Acrylic acid, monoester with propane-1,2-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are sufficient data available to assess the endpoint toxicity to reproduction.

The results of an OECD 443 and OECD 422 study with hydroxypropyl acrylate and a 2-generation-study (OECD Guideline 416) with the structural analogues acrylic acid (CAS No. 79-10-7) in rats by the oral route and the continuous breeding study with the metabolic cleavage product propylene glycol gave no indication of a fertility impairing effect.

In addition no indications of a possible impairment of fertiliy were observed in the oral 90 d study with hydroxypropyl acrylate in respect to histopathology of the gonads, sperm morphology or differential counts and ovarian follicle count. (see 7.5.1." A 90-Day Study of Hydroxypropyl Acrylate by Oral Gavage in Wistar Han Rats", Hydroxy Acrylate REACH Task Force, 2018)

Link to relevant study records

Reference

Reference 1

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

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.

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

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.

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.

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

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: 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.

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

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.

Behaviour (functional findings):

not examined

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.

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.

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

Reproductive effects observed:

no

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Chales River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Age at study initiation: 11 weeks (male animals); 10 weeks (female animlas)
- Housing: During the pretreatment period of the study, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Ho
henpeißenberg, Germany. During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance solutions in drinking water 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.

Details on mating procedure:

In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water for a period of 7 days at room temperature were carried out.

Duration of treatment / exposure:

After the acclimatization period, the test substance was administered to the parental animals orally by gavage.

Frequency of treatment:

once daily at approximately the same time in the morning

Dose / conc.:

0 mg/kg bw/day (nominal)

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:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity,
pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions: Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20. Food consumption of the females which gave birth to a litter was determined for PNDs
1-4, 4-7, 7-10 and 10-13. Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

Body weight data
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
During the mating period the parental 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 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume;

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the
administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

Functional observational battery
A functional observational battery (FOB) was performed in the first five male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremors, convulsions, abnormal movements, gait, other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test

Motor activity assessment
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For the duration of the measurement the animals were placed in new clean polycarbonate cages with a small amount of bedding. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed into the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Clinical Pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. 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. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, mean corpuscular volume, mean corpuscular Hemoglobin, mean corpuscular Hemoglobin concentration, Platelet count, Differential blood, Reticulocytes, Prothrombin time.

Clinical chemistry
Parameters: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase), γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, choloesterol, bile acids.

Thyroid Hormones
Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
All generated serum samples were frozen at -80°C until measurement.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).
T4 ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Oestrous cyclicity (parental animals):

For all females in a pool of 50 animals, estrous cycle normality was evaluated before randomization (the estrous cycle data of these individuals are not reported and can be found in the raw data). For a minimum of 2 weeks prior to mating, estrous cycle length and normality was evaluated by daily analysis of vaginal smears for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, one additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Litter observations:

Litter observation:
Litter data
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 “Pup Necropsy observations“. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying
between PNDs 1-4, 5-7 and 8-13 were determined. 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 of birth (PND 0), and on lactation days 4, 7 and 13.

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.

Pup body weight data
The pups were weighed on the day after birth (PND 1) as well as on PND 4 (before
standardization), 7 and 13. 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). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females. „Runts“ were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

Necropsy
All 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.

Organ weights
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed) ,Uterus (with cervix).

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus.

Organ/tissue fixation
Parental animals
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde solution or in modified Davidson`s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides (modified Davidson`s solution), Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson`s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer`s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson`s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson`s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964), Vagina.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All gross lesion, Adrenal glands, Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer`s patches, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid , glands, Trachea, Urinary bladder, Uterus, Vagina.

Postmortem examinations (offspring):

On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing.

Statistics:

Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Reproductive indices:

The following indeces were determined: mating and fertility indes for both males and females, gestation index, live birth index and postimplantation loss for female.

Offspring viability indices:

The following indeces were determined: viability index, survival index, sex ration, anogenital index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Most animals of test group 3 as well as several animals of test group 2 (150 and 50 mg/kg bw/d) show ed salivation after treatment (grade: slight to severe) at some occasions during the study period. In female animals the incidence was generally higher during gestation and lactation periods, affecting most test group 3 and several test group 2 females. These observations were considered to be treatment-related.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups. Spontaneous findings were seen in one high-dose male (No. 33) which showed red discharge in the right eyeduring mating days 2 - 4, 8 - 11 and postmating days 0 – 1 as well as one sperm positive mid-dose female (No. 121) which did not deliver F1 pups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups showed no significant difference to the concurrent control during the entire study.
The statistically significantly lower body weight change in the mid-dose females during PND 7 - 10 was considered as spontaneous in nature and not as treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the F0 males in all dose groups (15, 50 and 150 mg/kg bw/d) as well as low and mid-dose females was not influenced by the treatment throughout the study.
Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values during the entire premating period (up to 7%) while it remained unchanged during gestation and lactation periods.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males of test group 3 (150 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significant ly reduced, but the mean was within the historical control range (males, HQT 37.4 - 40.9 sec). Therefore, this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males of test groups 1 and 2 (15 and 50 mg/kg bw/d) cholesterol values were significantly changed (test group 1 lower, test group 2 higher) compared to controls, but the alteration was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatmentrelated.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational battery (FOB)
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations: Two male animals of dose group 3 (Nos. 33 and 34 - 150 mg/kg bw/d) showed slight (area around the
mouth was moist) and severe (mouth very wet, wet paws) salivation, respectively. All other male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) did not show any abnormalities.
Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test
groups. The statistically significantly higher value of the landing foot splay test in females of dose group 2 was considered as spontaneous in nature and not treatment related.
Motor activity measurement (MA)
No treatment-related changes of motor activity data was observed in all male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) in comparison to the concurrent control group. Overall activity levels and habituation to the test environment corresponded to the age of these animals, if usual biological variation inherent in the strain of rats used for this experiment was considered.
The isolated statistically significantly decreased numbers of beam interrupts in the males of dose groups 1 and 2 during interval 12 were not related to the dose and did not influence the overall activity levels and habituation. Thus, they were not considered to be related to the test substance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in forestomach and duodenum of male and female animals of test groups 2 and 3.
Duodenum: In the duodenum, a thickening of the mucosa characterized by an increase in villus height, was observed, which correlated to the macroscopic finding “dilation”. One female animal of test group 3 showed a focal hyperplasia of the duodenal mucosa.
Forestomach: Diffuse squamous hyperplasia (correlating with the macroscopic findings “margo plicatus, thickened”) and the presence of erosion/ulcer (correlating often with the macroscopic finding “focus”) were noted in the forestomach of male and female animals.
No treatment-related findings were noted in the glandular stomach and no correlate was found for the macroscopically observed discoloration in two test group 3 male animals. 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.The morphology of the testicular interstitium and the stages of spermatogenesis in the testes of males of the high dose (150 ppm) were comparable to those of the controls.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 4.0 days in test groups 0 - 3, respectively.

Reproductive performance:

no effects observed

Thyroid hormones:
In the F0 parental males of test groups 2 and 3 (50 and 150 mg/kg bw/d) T4 values were significantly higher compared to controls. However, the means were within the historical control range (males T4 44.87-88.29 nmol/L). Additionally, neither a change of thyroid weight nor any histopathological findings in the thyroids were noted. Therefore, the higher T4 values in parental males were regarded as incidental and not treatment-related.
In male and female pups at PND13 (test groups 11, 12 and 13; 15, 50 and 150 mg/kg bw/d), no treatment-related alterations of T4 levels were observed.

