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Toxicity to reproduction

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Administrative data

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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

According to ECHA Compliance Check Decision number CCH-D-2114382275-45-01/F
- Premating exposure duration for parental (P0) animals : Ten weeks premating exposure duration for the parental (P0) generation, according to ECHA Compliance Check Decision.
- Basis for dose level selection : based on the results of the OECD 422 (2018) (Dose level setting shall aim to induce systemic toxicity at the highest dose level, according to ECHA Compliance Check Decision)
- Exclusion of extension of Cohort 1B, according to ECHA Compliance Check Desision
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B , according to ECHA Compliance Check Decision
- Inclusion of developmental immunotoxicity Cohort 3, according to ECHA Compliance Check Decision
- Route of administration : oral (gavage), according to ECHA Compliance Check Decision
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

Test material

Constituent 1
Chemical structure
Reference substance name:
Acrylic acid, monoester with propane-1,2-diol
EC Number:
247-118-0
EC Name:
Acrylic acid, monoester with propane-1,2-diol
Cas Number:
25584-83-2
Molecular formula:
C6H10O3
IUPAC Name:
1-hydroxypropan-2-yl prop-2-enoate; 2-hydroxypropyl prop-2-enoate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany, Sulzfeld,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 weeks
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex.
- Housing: During the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany with the following exceptions:During overnight mating (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany).
Dams and their litters were housed together until PND 21/22 in Polycarbonate cages type III.
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland (ad libitum)
- Water (e.g. ad libitum): drinking water (ad libitum)
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer until it was completely dissolved. During administration, the preparations were kept homogeneous with a magnetic stirrer.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy:sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in drinking water over a period of 7 days at room temperature has been verified prior to the start of the study in a similar batch. At the beginning of administration, towards the middle and towards the end of administration each 1 sample was taken from the low, mid and high concentration for a concentration control analysis.
Duration of treatment / exposure:
F0 males: 10 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 10 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Cohort 1A and 1B): post weaning until an approx. age of 12-13 weeks
F1 animals (Cohort 3): post weaning until an approx. age of 8-9 weeks
Frequency of treatment:
once daily
Females in labor were not treated.
Details on study schedule:
F0 generation animals and their progeny:
The male and female rats were about 4 weeks old when they arrived from the breeder. During an acclimatization period of about 6 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 100 male and 100 female animals required for the study were about 5 weeks old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex. The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight one day before the beginning of the administration period (day -1). After the acclimatization period, the test substance was administered to the animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (drinking water), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight. After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated, overnight at a ratio of 1 : 1 (for details see: Pairing of F0 generation parental animals). The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 3 different cohorts which were subjected to specific postweaning examinations. On PND 4 blood samples were collected from 10 surplus (culled) F1 pups per sex and group. On PND 22 blood samples were collected from 10 surplus F1 pups per sex and group. Urine samples were taken from 10 selected F0 parents and all cohort 1A animals shortly before sacrifice. Blood samples were taken from 10 animals per test group of the F0 parental animals and cohort 1A animals. Before weaning of the F1 pups the F0 generation parental male animals were sacrificed. After weaning of F1 pups the F0 generation parental female animals were sacrificed.

F1 rearing animals:
Before weaning of the F1 generation pups on PND 21, 55 male and 55 females per group were randomly selected (selection see below), to be placed into cohorts according to the scheme. Obvious runts (those pups whose body weight was greater than 25% below the mean body weight of the control group, separate for sexes) were not included.
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Cohort 3: One male or one female/litter (10/sex/group)
Selected F1 offspring received the test substance daily by gavage until one day before sacrifice. In addition, 10 male and 10 female pups were randomly selected from the control group to build test group 14 (positive control group).

Standardization of litters (culling) of F1 generation:
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 5 male and 5 female pups (always the first 5 pups/sex and litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Surplus animals were sacrificed according to 3.8.2.7. Standardization of litters was not performed in litters with <= 10 pups.

Pups after standardization/weaning:
With the exception of those F1 generation pups, which were chosen as F1 rearing animals, and those F1 pups, which were chosen for blood sampling on PND 4 and 22, all pups were sacrificed under isoflurane anesthesia with CO2 after standardization or weaning. The pups chosen for blood sampling were sacrificed by decapitation under isoflurane anesthesia. All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a caseby-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0: 25 animals per sex per dose
F1 cohort 1A: 20 animals per sex per dose
F1 cohort 1B: 25 animals per sex per dose
F1 cohort 3: 10 animals per sex per doese
F1 positive control for immuotox: 10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Based on the results of the OECD 422:
15 mg/kg body weight/day as low dose level
50 mg/kg body weight/day as mid dose level
150 mg/kg body weight/day as high dose level
Positive control:
Cyclophosphamide monohydrate (control group for immunotoxiciy):
The positive control substance solution in drinking water was prepared before the beginning of the administration period. For the preparation of the administration solution the positive control substance was weighed in a weighing boat depending on the dose group and transferred quantitatively in a graduated flask, topped up with drinking water and subsequently thoroughly mixed by a magnetic stirrer until it was completely dissolved. Thereafter the positive control preparation was split in “Nalgene Dosen” (40 ml each) and frozen by -18°C.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were performed in all F0 parental animals (once before the beginning of the administration period on day 0) and F1 animals in cohorts 1A, 1B and 3 at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed: Abnormal behavior in handling; Fur; Skin; Posture; Salivation; Respiration; Activity/arousal level; Tremors; Convulsions; Abnormal movements; Gait abnormalities; Lacrimation; Palpebral closure; Exophthalmos (Protruding eyeball); Assessment of the feces excreted during the examination (appearance/consistency); Assessment of the urine excreted during the examination; Pupil size

BODY WEIGHT: Yes / No / No data
In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning), with the following exceptions:
• During the mating period of the F0 parental animals, the females were weighed on the day of positive evidence of sperm GD 0 and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition PND 0 and on PND 1, 4, 7, 10,14, 18 and 21.
The body weight change of the animals was calculated from these results. Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22), were weighed once a week together with the males. These body weight data were solely used for the calculations of the dose volume; therefore these values are not reported in the Summary but in the Individual Tables.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Food consumption was not determined after the 10th premating week (male F0 animals).
• During pregnancy, food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Generally, water consumption was determined once a week (over a period of 3 or 4 days) for male and female F0 parental animals and F1 rearing animals (except of the positive control animals, test group 14), with the following exceptions:
• Water consumption was not determined after the 10th premating week (male F0 animals) • During pregnancy, water consumption of the F0 females with evidence of sperm was determined for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• During lactation, water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period, in the females after weaning.

