versus inhouse mated: mean = 11.6 (Range 9.9 – 12.7). These data further substantiate a dependency of both parameters to maternal stress conditions due to transportation of pregnant animals, which is of a comparable magnitude to the changes observed after administration of the test article. Since the reduction of implantation sites occurred at a maternally toxic dose level, a dependency between these effects cannot be excluded.

 

However, since it cannot be completely proven at this point that the reduced numbers number of implantation sites are solely triggered by maternal toxicity and maternal stress, the test article is classified for reproduction toxicity with category 2, following the precautionary principle. The personal protection equipment required to handle Category 2 reprotoxicants in industrial and profesional settings will assure that the potential hazard to reptotoxicity is well under control. In addition, the general population does not come into contact of the pure substance, exposure is limited to articles containing the test article embedded in a matrix with negligible exposure potential. Since the above described effects were adverse only at the high-dose level, classification with category 2 is considered a more than sufficient precaution.

Reference:

BURKUŠ J. et al., 2015. Stress exposure during the preimplantation period affects blastocyst lineages and offspring development. Journal of Reproduction and Development, Vol. 61, No 4.