Male reproduction data:
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 test groups 0 - 3. Fertility was proven for most of the F0 parental males within the scheduled mating interval for
F1 litter.
One mid-dose male (50 mg/kg bw/d - No. 21) did not generate pregnancy.
Thus, the male fertility index ranged between 90% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until copulation was confirmed (GD 0) varied between 2.1 and 5.3 days without showing a statistically significant difference. The reason for the higher average in test group 3 were 3 mating pairs with copulation confirmed after 12 or 14 days of pairing, respectively. All 3 affected females had normal estrous cycles and normal pregnancies, and no findings were noted in the reproductive organs of the affected males and females. The very extensive period before copulation fits best to artificial pseudo-pregnancies provoked by the daily manipulations during vaginal lavage. None of this is considered to be treatment-related.
All female rats delivered pups or had implants in utero with the following exceptions: Mid-dose female No. 121 (mated with male No. 21) did not become pregnant.
The fertility index varied between 90% in test group 2 and 100% in the control and test groups 1 and 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non-pregnant female had no relevant gross lesions or microscopic findings.
The mean duration of gestation values varied between 21.9 (test group 1), 22.2 (control and test group 2) and 22.4 (test group 3), not indicating any influence by the test substance.
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 (12.8 / 13.0 / 13.2 and 14.3 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.1 / 12.6 / 12.6 and 13.9 pups/dam in test groups 0 - 3, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 100% and 99.3% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance Hydroxypropylacrylate did not adversely affect reproduction and delivery of the F0 generation parental animals.

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed.

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: irritation in forestomach and duodenum

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

PND 0: 1 male pup died in test group 0 and 3
PND 1-4: 1 male and 1 female pup died in test group 1; 2 male pups died in test group 2

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1 - 3 (15, 50 and 150 mg/kg bw/d).
One female runt was seen in the control, one male and two female runts were seen in test group 1, three male and two female runts were seen in test group 2 and one male and two female runts were seen in test group 3. These are spontaneous findings unrelated to the treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored testis (left, red) and dilated renal pelvis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Litter data
Pup number and status at delivery
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
Pup viability
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100%/ 98.6% /97.9% and 100% in test groups 0 - 3, respectively. The survival index indicating pup mortality until mid-lactation (PND 4 - 13) was 100% in all test groups.
Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Anogenital distance/anogenital index
No test substance-related effects were noted on anogenital distance or anogenital index in all treated F1 offspring (test groups 1 - 3 [15, 50 and 150 mg/kg bw/d]).
Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL for fertility and reproductive performance was 150 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/d.

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From May 19, 1992 to April 19, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats (Chbb = THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: (P): 35 ± 1 days
- Weight at study initiation: (P) Males: 140.0 (127 - 154) g; Females: 118.8 (106 - 130) g
- Housing: Single in type DK III stainless steel wire mesh cages, with the following exceptions: during mating periods, the males designated for mating were kept individually in Makrolon cages, type M III; for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages.
- Diet (ad libitum): Kliba maintenance diet rat/ mouse/hamster GLP 343 meal (KLINGENTALMUEHLE AG, Kaiseraugst, Switzerland)
- Water (ad libitum): Tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 3 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of Acrylic acid in the aqueous solutions was determined by gas chromatography.

Duration of treatment / exposure:

Exposure period: F0: at least 70 days before the mating and afterwards during the gestation and lactation periods.
F1: at least 98 days before the mating and afterwards during the gestation and lactation periods.
Premating exposure period (males): at least 70 days
Premating exposure period (females): at least 70 days
Duration of test: approx. 12 months

Frequency of treatment:

continuously

Dose / conc.:

500 ppm

Remarks:

corresponding to approx. 53 mg/kg bw/day

Dose / conc.:

2 500 ppm

Remarks:

corresponding to approx. 240 mg/kg bw/day

Dose / conc.:

5 000 ppm

Remarks:

corresponding to approx. 460 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Positive control:

none

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. Particular attention was given to the nesting, littering and lactation behaviour of the dams, but only special findings were documented.

BODY WEIGHT:
- Parental animals: generally, body weight was determined once weekly until the end of the study, and at the time of necropsy.
- F0 and F1 fertilized females and females with litter: body weight was determined on the day of sperm evidence in the vaginal smear and thereafter on days 7, 14 and 20 of gestation, one day after parturition, and on days 7, 14 and 21 post-parturition.
- Females without positive evidence of sperms: body weight was not determined during the mating interval.
- Females without litter: body weight was not determined during the lactation phase.

FOOD CONSUMPTION:
- F0 and F1 parental animals: food consumption was determined once weekly (over 7 days) during the period prior mating.
- Pregnant females: food consumption was determined for days 0-7, 7-14, 14-20 post coitum (pc).
- Lactating females: food consumption was determined for days 0-4, 4-7, 7-14 post parturition (pp).
- F0 and F1 dams between day 14 and 21 pp: food consumption was not determined for the F0 and F1 dams between day 14 and 21 pp, since during this period the pups started consumption of solid food; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: food consumption was not determined respectively during mating period, gestation period or lactation phase.


WATER CONSUMPTION:
- F0 and F1 parental animals: water consumption was determined once weekly (over 3 days) during the period prior mating.
- Pregnant females: water consumption was determined for days 0-1, 6-7, 13-14, 19-20 post coitum (pc).
- Lactating females: water consumption was determined for days 1-2, 3-4, 6-7, 13-14 post parturition (pp).
- F0 and F1 dams from day 20 and 21 pp: water consumption was not determined for the F0 and F1 dams for days 20 - 21, since during this period the pups started consumption of water; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: water consumption was not determined respectively during mating period, gestation period or lactation phase.


INTAKE OF TEST SUBSTANCE:
The intake of test substance (IT, in mg/kg bw/day) was calculated according to the following formula:

ITx = WCx * D / BWy

D = dose in ppm
WCx = daily water consumption on day x; in g
BWy = body weight on day y; in g

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

The pups (F1 and F2 litters) were examined as soon as on their day of birth for the determination of the total number of pups and the number of liveborn and stillborn pups (pups died on day of birth prior the first examination). Thereafter the pups were checked twice daily on workdays (once a day on week ends and public holidays) for mortality (i.e. dead and moribund pups) and the mortality (number and percentage) was determined for the day of birth (i.e. day 0) and for the periods: days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of lactation. Pups that died accidentally and had to be sacrificed because of maternal death were not considered for calculation. The number of surviving pups was determined for days 0, 4, 7, 14 and 21 of lactation and served for the calculation of the viability index and the lactation index.

The sex of the pups was determined on day 0 and day 21 (measurement of the anogenital distance, which is known to be greater in male pups than in females), and the sex ratio was calculated according to following formula:

- Sex ratio = number of live male or female pups on day 0/21 * 100 / number of live male and female pups on day 0/21

The pups were weighed on days 1, 4, 7, 14 and 21 after birth, and they were examined daily for clinical symptoms or gross morphological abnormalities. The determination of the relative organ weight was based on the pup body weight on day 21 after birth. The bodies of the sacrificed pups were examined for external abnormalities and the organs also were subjected to gross pathology; skeletal staining according to the modified Dawson´s method and/or further processing of the head according to Wilson´s method was done in case of abnormal findings. Stillborn pups as well as pups that died during weaning also were subjected to necropsy.

Development stages / Behavioral tests:
Physical development was assessed by monitoring pinna unfolding, opening of the auditory canal and opening of the eyes. Additional tests
were performed to assess grip reflex, hearing and pupillary reflex as follows :
- Grip reflex: Tested on day 13 after birth by placing front paws onto 3-mm diameter rod. For a positive response, the animal had to grip the bar and pull itself up.
- Hearing test: On day 21 after birth, animals were placed in a soundproof box and exposed to a sound (0 .1 sec, 5000 Hz, about 90 dB); a startle reflex was considered a response to this stimulus.
- Pupillary reflex: On day 2l after birth, pupillary constriction reflex was assessed by shining a penlight on the eye and observing the reaction.

Postmortem examinations (parental animals):

SACRIFICE
Parental animals were killed by decapitation under CO2 anaesthesia and examined macroscopically.

GROSS NECROPSY
Terminal body weights as well as the weights of liver, kidneys, epididymides and testes were recorded .

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys and stomach (non-glandular and glandular).

Postmortem examinations (offspring):

SACRIFICE
Pups were killed by CO2 asphyxiation, examined externally, eviscerated and their organs assessed macroscopically.


GROSS NECROPSY
External and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY
Vagina, cervix, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulation gland, oesophagus and duodenum.

Statistics:

The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: Dunnett test, Fisher`s exact test and Wilcoxon test.

Reproductive indices:

Mating and fertility indices were calculated according to following formulas:

- Male mating index (%) = number of males with confirmed mating * 100 / number of males placed with females

- Male fertility index (%) = number of males proving their fertility * 100 / number of males placed with females

Remark:
Males were defined as “with confirmed mating” by the presence of vaginal sperm in the female, or by the production of a litter, or by the presence of fetuses in the uterus.
Males were defined as “proving their fertility” by female giving birth to a litter or having pups or fetuses in the uterus.