CLIINICAL PATHOLOGY in F0 parental animals:
Samples were withdrawn from 10 F0 parental males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a random- ized sequence. The list of randomization instructions was compiled with a computer. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instruc- tions was compiled with a computer). The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.
Hematology:
The following parameters were determined :
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)
Clinical chemistry:
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
Hormones:
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisas was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
Urinalysis:
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHO
Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.
Sperm parameters (parental animals):
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
Pup number and status at delivery:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.
Pup viability/mortality:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section “Pup necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21.
Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.
Pup clinical observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. In the summary tables pup body weights and pup body weight gains are listed for males, females and males + females. Additionally, the body weight of all F1 rearing animals was determined on the day of vaginal opening and preputial separation.
Anogenital distance:
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.
Nipple/areola anlagen:
All surviving F1 male pups were examined for the presence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. The number of nipple/areola anlagen will be counted.
Vaginal opening:
All female F1 pups selected to become the F1 rearing animals (cohort 1A, 1B and 3) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.
Preputial separation:
All male F1 pups selected to become the F1 rearing animals (cohort 1A, 1B and 3) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.

CLINICAL PATHOLOGY in cohort 1A animals:
Samples were withdrawn from 10 cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a random- ized sequence. The list of randomization instructions was compiled with a computer.
In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instruc- tions was compiled with a computer). The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.
Hematology:
The following parameters were determined :
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)
Clinical chemistry:
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
Hormones:
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisas was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
Hormones in PND 4 and 22 F1-offspring:
Blood sampling
Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis. Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group. The blood samples was collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisas was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
Urinalysis:
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHO

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes
Developmental immunotoxicity examinations in cohort 3 animals and positive control animals
Cyclophosphamide dependent immune system response:
Ten male and ten female offspring derived from test group 00 (as far as possible from different litters) were selected at weaning to become a positive control group in this study. These animals were treated with Cyclophosphamide monohydrate to prove the functional responsiveness of major components of the immune system of the rats against an immunosuppressant. The animals were treated by daily oral gavage from PND 35 onwards, for about four weeks. The following dose level of Cyclophosphamide monohydrate was selected to be sufficient to cause immunosuppressive activity as positive control substance: 4.5 mg/kg body weight/day: as dose level with expected immunosuppressive effects The oral route was selected since oral administration of Cyclophosphamide effectively causes the desired immunosuppressive effect.
POSITIVE CONTROL SUBSTANCE PREPARATIONS AND ADMINISTRATION:
Oral administration by gavage using 3 or 5 mL syringes, once daily. 10 mL/kg body weight; the body weight determined most recently were used to calculate the administration volume. Duration ofadministration about 4 weeks. Six days after immunization blood samples were taken by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.

T-cell dependent antibody response:
All males and females of cohort 3 and the positive control animals were used to assess the functional responsiveness of major components of the immune system to a T-cell dependent antigen, sheep red blood cells (SRBC). For this purpose, the Anti SRBC-IgM ELISA of Life Diagnostics Inc, West Chester, USA (cat. no. 4200-2), was performed. Each sample was di- luted 1:500. SRBC-IgM concentrations outside the standard curve range were measured in a second test run with an appropriate dilution. Generally, two in-house controls were measured with each test run. The ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maenne- dorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Splenic lymphocyte subpopulation analysis:
The immunophenotyping was performed with a FACSLyric flow cytometer (Becton Dickinson, Heidelberg, Germany). 10 males and 10 females of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis using one half of the spleen.
Parameter: B lymphocytes (B_SPL); T lymphocytes (T_SPL), CD4+ lymphocytes (CD4_SPL); CD8+ lymphocytes (CD8_SPL); Natural killer cells (NK_SPL)

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE / NECROPSY
All F0 generation parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.) The female animal No. 139 (F0 generation, parental animals, test group 02) died intercurrently they were necropsied and assessed by gross pathology as soon as possible after their death.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Caudae epididymides
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

Organ/tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens
The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution. The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
For technical reasons, after about 24 hours fixation the ovaries of all F0 females of all test groups were transferred to 70% ethanol.
The uteri of all cohabited female F0 generation parental animals were examined for the pres- ence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

Peer review:
After completion of the histopathological assessment by the study pathologist an internal peer review was performed by Dr. Maria Cecilia Rey Moreno (BASF SE, Ludwigshafen) including forestomach and glandular stomach of all examined male and female animals of the F0 parental generation and of the F1 cohort 1A rearing animals. Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Postmortem examinations (offspring):
SACRIFICE / NECROPSY
All animals of F1 generation, rearing animals, cohort 1A were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were nec- ropsied and assessed by gross pathology; special attention being given to the reproductive organs.) The female animal No. 374 (F1 generation, rearing animals, cohort 1A, test group 13) was sacrificed moribund, they were necropsied and assessed by gross pathology as soon as possible after their death.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Caudae epididymides
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

Organ/tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens
The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution. The left testis and left epididymis of all Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
For technical reasons, after about 24 hours fixation the ovaries of all cohort 1A females of all test groups were transferred to 70% ethanol.
Spleens of 10 animals per sex per group of cohort 1A were split in two comparable parts (transversally). One part of the spleen was fixed in 4% neutral buffered formaldehyde and afterwards embedded in paraplast. The other part of the spleen was frozen at -80o C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).