Reevaluation of fertility:
If an animal of the F0 or F1 generation parental animals had not produced any offspring after the scheduled mating of F0 parents (to get F1 litter) or after the scheduled mating of F1 parents (to get of F2 litter), those animals treated with the test substance were mated with fertile animals of the control groups. Animals of the control groups which seemed to be infertile were mated with mating partners with proven fertility of the controls.
After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological and histopathological assessments. The uteri of the females reevaluated for fertility were examined for live and dead implantations. In the case of an apparently non-pregnant animal or of an empty uterus horn in the case of single-horn pregnancy, the uterus was stained with sodium sulfide and assessed for early implantations. Then the uteri were rinsed carefully under running water. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation and evaluation.

Offspring viability indices:

- Viability index (%) = number of live pups on day 4 after birth * 100 / number of liveborn pups on the day of birth

- Lactation index (%) = number live pups on day 21 after birth * 100 / number of live pups on day 4 after birth

Remark:
Day 4 after birth preceded standardization of the litters.
Day 21 after birth followed standardization of the litters.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

**** F0 GENERATION PARENTAL ANIMALS ****

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no mortalities in any of the F0 generation parental animals in any of the groups.
- No clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals. The 3 concentrations administered in the drinking water did not lead to disturbances of the general behavior in any of the F0 parental animals.
 - There were no particular substance-related clinical findings in F0 females during the gestation period for F1 litter. Insufficient nesting activity was observed for several dams of all groups including the controls.
 - No substance-related clinical findings were recorded for the F0 dams during the F1 lactation period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
In general, the food consumption of the males (during the premating period) and of the females (during premating, gestation and lactation periods) of all test groups was not influenced by the test substance administration. It was, however, slightly, but statistically significantly reduced in the high dose males during the first week of the premating period and in the females of 5000 ppm test group during the second week of the lactation period.
The sporadic and only marginal reductions in food consumption of the 5000 ppm rats are probably related to the reduced consumption of aqueous acrylic acid solutions of these animals and thus are likely indirectly associated with the administration of the test substance. All other observable differences between the groups are without biological relevance, because they are not dose-related; this includes the statistically significantly increased food consumption of the low dose females during study weeks 0-1 and 6-7 of the premating period.

BODY WEIGHT (PARENTAL ANIMALS)
In the F0 males statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) from week 12 until week 20 of the study period. Body weight changes of the high dose males were statistically significantly diminished only at certain study intervals (weeks 0-1, 6-7, 11-12); if calculated for the total study period (weeks 0-20), body weight gain of the high dose males was about 9% lower than that of the respective controls.
Body weights and weight gains of the substance-treated females were similar to control values during the premating period and during gestation and lactation. Only during the first week of gestation did the high dose dams gain statistically significantly less weight than the corresponding controls.
Finally the impairments in body weight/body weight gain in the high dose F0 males and - to a lesser extent - in the F0 females are assessed as being substance-related effects. All other statistically significant differences in body weights and body weight gains are considered unrelated to the test substance because the values were not influenced in a dose-dependent manner and/or are within the biological range of variation.

WATER CONSUMPTION (PARENTAL ANIMALS)
The water consumption of the high dose male and female animals was clearly reduced. This reduction was statistically significant during the premating period. It was also diminished in the 5000 ppm females during gestation and lactation of F1 litter. In total the 5000 ppm males consumed about 11% and the high dose females about 13% less drinking water (aqueous acrylic acid solutions) then the respective controls during the first 10 study weeks. The marked reduction in the drinking water consumption of the high dose rats was associated with the test substance administration. All other observable differences between the groups in respect to water consumption are without biological relevance, because they are not dose-related; this includes the statistically significantly increased water consumption of the 500 ppm female animals during premating weeks 6-7 and 9-10.

MALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
For all F0 males which were placed with females to generate F1 pups mating was confirmed; thus, the male mating index was 100% in all groups. For nearly all F0 males fertility could be confirmed within the scheduled mating interval; the fertility index varied between 92% and 96% with no treatment related effect. Thus, the fertility of the F0 generation parental males was not adversely influenced by the administration of aqueous acrylic acid solutions.

FEMALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
The female mating index calculated after the mating period for F1 litter was 100% for all groups. The mean duration until sperm was detected (day 0 pc) varied between 1.8 and 3.8 days and was statistically significantly longer for the high dose dams; the high dose value (3.8 days), however, is substantially similar to the mean cohabitation time value of the control group (3.2 days) of the second parental generation (F1 animals) and therefore was not considered treatment-related. Only one or two females in all groups, including the controls, did not become pregnant within the scheduled mating interval. The fertility index varied between 92% and 96% without any dose-response relationship. All females in question except the 2 low dose females proved to be fertile after being mated again with control males. The mean duration of gestation was similar in all groups and the gestation index reached 100% for all groups. The mean number of pups delivered/dam was uninfluenced by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups, and the live birth index was 98% in test groups. Thus, the administration of aqueous acrylic acid solutions did not adversely affect reproduction and delivery data of the FO generation parental females.


GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

Dose descriptor:

NOAEL

Effect level:

240 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: fertility

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Critical effects observed:

no

**** F1 GENERATION PARENTAL ANIMALS *****

MORTALITY / CLINICAL OBSERVATIONS
- One male animal of 2500 ppm test group had to be sacrificed in a moribund state due to a severe skin lesion on the base of the tail. There were no other unscheduled mortalities in any of the test groups.
 
- No clinical signs which might have been attributed to the test substance administered were detected in male or female F1 generation parental animals. The 3 doses administered in drinking water did not lead to disturbances of the general behavior in any of the F1 parental animals. One male animal of 2500 ppm test group developed a severe skin lesion on the base of the tail and was sacrificed in a moribund state. Another male of the same dose group showed unilateral chromodacryorrhea. The clinical findings which occurred in just two intermediate dose males were spontaneous in nature.
 
- No particular clinical findings were noted for F1 dams with positive sperm detection except insufficient or no nesting activity, which was recorded for several dams of all groups (0, 500, 2500 and 5000 ppm) and which occurred without a clear dose-response relationship. One female of the low dose group showed vaginal hemorrhage towards or after the end of the gestation period (days 23 - 26 pc), and was not able to deliver the pups, which were palpable earlier in the abdomen of this dam. After day 26 pc, no pups could be palpated for this dam.
 
- There were no substance-related clinical findings in the F1 dams during the lactation of F2 litters. Only one dam of the low dose group and one dam of the high dose group did not nurse their pups properly; all pups of high dose dam were cannibalized and/or died intercurrently. Furthermore, another low dose dam showed blood in bedding during the first days of the lactation period, and was not able to deliver its litter completely. It delivered only 2 pups which were cannibalized on day 1 pp.

FOOD CONSUMPTION
The mean food consumption of the males and females of 5000 ppm test group was clearly reduced during the premating period, the differences in comparison to the controls being statistically significant at most time intervals. In total, the high dose males consumed about 9% and the females about 8% less food than the respective control animals during the premating phase. Food intake was also statistically significantly diminished in the females of this test group (5000 ppm) during the gestation period (days 7-20 pc) and during the lactation period (days 7-14 pp only). The reduction in food consumption of the 5000 ppm males and females was considered to be related to the administration of the test substance. All other differences in food consumption between the groups are without any biological relevance.
 
WATER CONSUMPTION
In comparison to the respective control values the water consumption of the 2500 and 5000 ppm F1 males and females was distinctly lower during the premating period, the differences being statistically significant at all intervals in the high dose level and in several but not all intervals at the intermediate dose. In total a clear dose-response relationship was observed: high dose males consumed about 18%, intermediate dose males about 9% less water than control males; for high dose females about 27% and for intermediate dose females about 13% less water intake than in the female controls was recorded. Water consumption was also reduced during gestation and lactation periods in these test groups, again more pronounced in the high than in the 2500 ppm group. The water consumption of the animals of the low dose group (500 ppm) reached or even exceeded the relevant control values during premating, gestation and the lactation periods. The distinct reductions in the drinking water consumption in both sexes at 2500 and 5000 ppm are considered treatment-related, whereas the differences in water consumption between the low dose group and the control are considered to be without toxicological relevance.
 
BODY WEIGHT
In the F1 males, statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) throughout the total study period (about 87% of the control value at the end of this study interval). Body weight gains of the 5000 ppm males, however, were generally similar to the respective control values. In total, the weight gain of the high dose F1 males was only about 5% lower than the body weight gain of the control males. Body weights of the 5000 ppm females were also statistically significantly reduced during the premating period (about 89% of the control value at the end of the premating phase). During gestation and lactation of F2 litter, mean body weights of the high dose F1 dams were statistically significantly lower than the corresponding control values. During premating, gestation and lactation periods body weight gain of the high dose females reached or even exceeded body weight gain of the controls.
The statistically significantly lower body weights recorded for the 5000 ppm F1 males and females were considered to be related to the test substance administration. A lower body weight was also recorded for these animals at the pup stage; during the following premating period the F1 parental animals of the high dose group gained substantially as much weight as the controls, but the body weights of the 5000 ppm rats were still reduced. All differences between the controls and 500 or 2500 ppm groups concerning body weights/weight gains, however, were regarded as spontaneous in nature.
 
MALE REPRODUCTIVE FUNCTION
For all F1 males which were placed with females to generate F2 pups, mating was confirmed. The male mating index was 100% in all groups.
 
FEMALE REPRODUCTIVE FUNCTION
The female mating index reached 100% in all groups. The mean duration until sperm was detected (day 0 pc) varied between 2.1 and 3.2 days and was highest for the control group, because one dam of this group had a prolonged cohabitation time. In the scheduled mating interval (F2), 2 control females did not become pregnant. Therefore, the fertility index was lowest in the control group (92%), whereas it was 100% in all substance-treated groups. There were no biologically relevant differences between test groups and the controls concerning the mean duration of gestation and the number of liveborn and stillborn F2 pups. All pregnant females - except one low dose female which had palpable pups in the uterus but did not deliver - gave birth to litters with liveborn pups. Consequently, the gestation and the live birth indices were not influenced by the administration of the test substance. The mean number of delivered pups/dam was not influenced by the test substance administered.

GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: fertility

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Dose descriptor:

NOAEL

Effect level:

53 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

**** F1 GENERATION PUPS/LITTERS ****

VIABILITY
No substance-induced effects on pup mortality/viability were recorded during the lactation period. Both the viability index, as an indicator of the viability of the pups during the first 4 days after birth, and the lactation index, as an indicator how the pups were nursed during the rest of their rearing, do not show differences of biological relevance.
 
SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 post parturition (pp) did not show any substantial difference between controls and treated groups; all differences observed are regarded as spontaneous.
 
BODY WEIGHT
Mean body weights of F1 male and female pups were clearly reduced in 5000 ppm test group from day 14 pp onwards and impaired in the intermediate dose (2500 ppm) on day 21 pp when compared to the controls. On day 21 pp the pup weights (both sexes combined) in the high dose group were about 35% and those of the 2500 ppm pups about 11% lower than the corresponding control values. Body weight gains of the 2500 ppm and 5000 ppm pups were also statistically significantly decreased from days 7 (5000 ppm) or 14 pp (2500 ppm) up to weaning (day 21 pp). The reductions in pup body weights/body weight gains in the 5000 and 2500 ppm groups were attributed to the test substance administration. All other differences concerning pup body weights/body weight gains are without any biological relevance and lie within the biological range of variation.
 
CLINICAL OBSERVATIONS
- None of the F1 pups of any one group showed abnormal clinical findings during the lactation period.
- There were no biologically relevant differences between the control and the substance-treated F1 pups in the several morphological development stages monitored up to weaning.
- No remarkable differences between the groups were observed in the different behavioral tests which the pups underwent up to weaning.
- Only spontaneous findings were seen at necropsy (e.g. incisors sloped, hernia diaphragmatica, dilated renal pelvis) in very few of the pups examined. All findings were present in the concurrent control at a comparable frequency and/or did not show a clear relation to dosing.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

53 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

460 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: fertility

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Critical effects observed:

no

**** F2 GENERATION PUPS/LITTERS ****

PUP NUMBER AND STATUS
The mean number of delivered F2 pups/dam in the treatment groups was similar to the relevant control value; moreover, the percentages of liveborn and stillborn F2 pups were comparable between the groups; all differences between the groups are in the range of biological variation.
 
VIABILITY/MORTALITY
During the lactation period a statistically significant increase in pups cannibalized by dams was noted in the high and intermediate dose groups. The increased cannibalization rate was predominantly caused by just one intermediate dose dam and two high dose dams. One Female of the high group, however, neglected her pups during the lactation period, thus nearly all pups of this dam died before schedule and/or were cannibalized. Occasionally insufficient nursing behavior and cannibalism occured also in control females, and thus the higher rate of cannibalized pups at these dose levels was not considered treatment-related.
There were no differences in biological relevance between the control and the 500, 2500 and 5000 ppm F2 pups concerning viability and mortality; consequently the viability and lactation indices were not affected by the test substance administration (although some statistically significant differences existed). All relevant values are inside the historical control range and/or do not show a clear relation to dosing; moreover it had to be taken into consideration, that the high dose dams delivered on average distinctly more pups (14.0 pups/dam) than the controls (12.9 pups/dam).
 
SEX RATIO
No remarkable differences between the control and the substance-treated groups were found in respect to the sex ratio of the F2 pups. The observable differences are in the range of biological variation.
 
BODY WEIGHT
Mean body weights/body weight gains of the F2 male and female pups of the 5000 ppm and 2500 ppm groups were clearly influenced by the test substance administration. Mean pup body weights of the 5000 ppm pups were statistically significantly lower than the corresponding control values from days 14 (males and females) until weaning on day 21 pp, when the high dose pups (both sexes combined) weighed about 32% less than the controls. Mean pup body weights of the 2500 ppm pups were statistically significantly (about 12%) lower than the corresponding control values on day 21 pp (both sexes combined). Weight gains of the pups of the 2500 and 5000 ppm test groups were also statistically significantly reduced from the second week of the lactation period onward, the reduction more pronounced in the 5000 ppm than in the 2500 ppm pups. All differences between the control group and the 500 ppm group concerning pup body weight data of the F2 generation were considered spontaneous in nature.
 
CLINICAL OBSERVATIONS
- F2 generation pups did not show any clinical signs up to weaning which could be attributed to the treatment. Hydrocephaly, which occurs also occasionally in control pups was recorded in one 500 ppm pup.
- Development stages: There was a statistically significantly lower incidence of F2 pups/litter with auditory canal opening on time in the intermediate dose group and with eye opening on time in the 5000 ppm group. The relevant values were within the historical control ranges and a clear relation to dosing was not observed. These effects must be considered in conjunction with the retarded weight gain of these pups and were therefore assessed as being possibly substance-related. There were no differences of biological relevance in different stages of development between the low dose and the control pups.
- No substantial differences could be noted between the F2 pups of all test groups and the control pups in the different behavioral tests. The observable differences were without biological relevance.
- The examinations of F2 pups at necropsy did not reveal any differences considered to be of biological relevance between the controls and the substance-treated groups either in the type or in the number of pup necropsy observations. A few pups of the different groups showed some spontaneous findings like hernia diaphragmatica, incisors sloped, dilated renal pelvis, hydroureter, hydrocephaly, focal liver necrosis, cardiomegaly, septal defect and post mortem autolysis.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