Differential Ovarian Follicle Count (DOFC) in cohort 1A females:
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E-stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).
Peer review:
After completion of the histopathological assessment by the study pathologist an internal peer review was performed by Dr. Maria Cecilia Rey Moreno (BASF SE, Ludwigshafen) including forestomach and glandular stomach of all examined male and female animals of the F0 parental generation and of the F1 cohort 1A rearing animals. Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.

NECROPSY Cohort 1B:
All cohort 1B animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Animals which died intercurrently (male animals No. 475 (test group 12) and No. 487 (test group 13) were necropsied as soon as possible after their death and assessed by gross pathology.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland (fixed)
8. Prostate (ventral and dorsolateral part together, fixed)
9. Testes
10. Seminal vesicles including coagulating gland (fixed)
11. Uterus (with cervix)
All paired organs were weighed together (left and right)

Organ/Tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymides (fixed in modified Davidson ´s solution)
6. Liver
7. Ovaries (fixed in modified Davidson´s solution)
8. Pituitary gland
9. Prostate
10. Seminal vesicles including coagulating glands
11. Stomach (forestomach and glandular stomach)
12. Testes (fixed in modified Davidson ´s solution)
13. Uterus
14. Vagina
The testes and epididymides of the animals that died (Nos. 475, test group 12 and 487, test group 13) were fixed in 4% buffered formaldehyde solution. For technical reasons, after about 24 hours fixation the ovaries of all cohort 1B females of all test groups were transferred to 70% ethanol.

Histotechnical processing and examination by light microscopy was not performed. For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.

NECROPSY Cohort 3 and positive control:
All Cohort 3 animals and the animals of the positive control were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.
HISTOPATHOLOGY / ORGAN WEIGHTS:
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Spleen
3. Thymus

Organ/Tissue fixation.
The following organs or tissues were fixed in 4% buffered formaldehyde solution:
1. All gross lesions
2. Spleen
3. Thymus

Histotechnical processing and examination were not performed.

NECROPSY surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts).

All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.
HISTOPATHOLOGY / ORGAN WEIGHTS:
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Spleen
4. Thymus (fixed)

Organ/Tissue fixation.
The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutralbuffered formaldehyde solution:
1. All gross lesions
2. Brain
3. Mammary gland (male and female)
4. Spleen
5. Thymus
6. Thyroid glands

Histotechnical processing and examination was not performed.
Statistics:
Water consumption (parental and rearing animals), food consumption (parental and rearing animals), body weight and body weight change (parental animals, rearing animals and pups; for the pup weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal opening, days to
preputial separation), anogenital distance, anogenital index: DUNNETT test (twosided)
Male and female mating indices, male and female fertility indices, gestation index, females mated, females pregnant, females delivering, females with liveborn
pups, females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided)
Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal Loss, nipple development: WILCOXON test (one-sided+)
Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, lactation index: WILCOXON test (one-sided-)
% live male day x, % live female day x: WILCOXON test (twosided)
Number of cycles and Cycle Length: KRUSKAL-WALLIS test (two-sided), WILCOXON-test (twosided)
Blood parameters and splenic lymphocytes subpopulations: Non-parametric one-way analysis using KRUSKAL-WALLIS test., WILCOXON-test (two-sided), WILCOXON-test (one-sided)
Urinalysis parameters (apartfrom pH, urine volume, specific gravity, color and turbidity: WILCOXON-test (one-sided)
Urine pH, volume and specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test, WILCOXON-test (two-sided)
Urine color and turbidity: Urine color and turbidity are not evaluated statistically.
Spermanalysis parameters:WILCOXON-test (one-sided)
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided), WILCOXON-test (two-sided), WILCOXON-test (two-sided) f
DOFC (differential ovarian follicular count): WILCOXON-test (one-sided-)
Reproductive indices:
Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100
Female mating index (%) = number of females mated / number of females placed with males x 100
Female fertility index (%) = number of females pregnant / number of females mated x 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100
Offspring viability indices:
Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high-dose and nearly all mid-dose males showed salivation at least once during the study. Likewise, salivation was observed in all high-dose females as well as about half of the middose females at least once during the study (including premating, mating, gestation and lactation periods). In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.

Clinical observations for males and females (except gestation and lactation period):
One mid-dose male animal (No. 60) showed eye discharge (left, red) during study days 20 - 114. One control female animal (No. 119) had a protruding eyeball (right) during premating days 20 – 75 and during the mating period (days 1 - 3). One low- and one mid-dose female (Nos. 130 and 154, respectively) showed reduced nutritional condition (grade: slight to severe with indrawn flanks and grade: slight to moderate with indrawn flanks, respectively) during premating days 47 - 70 and 52 - 75, respectively. These observations were considered not to be associated with the test compound.

Clinical observations for females during gestation of F1 litters:
One control female animal (No. 119) had a protruding eyeball (right) during the entire gestation period. One low-dose female animal (No. 146) had vaginal discharge (reddish) on GD 18. One mid-dose female (No. 154) had piloerection, reduced feces and reduced nutritional condition during GD 8 - 57.
One sperm positive low-dose female (No. 130) and one sperm positive mid-dose female (No. 154) did not deliver F1 pups and had no implants in the uterus. These observations were considered not to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters: One control female animal (No. 119) had a protruding eyeball (right) during lactation period.