53 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

460 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: fertility

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Critical effects observed:

no

Reproductive effects observed:

no

Mean Body Weight Changes (F0 parental animals), grams

Treatment week	0 mg/kg bw/d		53 mg/kg bw/d		240 mg/kg bw/d		460 mg/kg bw/d
	male	female	male	female	male	female	male	female
0 - 1	52.6	23.8	52.2	24.8	52.3	23.6	46.7^**	23.1
1 - 2	53.7	22.1	55.5	22.0	54.4	22.3	51.6	21.8
2 - 3	47.2	16.2	45.8	17.2	46.9	17.5	44.8	14.6
3 – 4	35.4	12.9	34.8	17.1^*	35.1	14.9	32.4	17.8^*
4 – 5	29.1	15.7	27.6	13.3	28.6	12.6	27.0	13.7
5 – 6	21.0	9.4	21.8	8.8	23.3	12.5	23.2	11.0
6 – 7	23.2	8.5	24.7	11.7	21.2	10.1	19.3^*	10.7
7 – 8	20.4	7.7	17.7	10.3	19.8	7.8	18.5	8.6
8 – 9	19.3	9.8	19.1	7.1	18.0	7.2	16.6	8.1
9 – 10	13.1	4.7	13.0	4.5	14.6	7.9^*	11.9	6.0
10 – 11	-7.0		-2.8		-2.8		-2.8
11 – 12	21.8		18.5		18.4		14.7^**
12 – 13	14.8		13.3		13.7		11.1
13 – 14	8.0		10.7		9.7		6.1
14 – 15	9.2		7.0		7.5		8.1
15 – 16	10.5		10.4		8.0		9.9
16 – 17	5.7		7.9		7.3		4.6
17 – 18	2.7		4.6		1.7		1.4
18 – 19	1.1		0.2		1.3		1.6
19 - 20	4.1		6.7		5.8		5.0

^*P<0.05

^**P<0.01

Reproduction and litter data for F0 parents /F1 pups

	0 ppm	500 ppm	2500 ppm	5000 ppm
Parents
Females mated	25	25	25	25
Females pregnant	24	23	23	24
Females with delivery	24	23	23	24
Mean duration of gestation (d)	22.0	21.9	21.9	21.9
Litter means
Live births/litter	13.8	13.8	13.7	14.3
Survivors day 4 preculling	13.3	13.5	13.4	13.8
Survivors day 4 postculling	7.5	7.9	7.9	7.9
Survivors day 21	7.5	7.9	7.8	7.8
Weight at day 1 (g) M/F	6.4/6.1	6.6/6.2	6.5/6.2	6.5/6.2
Weight at weaning (g) M/F	52.3/50.01	52.1/49.4	46.6^**/44.6^**	34.2^**/32.7^**
Sex ratio of live newborns % M/F	51/49	49/51	55/45	53/47
Selected as parents for the next generation M/F	25/25	25/25	25/25	25/25

^**P≤0.01

Reproduction and litter data for F1 parents /F2 pups

	0 ppm	500 ppm	2500 ppm	5000 ppm
Parents
Females mated	25	25	25	25
Females pregnant	23	23	23	24
Females with delivery	23	23	23	24
Mean duration of gestation (d)	22.0	21.9	21.9	21.9
Litter means
Live births/litter	12.5	11.6	12.0	13.8
Survivors day 4 preculling	11.8	10.6	10.8	12.5
Survivors day 4 postculling	7.7	7.0	7.5	7.8
Survivors day 21	7.7	6.9	7.5	7.4
Weight at day 1 (g) M/F	6.3/5.9	6.5/6.2	6.1/5.8	6.4/6.1
Weight at weaning (g) M/F	50.4/48.4	52.1/49.4	44.6^**/42.4^**	34.5^**/33.2^**
Sex ratio of live newborns % M/F	47/53	55/45	53/47	49/51

^**P≤0.01

Development landmarks in the F2 (mean % of pups reaching criteria/litter)

Parameter	0 ppm	500 ppm	2500 ppm	5000 ppm	Historical control range
Pinna unfolding	93.6 (16.65)	93.5 (20.87)	80.7 (33.37)	86.9 (26.87)	74-100
Auditory canal opening	98.8 (3.87)	95.1 (20.90)	91.4^*(18.09)	94.2 (12.26)	81-100
Eye opening	93.2 (18.08)	92.4 (21.89)	92.4 (21.89)	86.5^*(21.15)	85-100

^*P≤0.05

Figures in parentheses indicate standard deviations.

Reference 4

Endpoint:

two-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, published in peer-reviewed literature, minor restrictions in design and reporting, but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

other: NTP Reproductive Assessment by Continuous Breeding (RACB)

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY.
- Age at study initiation: 6 weeks
- Housing: group housed by sex in solid-bottom polypropylene or polycarbonate cages with stainless-steel wire lids, during the quarantine and the 1-week premating periods. subsequently, the animals were housed as breeding pairs or individually
- Diet (e.g. ad libitum): ground rodent chow, ad libitum
- Water (e.g. ad libitum): deionized/filtered water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Photoperiod (hrs dark / hrs light): 14/10

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each concentration was mixed separately at periodic intervals.

VEHICLE
Water
- Concentration in vehicle: 1.00, 2.50 and 5.00%

Details on mating procedure:

In the continuous breeding study mice were exposed to the test substance for 7-day premating period, and were then randomly grouped as mating pairs and cohabited and treated continuously for 98 days. Data were collected on all newborns during this period within 12 hours of birth, after which each litter was discarded. After the 98-day cohabitation, the pairs were separated but continued on treatments. During the next 21 days, any final litters were delivered and kept for at least 21 days (weaning). The mother was dosed through weaning and F1 mice were dosed until mated at 74 ± 10 days of age. For this, male offspring were mated to female off-spring from the same treatment group (n = 20/group/sex) and the F2 litters were examined for litter size, sex and pup weight.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days in the dose range-finding study; 7 days pre-mating period, 98 days (14 weeks) cohabitation, 21 days post-cohabitation. Any litters delivered during these 21 days (second generation animals) were kept for at least 21 days (weaning) and continued receiving the chemical treatment initially through lactation (mother was dosed throughout) and then through drinking water. Second generation animals were mated at 74 ± 10 days of age.

Frequency of treatment:

Daily

Details on study schedule:

Other: A 14-day dose-setting study utilized one control group and 5 groups of dosed animals (8/sex/dose). The endpoints for this study were clinical signs, mortality, body weight gain and consumption of food and water.
In the main study, the litters were removed and examined as soon as possible after delivery was completed. The offsprings were sexed, weighed and killed so that the female may be impregnated immediately. This approach maximized the number of litters which could be produced in the 14-week breeding phase.

Remarks:

Doses / Concentrations:
0, 1.82, 4.80 and 10.10 g/kg bw/day (main study)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0, 0.5, 1.0, 2.5, 5.0 and 10% in water (dose-range finding study)
Basis:
nominal in water

Remarks:

Doses / Concentrations:
5% in water (second generation animals)
Basis:
nominal in water

No. of animals per sex per dose:

Dose range-finding study: 8/sex/dose
Main study: 20/sex/dose in each treatment group; 40/sex/dose in the control group
Second generation animals: 20/sex/dose (only the highest dose (5% in water) was tested).

Control animals:

yes

Details on study design:

- Dose selection rationale: based on a 14-day dose-selecting study
- Rationale for animal assignment: stratified randomization procedure based on body weights.

Other: when significant adverse effects on fertility were observed in the continuous breeding phase a crossover mating trial was usually performed to determine whether F0 males or females were more sensitive to the effects. High-dose animals of each sex were mated to control mice of the opposite sex to determine the affected sex. The high dose animals were selected to increase the possibility of detecting effects in the crossover mating. There were three conbinations of control and treated mice: control males with control females, high-dose males with control females, and control males with high-dose females. The offspring of the crossover matings were analyzed as in Task 4, and the parents were necropsied. Results of mating high-dose mice with control group partners were compared to matings within the control group to determine which sex was adversely affected. The crossover mating was not done if significant reproductive effects were not observed in the continuous breeding phase.
Necropsies were performed in this series of studies, usually on only F0 mice involved in th crossover mating trial, when there was evidence of an effect on reproduction or, at the least, in the second generation even if there was no effects on the F0 mice. Endpoints examined for the females included selected organ weights and histology. At necropsy, the endpoints of male reproductive function included selected organ weights and histology, percentage motile sperm, epididymal sperm concentration, and percentage abnormal sperm. These multiple measured of fertility (whole animal, organ) were designed to increase the sensitivity of the RACB protocol.

Positive control:

Diethylstilbestrol and ethylene glycol monoethyl ether

Parental animals: Observations and examinations:

Clinical signs, mortality, body weight gain and consumption of food and water were assessed during the 14-day dose-setting study.

Oestrous cyclicity (parental animals):

Not performed.