Detailed clinical observations (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One mid-dose male (No. 60) showed eye discharge (left, red) during DCO days 22 - 106. One control female (No. 119) had a protruding eyeball (right) during DCO days 22 - 127. One lowdose female (No. 130) had reduced nutritional conditions (grade: slight, with indrawn flanks) during DCO days 50 - 64. One low-dose female (No. 146) had vaginal discharge (reddish) on DCO day 99. One mid-dose female (No 154) showed reduced nutritional condition (grade: slight to moderate, with indrawn flanks) during DCO days 57 - 71 and 120 - 127 and piloerection during DCO days 120 - 127. These observations were considered not to be associated with the test compound.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test substance-related mortalities in any of the groups.
One low-dose female animal (No. 139) showed hypothermia, piloerection, and blood in bedding on GD 23, had semiclosed eyelids (both), all pups stillborn and pups still palpable in abdomen after delivery on PND 0, hypothermia, piloerection, pale skin (entire body) and hunched posture during PND 0 - 1, smeared fur (anogenital region, light yellow) on PND 1 and was found dead on PND 2. It showed an adenocarcinoma in the uterus and multifocal necrosis in the liver, which were unrelated to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant increases or decreases in body weight/body weight change in F0 animals of various dose groups, which were small, temporary, not dose-related and rather inconsistent and thus considered to be of no toxicological relevance:
• decreased body weight change in high-dose males on study days 21 - 49, 84 - 105 and 0 – 112
• decreased body weight change in mid-dose males on study days 21 - 28, 35 - 42, 49 - 56 and 0 - 112
• increased body weight change in low-dose males on study days 70 - 77
• decreased body weight change in mid-dose females on premating days 14 - 21 and 0 - 70
• decreased body weights in mid-dose males on study days 84 - 91 and on day 105
• decreased body weights in mid-dose females on premating days 28 - 35, 49 - 70 and on PND 1 and 14
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of all male and female animals of all test substance-treated groups was generally comparable to the concurrent control values throughout the entire study. There were statistically significant increases or decreases in food consumption in F0 animals of various dose groups, which were small, temporary and rather inconsistent and thus considered to be spontaneous in nature and not treatment-related:
• increase in high-dose males on study days 0 – 21
• increase in high-dose females on premating days 35 - 42 and 0 - 70
• decrease in low-dose males on study days 42 – 49
• decrease and in low-dose females on PND 4 – 7.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption of all male and female animals of all test substance-treated groups was generally comparable to the concurrent control values throughout the entire study. The statistically significantly increased water consumption of the high-dose females during premating days 0 - 3 (about 11%) was most likely spontaneous in nature and not treatmentrelated
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in F0 males of test group 03 (150 mg/kg bw/d) absolute and relative neutrophil cell counts were significantly increased whereas relative lymphocyte counts were significantly decreased. In parental females of test group 03 (150 mg/kg bw/d red blood cell (RBC) counts, hemoglobin and hematocrit values were significantly decreased whereas absolute reticulocyte counts were significantly increased. These changes were regarded as treatment related and adverse.
In parental males of test group 03 (150 mg/kg bw/d) absolute reticulocyte counts were significantly increased whereas relative basophil counts were significantly decreased, but the values were within historical control ranges (males, absolute reticulocytes 105.7-175.0 Giga/L, relative basophils 0.1-0.4 %). In males of test group 02 (50 mg/kg bw/d) hematocrit values were significantly higher compared to controls, but the alteration was not dose dependent. Therefore, these changes were regarded as incidental and not treatment related.
In F0 females, absolute lymphocyte counts were significantly increased, but the values were within the historical control range (females, absolute lymphocytes 1.93-3.07 Giga/L). Therefore, this change was regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period, in males of test group 03 (150 mg/kg bw/d) cholesterol values were significantly increased and they were above the historical control range (males, cholesterol 1.56-2.10 mmol/L). However, this is an isolated liver parameter change. Therefore, it was regarded as maybe treatment related, but non-adverse. In females of this test group urea and inorganic phosphate values were significantly increased whereas globulin values were significantly decreased. Inorganic phosphate levels were already significantly higher in females of test group 02 (50 mg/kg bw/d). However, all values among these females were within historical control ranges (females, urea 5.63-8.76 mmol/L, inorganic phosphate 0.98- 1.57 mmol/L, globulins 24.80-27.79 g/L). therefore, the mentioned changes were regarded as incidental and not treatment related.
In males of test group 02 (50 mg/kg bw/d) glucose values were significantly decreased and in males of the same test group calcium values were significantly decreased. However, the changes were not dose dependent, and therefore, they were regarded as incidental and not treatment related.
Endocrine findings:
no effects observed
Description (incidence and severity):
Neither the anogenital distance/index nor the check for the presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects.
A statistically significant delay in vaginal opening of about one day beyond the concurrent control was observed in the female F1 offspring of the high-dose group (150 mg/kg bw/d). The delay is, however, within the historical control range of the test facility. This apparent delay was mainly due to 1 high-dose individual entering puberty after the age of 43 days. This and the still rather small average difference to the control of about one day indicates that the later onset
of puberty in the high-dose group is not a specific effect on the timing of puberty. In addition, none of the other endocrine-sensitive parameters like anogenital distance, or estrous cyclicity in the F1A offspring brought beyond puberty, or the integrity of sexual organs in these females including differential ovarian follicle count, indicated any effect of the test item. Thus, the apparently later entry of high-dose offspring into female puberty is likely to be incidental.
Entry into male puberty was not influenced by the test substance as indicated by an unchanged timing of preputial separation.
Measurement of thyroid hormones revealed no effect caused by the test substance, neither in the F0 parental animals nor in the F1 offspring.
At the end of the administration period in F0 males of test groups 01, 02 and 03 (15, 50 and 150 mg/kg bw/d) T4 values were significantly higher compared to controls. The mean values of all three test groups were above the historical control range (males, T4 44.65-73.22 nmol/L). However, TSH values were not changed among these individuals. No histologic finding in the thyroids was observed although relative thyroid weight in males of test groups 02 and 03 were significantly increased, but they were within the historical control range. Therefore, the isolated T4 increase in F0 males of test groups 01, 02 and 03 was regarded as maybe treatment related, but non-adverse (ECETOC Technical Report No 85, 2002).
In parental females of test group 03 (150 mg/kg bw/d) T4 values were significantly increased. The mean value was above the historical control range (females, T4 24.28-43.26 nmol/L). However, TSH values were not significantly changed and no histologic finding in the thyroids was observed. Therefore, the isolated T4 increase in F0 females of test group 03 was regarded as maybe treatment related, but non-adverse (ECETOC Technical Report No 85, 2002).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the fore- and glandular stomach in male and female animals of test groups 02 and 03.
Secondary findings were noted in the pancreatic lymph node in 10/25 male animals of test group 03. This lymph node is not a protocol organ and was only collected in the animals where it was macroscopically enlarged.