Sperm parameters (parental animals):

As no adverse effects on fertility were observed in the continuous breeding study, the subsequent substudy (crossover mating with subsequent examination of male reproductive function in F0 animals) was not performed.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
mean No. litters per pair, mean No. live pups per pair, mean No. live male pups per litter, mean No. live female pups per litter, proportion of pups born alive, sex of pups born alive, mean live pup weight per litter, mean live male pup weight per litter, mean live female pup weight per litter; adjusted mean live pup weight per litter; adjusted mean live male pup weight per litter; adjusted mean live female pup weight per litter, body weight.

Postmortem examinations (parental animals):

As no adverse effects on fertility were observed during the continious breeding study, subsequent crossover mating with subsequent necropsies of F0 animals was not performed.

Postmortem examinations (offspring):

Not performed.

Statistics:

Dose-related trend in fertility: the Cochran-Armitage test (Armitage, 1971)
Pairwise comparisons involving mating and fertility indices: Fisher's exact test
Overall differences in number of litters, number of live pups, proportion of live pups and the sex ratio for dose group means: Kruskal-Wallis test (Conover 1980)
Ordered differences in number of litters, number of live pups, proportion of live pups and the sex ratio for dose group means: Jonckheere's test (Jonckheere, 1954)
Pairwise comparisons of treatment group means: Wilcoxon-Mann-Whitney U test
Average pup weight: Kruskal-Wallis test
Treatment differences in average pup weight: analysis of covariance (Neter and Wasserman, 1974)
Pairwise comparisons: two-sided t test
Organ weights: analysis of covariance F test (overall equality) and t test (pairwise equality)

Reproductive indices:

Fertility index was determined as (No. fertile/No.cohabited) x 100
Mating index was determined as No. females with plugs/No. cohabited

Offspring viability indices:

No data.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No chemical-related deaths were observed in either a dose-range finding study or during the 14 weeks cohabitation period in the main study. No significant chemical related clinical signs of toxicity were noted during the main study cohabitation phase.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the dose-range finding study, the untreated males, on the average, gained 5% of their original body weight. The correspoding value for animals in the 10.0% propylene glycol was 7%. The female CD-1 mice in the control and 10% dose groups gained 8 and 15% of their initial body weight, respectively. The comparison of average daily water consumption values revealed that animals in the 10.0% propylene glycol group consumed nearly 60% more than the control group.
In the main study, male mice in the control, 1.0, 2.5 and 5.0% dose groups gained an average of 8, 7, 10 and 7% of their original body weight after 14 weeks of treatment. Female mice responded similarly; group mean body weights for the females varied with the gestation phase. Dam weights at delivery were essentially the same for animals in the control and 3 treatment groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
In the dose-range finding study water consumption was greatly increased in the 10.0% group. It was suspected that treatment at 10.0% may result in significantly altered fluid balances and increased body weight realtive to the control group.
In the main study, the average daily consumption of dosed water by animals in the 3 treatment groups was consistently higher than the control values, especially in the highest dose group.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
For the second generation animals, estrual cyclicity was not sensitive to propylene glycol under the present experimental condition except a small decrease in the percent estrus phase in treated animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
For the second generation animals, sperm studies illustrated that propylene glycol treatment at the 5.0% dose level does not significantly affect (p > 0.05) sperm motility, sperm density (sperm count per g caudal tissue), or the incidence of abnormal sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All breeding pairs in the main study delivered at least 1 litter after 14 weeks of cohabitation. Propylene glycol administered in drinking water at up to the 5.0% level had no significant effect (p > 0.05) on any of the reproductive parameters. These included the number of litters per pair, the number of live pups per litter, the proportion of pups born alive, the sex ratio and both the absolute and adjusted live pup weights.
The cumulative number of days to litter were also uneffected by propylene glycol treatment.
For the second generation animals, the mating index (percent of plug positive/no. cohabited) for the control and treated animals was 85%. The fertility index (percent of no. fertile/no. cohabited) values for the control and treated pairs was 75 and 80%, respectively. No significant differences (p > 0.05) were noted with respect to litter size, proportion of pups born alive, sex ratio, as well as absolute and adjusted pup weights.

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

10 100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No effects reported at the highest dose tested.

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Propylene glycol had no apparent effect on pup survival.

BODY WEIGHT (OFFSPRING)
Propylene glycol had no apparent effect on pup body weight gain. For the second generation animals treated with 5% solution of propylene glycol, there was no significant difference (p > 0.05) in the body weight between the treated and control animals.

ORGAN WEIGHTS (OFFSPRING)
For the second generation animals, the absolute as well as adjusted organ weights for the liver, kidney, seminal vesicles, right cauda, prostate, right testis and right epididymis were not affected by propylene glycol treatment.

Dose descriptor:

NOAEL

Remarks:

fertility

Generation:

F1

Effect level:

10 100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No effects on fertility of F1 generation were observed at the highest dose.

Dose descriptor:

NOAEL

Remarks:

developmental effects

Generation:

F2

Effect level:

10 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No effects on litter size, sex and pup weight in F2 pups were observed at the highest dose.

Reproductive effects observed:

not specified

Reference 5

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Sep 2019 - July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PAU 0118813

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator (KS)

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 13-14 weeks (male) / about 13 weeks (female)
- Weight at study initiation: mean males= 374 g, mean females=212 g
- Fasting period before study: no
- Housing: Polysulfonate cages: - During pretreatment: up to 5 animals per sex and cage; - During premating: 2 animals per sex and cage; - During mating and postmating: 2 animals per cage (males only) Polycarbonate cages: 1 animal ;
Exceptions: - During overnight mating: 1 male/1 female per cage; - During rearing up to PND 13: 1 dam with her litter
- Diet: ad libitum (Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum (drinking water)
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water 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 ultrapure water and intensely mixed with a homogenizer.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: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Pregnant animals and their litters were housed together until PND 13. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All measured values for the test item were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. The stability of test substance in drinking water was demonstrated for a period of 7 days at room temperature.

Duration of treatment / exposure:

The duration of treatment covered a 5 weeks in-life period (males) including 14 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 14 days mating period, 9 days postmating period in one female (for no evidence of sperm), the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

Frequency of treatment:

once daily

Dose / conc.:

12 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

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

DETAILED CLINICAL OBSERVATIONS: Yes
- 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. inability 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.
Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and 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
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results.
During the mating period the parental 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 and 13.
Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly. 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: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals).
Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals)
Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

NEUROBEHAVIOURAL EXAMINATION: Yes
- A functional observational battery (FOB) was performed in the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence.
The already single-housed female animals were transferred to new cages before the test, nor was drinking water withdrawn. At least 30 minutes before the start of the FOB the male animals were transferred from group housing to single animal polycarbonate cages type III, for the duration of the test. Drinking water was provided ad libitum, but no food was offered during the measurements. Likewise, food was withdrawn from the female animals for the duration of the test.
The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined: Behavior on removal from cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Gait, Activity/arousal level, Feces (appearance/ consistency) within 2 minutes, Urine (amount/color) within 2 minutes, Rearing within 2 minutes, Other findings

Sensory motor tests/Reflexes
The animals were removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test

Motor activity measurement
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Oestrous cyclicity (parental animals):

For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization (the estrous cycle data of these individuals were not reported and can be found in the raw data). For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Sperm parameters (parental animals):

Parameters examined in F1male parental generations:
testis weight, epididymis weight, stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structures

Litter observations:

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

PARAMETERS EXAMINED
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. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. 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 (PND 0), and on lactation days 4, 7 and 13.
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 and 13. 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. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

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 male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.

Thyroid Hormones
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All 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.