Forestomach:
In the forestomach, erosion/ulcer were noted in many animals of both sexes of test group 03, correlating to the gross finding “focus”. The forestomach also showed a thickening of the squamous cell layer, most frequently affecting the region of the margo plicatus, which was recorded as squamous hyperplasia, margo plicatus, diffuse. This was seen in both test group 02 and with increased incidence and severity in test group 03 animals of both sexes. This finding correlated to the gross finding “margo plicatus thickened”. In a few cases, the thickening of the squamous layer extended throughout the whole section of the forestomach, this was recorded as hyperplasia squamous cell diffuse. This was only seen in few animals of both sexes of test group 03.

Glandular stomach:
Multifocal degeneration/regeneration was seen in many animals of both sexes in test group 03. It was characterized by a loss of the normal architecture of the glandular mucosa, which was replaced by more basophilic undifferentiated cells. This finding was especially prominent adjacent to the margo plicatus. Erosion/ulcer were seen with increased incidence in treated animals of both sexes.

Pancreatic lymph node:
The gross finding “enlarged” in test group 03 correlated with increased cellularity of plasma cells and lymphocytes and lymphoid cysts and in a few cases with sinus histiocytosis. This was assumed to be a reaction to the ulcers and erosions seen in the fore- and glandular stomach. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The female animals (No.111 (test group 00), 130 (test group 01), and 199 (test group 03)), which were not pregnant as well as their male mating partners (No.11 (test group 00), 30 (test group 01), 54 (test group 02), and 99 (test group 03)) did not show relevant histopathological findings consistent with impaired fertility. Female animal No 154 showed an atrophic uterus, cervix, and vagina which was assumed to have contributed to the impaired fertility of this mating pair.

Decedents:
The female animal No. 139 of test group 01 died spontaneously. It showed an adenocarcinoma in the uterus and multifocal necrosis in the liver, which was unrelated to treatment.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups 01-03. The mean estrous cycle duration was comparable: 4.0 / 4.1 / 4.1 and 4.1 days in test groups 01-03, respectively.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermanalysis:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatmentrelated effects were observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (00 - 03). Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One low-dose male (No. 30) and one mid-dose male (No. 54) did not generate F1 pups. Thus, the male fertility index ranged between 96% and 100% without showing a doseresponse. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until copulation was detected (GD 0) varied between 2.4 and 2.9 days without any relation to dosing. All female rats delivered pups or had implants in utero with the following exceptions:
• Test group 01: female No. 130 (mated with male No. 30) did not become pregnant
• Test group 02: female No. 154 (mated with male No. 54) did not become pregnant
Except of female animal No 154, which had an atrophic uterus, cervix, and vagina, the apparently infertile female rats did not show histopathological findings that could explain infertility.
The fertility index ranged between 96% and 100% without showing any relation to dosing. The mean duration of gestation was comparable in all test groups (i.e. between 22.2 and 22.4 days).
The gestation index was 96% / 95.8% / 100% and 96% in test groups 00 - 03.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (11.9 / 11.9 / 11.8 and 12.1 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (11.2 / 11.2 / 5.4 and 10.8 mean% in test groups 00 - 03, respectively)., and the mean number of F1 pups delivered per dam remained unaffected (11.4/ 10.6 / 11.1 and 11.2 pups/dam in test groups 00 - 03, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97.4% / 100% / 97.5% and 98.2% in test groups 00 - 03, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups. The rate of liveborn pups was not affected by the test substance, as indicated by live birth index of 99.3% / 94.5% / 98.9% and 97.4% in test groups 00 - 03. Moreover, the number of stillborn pups was comparable between the groups. Thus, the test substance Hydroxypropyl acrylate did not adversely affect reproduction and delivery of the F0 generation parental females.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fertility/reproductive performance
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A:
All high- and most of the mid-dose males and females showed salivation at least once during the study. In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.
One mid-dose male animal (No. 246) had an encrusted eye (right, reddish) during study days 0 - 64, one mid-dose male animals (No. 248) had a protruding eyeball (right) during study days 21 - 64 and one high-dose male animal (No. 280) had a protruding eyeball (left) during study days 20 - 64. These observations were considered not to be associated with the test compound.
Detailed clinical observations (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups. One female animal (No. 374) of test group 13 was sacrificed moribund prior to DCO day 0. One mid-dose male animal (No. 246) had an encrusted eye (right, reddish) during DCO days 0 - 56, one mid-dose male animals (No. 248) had a protruding eyeball (right) during DCO days 21 - 56 and one high-dose male animal (No. 280) had a protruding eyeball (left) during DCO days 21 – 56. None of these findings was considered to be associated with the treatment.