Weight parameters: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight); Epididymides; Ovaries; Prostate (ventral and dorsolateral part together, fixed); Seminal vesicles with coagulating glands (fixed); Testes; Thyroid glands (with parathyroid glands) (fixed); Uterus with cervix
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands (fixed); Brain; Heart; Kidneys; Liver; Spleen; Thymus (fixed)
Organ / Tissue fixation:
The following organs or tissues of all parental animals are fixed in 4% neutral buffered formaldehyde solution or modified Davidson’s solution: All gross lesions; Adrenal glands; Aorta; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Esophagus; Extraorbital lacrimal glands; Epididymides (modified Davidson’s solution); Eyes with optic nerve (modified Davidson’s solution); Femur with knee joint; Heart; Ileum; Jejunum (with Peyer’s patches); Kidneys; Larynx; Liver; Lungs; Lymph nodes (axillary and mesenteric); Mammary gland (male and female); Nose (nasal cavity); Ovaries (modified Davidson’s solution); Oviducts; Pancreas; Parathyroid glands; Pharynx; Pituitary gland; Prostate; Rectum; Salivary glands (mandibular and sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Target organs; Testes (modified Davidson’s solution); Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina

HISTOPATHOLOGY: Yes
All gross lesions; Adrenal glands; Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Epididymides; Eyes with optic nerve; Heart; Ileum; Jejunum; Kidneys; Liver; Lungs; Lymph nodes (axillary and mesenteric); Ovaries; Oviducts; Prostate; Peyer’s patches; Rectum; Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic, lumbar); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Testes; Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina

CLINICAL PATHOLOGY:
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. 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 Inter¬national System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

Hematology
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), 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)
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument, Clotting tests were carried out using a ball coagulometer
Clinical chemistry: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.), gamma-Glutamyltransferase (GGT) (gamma -glutamyl) peptide: aminoacid--glutamyl-transferase; EC 2.3.2.2.), sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total proteine, Albumine, Globulins, Triglycerides, Cholesterol, Bile acids

Thyroid Hormones
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Postmortem examinations (offspring):

SACRIFICE
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations.

The remaining pups were sacrificed under isoflurane with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and processed

Statistics:

Weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS H and WILCOXON test
Clinical pathology parameters: KRUSKAL-WALLIS and WILCOXON-Test
Food consumption (parental animals), water consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index: DUNNETT test (two-sided)
Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (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+) with BONFERRONI-HOLM
Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index: WILCOXON test (one-sided-) with BONFERRONI-HOLM
% live male day x, %live female day x: WILCOXON test (two-sided)
Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS and WILCOXON test (two-sided)
Number of cycles and Cycle Length: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)

Reproductive indices:

see "Any other information"

Offspring viability indices:

see "Any other information"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All mid-and high-dose males showed salivation during the entire study. Salivation was also observed in most of the high-dose females during the premating, mating and gestation period, in all high-dose females during the lactation period, in one mid-dose female during the premating period and in some mid-dose females during the gestation and lactation period.
The temporary salivation was considered to be test substance-induced. Salivation occurred immediately after dosing (up to 2 hours post dosing) in the mentioned animals during the treatment period. It is likely, that this temporary finding was induced by a local affection of the upper digestive tract.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 - 3; 12, 40 and 120 mg/kg bw/d) during the study.
One mid-dose male (No. 30) showed piloerection on study day 21. One high-dose male (No. 32) had an injury of his hindlimbs (toe) during study days 30 - 33. One mid-dose female (No. 122) had an injury of the anogenital region during premating days 2 – 7.
Two high-dose females (Nos. 132 and 138) did not nurse their pups properly during PND 1- 3 and PND 1 - 2, respectively. As a consequence, several pups of high-dose female No. 132 and the only pup of high-dose female No. 138 died within 3 days after they were born.

Further details / tables see attachment

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of all male and female parental animals and body weight change of all female parental animals in all test substance-treated groups were comparable to the concurrent control during the entire study period. Body weight gain was lower in all treated groups at the beginning of exposure (though statistically significant only in the high-dose parental males during study days 0 – 7), suggesting that the animals needed a few days to get adapted to the bolus gavage of this irritating compound. After this adaptation the mean body weight change was generally comparable to the concurrent control values during the rest of the study. The statistically significantly higher mean body weight change in the mid-dose males during study days 13 - 21 was considered to be spontaneous in nature and not treatment related since it was not related to dose.

Further details / tables see attachment

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose male animals was statistically significantly above the concurrent control values during study days 7 - 13 and 0 - 13 (about 42% and 21%, respectively). The most likely explanation for this is that the animals spilled food.
Food consumption of the high-dose female animals was below control throughout lactation, the difference became statistically significant during PND 7 - 10 (about 23%). Overall the high-dose females consumed about 14% less food than the control females during lactation. Food consumption of the low- and mid-dose males and females during the entire study, and of the high-dose females during the premating and gestation period was comparable to the concurrent control values.
Further details / tables see attachment

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 animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. Water consumption of the mid-dose females during the entire study, and of the low- and high-dose females during the premating and gestation period was comparable to the concurrent control values. Water consumption of the high-dose and low-dose females was statistically significantly below the concurrent control values during PND 6 - 7 (about 17% and 20%, respectively). As there was no dose-response no association to the treatment was assumed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At the end of the administration period, in males of test groups 1, 2 and 3 (12, 40 and 120 mg/kg bw/d) relative eosinophil counts were decreased (in test group 2 not statistically significantly). The same was true for significantly decreased mean corpuscular hemoglobin concentration (MCHC) in females of test group 3 (120 mg/kg bw/d). However, the values were within historical control ranges (males, relative eosinophils 1.3-2.7 %; females, MCHC 20.84-22.23 mmol/L). In females of test group 2 (40 mg/kg bw/d) total white blood cell (WBC) counts and absolute neutrophil counts were significantly decreased. In females of test group 1 (12 mg/kg bw/d) absolute neutrophil counts were already significantly lower compared to controls. However, the alterations were not dose dependent. Therefore, all mentioned hematology changes were regarded as incidental and not treatment related.

Further details / tables see attachment

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 females of test group 3 (120 mg/kg bw/d) creatinine values were significantly decreased, but the values were within the historical control range (females, creatinine 26.9-35.4 µmol/L). In males of test group 2 (40 mg/kg bw/d) inorganic phosphate levels were significantly increased, but this alteration was not dose dependent. Therefore, both mentioned alterations were regarded as incidental and not treatment related.
In parental males of test groups 2 and 3 (40 and 120 mg/kg bw/d) T4 values were significantly increased. However, the means were within the historical control range (males, T4 44.65-73.22 mmol/L). Corresponding TSH values were not changed and within the historical control range (males, TSH 4.32-9.80 µg/L).

Further details / tables see attachment

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. Apart from transient salivation in three high-dose males and two mid-dose males, the open field observations did not reveal any test substance-related findings in male and female animals of all test groups. One high-dose male had an injury of hindlimbs during the open field observations, which was unrelated to treatment. There were no test substance-related findings in male and female animals of all test groups.
No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly higher values of landing foot-splay test in males of test group 1 and 2 were considered as spontaneous in nature and not treatment-related, as there was no dose-response. In addition, the measured values were within the historical control range (HCD: 9.3 - 13.8 cm). Because of the hindlimbs injury of high-dose male No. 32 no measurement of grip strength of hindlimb and landing foot-splay test was performed with this animal.
No treatment-related, adverse change of motor activity (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values.
The mean number of beam interrupts of the high-dose parental males was statistically significantly above the concurrent control values during interval 1. However, the animals of the high-dose group habituated normally to the test conditions afterwards and all other groups including control were rather at the lower end of the historical control range for this interval (HCD: 989.6 – 1349.4). The high-dose value is within the historical control range, thus, considering the regular habituation, this slightly higher activity level is considered to be in the normal range of rat behavior.

Further details / tables see attachment

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 stomach of male and females of test groups 2 and 3.
Forestomach:
Diffuse squamous hyperplasia characterized by a thickening of the epithelial layer including the Margo plicatus and diffuse hyperkeratosis was noted. The lesion was correlated with the macroscopical findings of a thickened Margo plicatus and thickened wall. Additionally, erosions and ulcerations were present in multiple males and females of the test group 3. The epithelium surrounding the ulcerations and erosions displayed a pronounced squamous hyperplasia and hyperkeratosis. An inflammatory infiltrate, consisting of mainly neutrophils, lymphocytes and plasma cells was seen in the underlying tissue, as well as a submucosal edema. The squamous hyperplasia seen in one control male and one male/female each in test group 1 is regarded as incidental and not-treatment-related. In addition, the erosion/ulceration in one control male is a spontaneous background lesion.
Glandular stomach:
Multifocal edema and hyperemia of the lamina propria, often accompanied by an attenuation of the overlying epithelial layer was noted in male and female animals of test groups 2 and 3 and is assumed as consequence of a local irritating effect of the test substance. Additionally, ulcerations and / or erosions without a dose-dependency were found in the glandular stomach of males and females correlating with macroscopic findings. Since no dose-dependency could be observed and animals affected in test group 1 and 2 did not display the described test substance related histological lesions in the forestomach (diffuse squamous hyperplasia, submucosal edema and erosion/ulceration) and glandular stomach (edema/hyperemia in the lamina propria), this finding is regarded as a not treatment-related spontaneous background lesion.
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.