F1 generation parental animals, Cohort 1B:
Nearly all high-and most of the mid-dose males and females showed salivation at least once during the study. In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.
One mid-dose male animal (No. 451) had a semiclosed eyelid (right) on study day 0, two middose male animals (No. 457 and 473) had encrusted eyes (both, reddish) during study days 0- 56 and 0 - 59, respectively. One high-dose male animal (No. 498) had a protruding eyeball (right) during study days 0 – 7, a protruding eyeball (right, dark red) during study days 7 – 15, a protruding eyeball (right, light red) on study days 16, a small eye (right) on study day 17, a discolored eye (right, light red) on study day 17 and an injury (right, cornea bleb) during study days 17 - 59. These observations were considered not to be associated with the test compound.
Detailed clinical observations (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups. Two mid-dose male animals (No. 457 and 473) had encrusted eyes (both, reddish) during DCO days 0 - 49, respectively. One mid-dose male animal (No. 472) had an semiclosed eyelid (right) on DCO day 0. One high-dose male animal (No. 498) had a protruding eyeball (right) on DCO day 0, a protruding eyeball (right, dark red) during DCO days 7 - 14 and an injury (right, cornea bleb) during DCO days 21 - 49 (for details see 4.2.4.2.). None of these findings was considered to be treatment-related.

F1 rearing animals, Cohort 3:
All high- and most of the mid-dose males and females showed salivation during the entire study. In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.
One control male animal (No. 1010) had a protruding eyeball (left) during study days 27 - 38. One high-dose female animal (No. 1133) ploughed nose first into bedding on study day 3. These observations were considered not to be associated with the test compound.
Detailed clinical observations:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A:
There were no test substance-related mortalities in any of the groups. One female animal (No. 374) of test group 13 was sacrificed moribund because of apathy, respiration sounds, piloerection and abdominal position on study day 0. Histopathology revealed a gavage error as the most likely reason for its death.

F1 generation parental animals, Cohort 1B:
There were no test substance-related mortalities in any of the groups. One male animal (No. 475) of test group 12 and one male animal (No. 487) of test group 13 were found dead on study day 9 and study day 51, respectively. The cause of death for both decedents were most likely gavage errors.

F1 rearing animals, Cohort 3:
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A:
Mean body weights and body weight change of all male and all female animals in all test substance-treated groups were comparable to the concurrent control values during the entire study. The statistically significantly increased body weight change in the mid-dose females during study days 0 - 7 was considered to be spontaneous in nature and not treatment-related since there was no relation to dose.

F1 generation parental animals, Cohort 1B:
Mean body weights and body weight change of male and female animals in all test substancetreated groups were generally comparable to the concurrent control values during the entire study.
There were statistically significant decreases in body weight/body weight change in F1B animals of various dose groups, which were small, temporary and rather inconsistent and thus considered to be of no toxicological relevance:
• decreased body weight change in high-dose males on study days 21 - 28, 35 - 42 and 42 - 49
• decreased body weight change in mid-dose males on study days 21 – 28 and 35 - 42

F1 rearing animals, Cohort 3:
The mean body weights and body weight change of all test substance-treated male and female animals were comparable to the concurrent control values throughout the entire study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F1 rearing animals, Cohort 1A:
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

F1 generation parental animals, Cohort 1B:
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

F1 rearing animals, Cohort 3:
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A:
Water consumption of all male and female animals of all test substance-treated groups was generally comparable to the concurrent control values throughout the entire study. The statistically significantly increased water consumption of the mid- and high-dose females during study days 7 - 17 and 14 - 17 was most likely spontaneous in nature and not treatmentrelated.

F1 generation parental animals, Cohort 1B:
Water consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. The statistically significantly decreased water consumption in the high-dose males during study days 49 - 52 as well as the statistically significantly increased water consumption of the highdose females during study days 35 - 38 were considered to be spontaneous in nature and not treatment-related.

F1 rearing animals, Cohort 3:
Water consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. The statistically significantly decreased water consumption in the mid-dose females during study days 21 - 24 was considered to be spontaneous in nature and not treatment-related since there was no relation to dose.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals:
Cohort F1A:
No treatment-related, adverse change among hematology parameters was observed. At the end of the administration period, in males and females of test group 13 (150 mg/kg bw/d) absolute reticulocyte counts were significantly increased. Both values were above the historical control range (absolute reticulocytes, males 138.2-186.4 Giga/L; females 138.4-188.7 Giga/L). Neither any histologic finding in the spleen nor in the bone marrow were observed. The same was true for the isolated, but significant decrease of relative eosinophil cell counts in males of test group 13 with values below the historical control range (males, relative eosinophils 1.7-2.7 %). No other differential blood cell fraction was altered, and the absolute eosinophil counts were also not significantly changed and within the historical control range (males, absolute eosinophils 0.09-0.12 Giga/L). Therefore, the isolated reticulocyte count increases as well as the significant relative eosinophil decreases were regarded as maybe treatment related, but non-adverse (ECETOC Technical Report No. 85, 2002). In males of test groups 11 and 13 (15 and 150 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) were significantly shortened, but the values were within the historical control range (males, HQT 32.6-38.3 sec). Therefore, this change was regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort F1A:
At the end of the administration period, in females of test group 13 (150 mg/kg bw/d) urea and inorganic phosphate levels were significantly increased. These changes were regarded as treatment related and adverse. In females of test group 12 (50 mg/kg bw/d) urea and inorganic phosphate values were already significantly higher compared to controls. Inorganic phosphate values were within the historical control range whereas urea values were slightly above this range (females, urea 5.63-6.34 mmol/L, inorganic phosphate 1.35-1.63 mmol/L). However, urea was the only changed parameter among these individuals and therefore, this alteration was regarded as treatment related, but non-adverse (ECETOC Technical Report No. 85, 2002), whereas the inorganic phosphate increase was regarded as incidental and not treatment related. In males of test group 13 (150 mg/kg bw/d) inorganic phosphate values were significantly increased whereas in females of this test group total bilirubin values were significantly decreased. However, both values were within historical control ranges (males, inorganic phosphate 1.60-2.04 mmol/L; females, total bilirubin 1.19-2.07 μmol/L). In males of test groups 11 and 12 (15 and 50 mg/kg bw/d) albumin values were significantly higher compared to controls, but the change was not dose dependent. Therefore, the alterations in this paragraph were regarded as incidental and not treatment related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalyses (cohort F1A):
No treatment-related, adverse changes among urinalysis parameters were observed.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio:
The sex distribution and sex ratios of live F1 pups on the day of birth and at weaning did not show substantial differences between the control and the test substance-treated groups; slight but statistically significant differences were regarded to be spontaneous in nature. All values were well in the PND 0 historical control range.

Vaginal opening:
Each female F1 pup, which was selected to become a rearing female (across all cohorts which have been taken beyond this age), was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 36. The mean number of days to reach the criterion in the control and 15, 50 and 150 mg/kg bw/d test groups was 30.6, 30.7, 30.9 and 31.7* (* = p≤0.05) days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 95.2 g, 96.4 g, 96.3 g and 97.8 g in test groups 00-03. The slightly higher high-dose time to puberty was mainly driven by one single female pup (194-04) which became sexually mature only on PND 43. As the average differed from control only by one day, the value was within the historical control range (29.5 – 38.8 days) and there were no corroborative findings in any endocrine sensitive tissues, no specific effect of the test compound on the timing of female puberty was assumed.

Preputial separation:
Each male F1 pup, which was selected to become a rearing male (across all cohorts which have been taken beyond this age), was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 39, the last was PND 47. The mean number of days to reach the criterion in the control and 15, 50 and 150 mg/kg bw/d test groups was 41.6, 41.3, 41.4 and 41.5 days, respectively. The mean body weight on the day, when preputial separation was recorded, amounted to 174.4 g, 173.7 g, 173.9 g and 172.1 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13 and during the re-examination on PND 20 in any male pups of all test groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 generation, rearing animals Cohort 1A:
Absolute organ weights:
- Liver (males): 110%* / 100% / 105% versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 109%** / 107%** / 113%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
- Liver (males): 110%** / 103% / 108%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 108%** / 108%** / 117%** versus ctrl in low, mid and high dose groups, respectively
- Thymus (females): 98% / 106% / 109%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10. As the thymus weight was not significantly changed in the immunotoxicity cohort 3 it was considered to be incidental.

F1 generation, rearing animals, cohort 1B
Absolute organ weights:
- Terminal body weithgt (males): 100% / 97% / 94%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 107%* / 109%** / 114%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
- Liver (males): 102% / 101%* / 105%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 106%** / 109%** / 115%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 106% / 115%** / 105% versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
The statistically significantly increased mean relative prostate weight in test group 12 was regarded to be incidental as there was no dose response relationship, the weight in test group 13 was not significantly changed and the weight in test group 12 (0.262%) was only very minimally (3rd decimal place) above historical controls (0.213 – 0.26%).

Comparison of liver weights in the F1 generation, rearing animals, cohort 1A and B with each other and with historical control data.The increased mean relative liver weights in males and females of test group 13 were regarded to be possibly treatment-related.

Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts):
Absolute and relative organ weights:
None of the mean absolute and relative weight parameters of test groups 01, 02, and 03 showed significant differences when compared to the control group 00.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort F1A:
In the forestomach, an increased incidence of the following findings was noted:
Focus:
male: 6/20 (150 mg/kg bw/d)
female: 3/20 (150 mg/kg bw/d)
Margo plicatus thickened:
male: 6/20 (50 mg/kg bw/d); 20/20 (150 mg/kg bw/d)
female: 2/20 (50 mg/kg bw/d); 19/20 (150 mg/kg bw/d)

In the duodenum thickening of wall was seen in few test group 13 animals.
Thickening of wall:
male: 4/20 (150 mg/kg bw/d)
female: 5/20 (150 mg/kg bw/d)
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 generation, rearing animals, cohort 1B:
In the forestomach, an increased incidence of the following findings was noted:Focus:
male: 4/25 (150 mg/kg bw/d)
female: 1/25 (50 mg/kg bw/d), 3/25 (150 mg/kg bw/d)
Margo plicatus thickened:
male: 6/25 (50 mg/kg bw/d); 20/25 (150 mg/kg bw/d)
female: 24/25 (50 mg/kg bw/d); 22/25 (150 mg/kg bw/d)
Thickening of wall:
male: 1/25 (150 mg/kg bw/d)
female: 1/25(50 mg/kg bw/d)
In a few animals, thickening of the wall of the duodenum was observed:
Thickening of wall:
male: 5/25 (150 mg/kg bw/d)
female: 3/25 (150 mg/kg bw/d)

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Decedents:
The animal No. 475 (male test group 12), died spontaneously 8 days after start of exposure and showed a red discoloration of the lungs. Male animal No. 487 (test group 13) died spontaneously 50 days after start of exposure; it showed a white deposition on the lungs on the right caudal lobe and at the transition to the diaphragm, as well as a thoracic cavity effusion with clear fluid). No histopathological evaluation was performed on these animals. The death was assumed to be likely a misgavage and not related to the test item.

Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts):
A single finding was noted in test group 02, it was considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort F1A:
In the forestomach, erosion/ulcer were noted in many animals of both sexes of test group 13, correlating to the gross finding “focus”.
The forestomach also showed a thickening of the squamous cell layer, most frequently affecting the region of the margo plicatus, which was recorded as squamous hyperplasia margo plicatus, diffuse. This was seen in both test group 12 and with increased incidence and severity in test group 13 animals of both sexes. This finding correlated to the gross finding “margo plicatus thickened”. In a few cases the thickening of the squamous layer extended throughout the whole section of the forestomach, this was recorded as hyperplasia squamous cell diffuse.
This was only seen in few male animals of test group 13 and one male in test group 12. Incidences and gradings of these findings are shown in the table below.
Glandular stomach:
Multifocal degeneration/regeneration was seen in many animals of both sexes in test group 13. It was characterized by a loss of the normal architecture of the glandular mucosa and was replaced by more basophilic undifferentiated cells. This finding was especially prominent adjacent to the margo plicatus. Unlike in the F0 generation, no treatment-related effect could be detected regarding erosion/ulcer in the glandular stomach.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. No histopathological correlate to the gross finding “Thickening of wall” in the duodenum could be observed.

Decedents:
The female animal No. 374 (test group 13) which was sacrificed moribund showed a white deposition on the thymus and partly red discoloration macroscopically. Histopathologically there was slight multifocal inflammation on the pleura of the lungs and on the pericardium with bacterial colonies. On the thymus, correlating to the gross findings, it showed marked hemorrhage and slight multifocal inflammation admixed with bacterial colonies and foreign material. The inflammation extended to the surrounding tissue of the thyroid gland where bacterial colonies were also found. The cause of death was therefore assumed to be a gavage accident.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
F1 generation, cohort 3 animals (immunotoxicity cohort) and animals of the positive control:

T-cell dependent antibody response (cohort 3):
No treatment-related changes regarding the anti-SRBC IgM antibodies occurred in the F1 test groups at PND 63 whereas in males and females of the positive control group with administered cyclophosphamide (test group 14) a significant decrease of anti-SRBC IgM antibodies was observed.

Weight parameters
Hydroxypropyl acrylate:
Absolute and relative organ weights: None of the mean absolute and relative weight parameters of test groups 11, 12, and 13, showed significant differences when compared to the control group 10.
Cyclophosphamide monohydrate
Absolute and relative organ weights: When compared to the control group 10 (set to 100%), the mean absolute and relative weight parameters of test group 14 (positive control) were significantly decreased.
Spleen:
male: 64%** (absolute) / 67%** (relative)
female: 73%** (absolute) / 74%** (relative)
Thymus:
male: 71%** (absolute) / 74%** (relative)
female: 68%** (absolute) / 68%** (relative)
*p <= 0.05; **p <= 0.01
A significant decrease in absolute and relative weights of the spleen and thymus occurred in the positive control male and female animals. This result was expected.

Gross pathology:
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Details on results (F1)

F1 generation pups/litters:
Pup number and status at delivery:
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the rat strain used in this study.

Pup viability/mortality
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.2% / 100% / 98.6% and 99.3% in test groups 00 - 03, showing no treatment-related effect. The lactation index indicating pup survival during PND 4 - 21 varied between 99.2% / 100% / 99.6% and100% in test groups 00 - 03, showing no treatment-related effect. Thus, the test substance did not influence pup survival in any of the treated groups (01 - 03).

Pup clinical observations:
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
For one male pup (No. 1) of test group 02 (dam No. 166) reduced nutritional condition (grade: severe) was observed on PND 7. For one female pup (No. 8) of test group 00 (dam No. 123) unsteady gait (grade: severe, abnormal position, left forelimb), semiclosed eyelid (both), reduced nutritional condition (grade: severe), piloerection and hunched posture was recorded during PND 14 - 19. These observations were considered not to be associated with the test compound.
Pup body weight data:
The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.

Pup necropsy observations in culled pups and decedents:
A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored liver lobe and empty stomach. These findings occurred without any relation to dosing. Thus, all these findings were not considered to be associated to the test substance.

F1 rearing animals, Cohort 1A:
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups 10 - 13. The mean estrous cycle duration was comparable between the groups: 4.0 / 3.9 / 4.0 and 4.1 days in test groups 10 - 13, respectively.

Thyroid hormones (surplus pups PND4, PND22 and cohort F1A):
In F1 PND4 male and female pups (test groups 01, 02 and 03; 15, 50 and 150 mg/kg bw/d) no treatment related alterations of T4 and TSH levels were observed. In F1 PND22 male and female pups (test groups 01, 02 and 03; 15; 50 and 150 mg/kg bw/d) no treatment related alterations of T4 and TSH levels were observed. In cohort F1A males and females of test groups 11, 12 and 13 (15, 50 and 150 mg/kg bw/d) no treatment related alterations of TSH values as well as in males of the mentioned test groups no change of the T4 values were observed. In F1A females of test group 13 (150 mg/kg bw/d) T4 values were significantly increased, but the values were within the historical control range (F1A females, T4 26.66-60.54 nmol/L). Therefore this alteration was regarded as incidental and not treatment related.

Lymphocyte subpopulations in spleen (cohort F1A):
No alterations in the absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) were observed in the F1 generation at PND90 in both sexes.

Spermanalysis (cohort F1A):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epidi-
dymidis as well as sperm head counts in the testis and in the cauda epididiymidis, no treatmentrelated effects were observed.

Differential ovarian follicle count (cohort F1A):
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and test group 13.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1 (cohort 3)
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 50 mg/kg bw/d for the F0 parental as well as F1 adolescent animals, based on changed clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 150 mg/kg bw/d. The NOEL (no observed effect level) for this study is below 15 mg/kg bw/d, based on clinical and pathological evidence of distinct local toxicity in the upper digestive tract, at all tested dose levels. These local effects were definitely dose-limiting.

The NOAEL for fertility and reproductive performance for the F0 parental rats is 150 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity in the F1 progeny is 150 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental immunotoxicity for the F1 progeny is 150 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.