Further details / tables see attachment

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was equal: 4.0 days in all test groups 0 - 3, respectively.

Further details / tables see attachment

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.

Further details / tables see attachment

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 (0 - 3). Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was 100% in all test groups. The female mating index calculated after the mating period for F1 litter was 100% in all test groups (0 - 3). All female rats delivered pups or had implants in utero: The fertility index was 100% in all test groups. The mean duration of gestation values varied between 22.0 / 22.0 / 22.0 and 22.4 in test groups 0 - 3, respectively. The gestation index was 100% in all test groups 0 - 3.
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 (11.4 / 11.2 / 12.8 and 12.4 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups (3.8 / 0.8 / 4.7 and 11.6 mean% in test groups 0 - 3, respectively), and all values were below or within the historical control range of the test facility (0.9 – 16.8 mean%) and the mean number of F1 pups delivered per dam remained unaffected (10.9 / 11.1 / 12.3 and 11.5 pups/dam in test groups 0 - 3, respectively).

Further details / tables see attachment

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects in highest dose observed

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced food consumption, increased liver weights

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: gastrointestinal pathology

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups

Further details / tables see attachment

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.1%/ 100% / 97.7% and 83.6% in test groups 0 - 3, respectively. The apparently lower pup survival rate in the high-dose group was caused by the two individual dams 132 and 138. Dam 132 had a large litter (14 pups) and was not able to nurse all her pups properly. Consequentially 5 pups died from undernutrition within 3 days after they were born. Dam 138 had only one pup, which also died from undernutrition 3 days after it was born. The pups surviving index indicating pup survival during lactation (PND 4 - 13) was 100% in all test groups.
Further details / tables see attachment

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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.

Further details / tables see attachment

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In male and female pups at PND13 (test groups 11, 12 and 13; 12, 40 and 120 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Further details / tables see attachment

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

In general, the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature. The statistically significantly shifted in sex ratio in the high-dose group on PND 0 (males 62.8*[*p<=0.05] versus [vs.] 43.8 in control and females 37.2* vs. 56.2 in control) was slightly outside the historical control range (HCD: 36.6% - 60.5% (males), 41.5% - 63.4% (females)). Nevertheless, it was assessed as not treatment-related, because pup necropsy at PND 13 revealed no morphological evidence for virilization and none of the other endocrine-sensitive parameters (e.g. pup weight, AGD, nipple retention) gave any indication of a test substance-related effect in any of the dose levels (see below). Thus, these values were most likely outliers and considered to be spontaneous in nature and not treatment related.

Further details / tables see attachment

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.

Further details / tables see attachment

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Further details / tables see attachment

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few individual pups showed spontaneous findings at gross necropsy, such as discolored testis, empty stomach, dilated renal pelvis, post mortem autolysis, partly cannibalized, absent mouth, absent jaw and dilated ureter. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Further details / tables see attachment

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at highest dose

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

The results in tabular form are provided in the attached background material as pdf. Results for all organ weights (absolute and relative) are provided as separate pdf.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD Guideline 433:

Hydroxypropyl acrylate was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 15, 50 and 150 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group.Pups of the F1 litter were selected (F1 rearing animals) andassigned to 3 different cohorts (1A, 1B and 3) which were subjected to specific postweaning examinations.The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle(drinking water). The parents' and the pups' state of health was checked each day and parental animals were examined for their mating and reproductive performances. Water consumption of the F0 parents and F1 rearing animals (except of the positive control animals, test group 14) was determined regularly once weekly over a period of 3 or 4 days (except water consumption of the F0 males wasdetermined after the 10^th premating week) and weekly during gestation days (GD) 0-1, 3-4, 7-8, 10-11, 14-15, 17-18, 19-20 and lactation days (PND) 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.Food consumption of the F0 parents and F1 rearing animals was determined regularly once weekly over a period of 7 days (except food consumption of the F0 males was determined after the 10^th premating week) and weekly during GD 0-7, 7-14, 14-20 and PND1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on GD 0, 7, 14 and 20 and on PND 0, 1, 4, 7, 10, 14, 18 and 21.A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A, 1B, 2A and 3 at weekly intervals. Estrous cycle data were evaluated for F0 females over a three weeks period prior to mating until evidence of mating occurred.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. Moreover, the estrous stage of each F0, 1A and 1B female was determined on the day of scheduled sacrifice. The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 rearing animals was recorded. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one PND 20. The number of nipple/areola anlagen were counted. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all liveF1 pupson PND 1. Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Blood samples for clinical pathological investigations were withdrawn from 10 F0 parental and cohort 1A animals per sex and group. Furthermore, blood samples were taken from all surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 and 1A males at scheduled sacrifice or after appropriate staining. All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females of cohort 1A.

 All F1 rearing animals were assessed by pathological examinations. 

The various analyses:

·        Demonstrated the stability of the test substance preparations over aperiod of 7 days at room temperature.

·        Confirmed the homogeneous distribution of the test substance indrinking water.

·        Verified correct concentrations of the test substance in drinking water  

The following test substance-related adverse effects/findings were noted:

150 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Salivation after gavage dosing in almost all male and female animals across all studyperiods

·       Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in females

·       Increased absolute reticulocyte counts in females

·       Increased absolute and relative neutrophil cell counts in males

·        Decreased relative lymphocyte counts in males

·       Gross finding “focus” in the forestomach of 18 male and 6 female animals

·       Gross finding “margo plicatus thickened” in the forestomach 17 males and 25 females

·       Gross finding “focus” in the glandular stomach of 4 male and 7 female animals

·       Erosion/ulcer in the forestomach in 19 males and 6 females, correlating to the gross finding “focus”

·       Hyperplasia of the squamous cell layer of the margo plicatus in the forestomach of 16 males (graded up to moderate) and 24 females (graded up to marked), correlating to the gross finding “margo plicatus thickened”

·       Hyperplasia squamous cell diffuse in the forestomach in 6 males and 11 females, mostly graded minimal

·       Multifocal degeneration/regeneration of the glandular stomach in 15 male and 12 female animals, mostly graded minimal to slight

·       Erosion/ulcer in the glandular stomach in 6 males and 12 females

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

·        No test substance-related adverse findings 

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Salivation after gavage dosing in all male and female animals during the entire study

·        Increased urea and inorganic phosphate levels in females

·        Gross finding “focus” in the forestomach of 6 male and 3 female animals

·        Gross finding “margo plicatus thickened” in the forestomach of 20 males and 19 females

·        Erosion/ulcer in the forestomach of 6 males and 4 females, correlating to the gross finding “focus”

·        Hyperplasia of the squamous cell layer of the margo plicatus in 20 males and 18 females graded up to moderate in males and slight in females, correlating to the gross finding “margo plicatus thickened”

·        Minimal diffuse hyperplasia squamous cell in the forestomach of 6 male animals

·        Mostly minimal multifocal degeneration/regeneration in the glandular stomach of 7 male and 3 female animals

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ PATHOLOGY

·        Salivation after gavage dosing in nearly all male and all female animals during the entire study

·        Gross finding “focus” in the forestomach of 4 male and 3 female animals

·        Gross finding “margo plicatus thickened” in the forestomach of 24 males and 22 females

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/CLINICAL PATHOLOGY/ PATHOLOGY

·        Salivation after gavage dosing in all male and female animals during the entire study

50 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Salivation after gavage dosing in about half of the animals across all study periods

·        Gross finding “focus” in the glandular stomach of 2 male and 12 female animals

·        Gross finding “margo plicatus thickened” in the forestomach of 24 males and 22 females

·        Erosion/ulcer in the glandular stomach of 6 males and 12 females, correlating to the gross finding “focus”

·        Minimal hyperplasia of the squamous cell layer of the margo plicatus in the forestomach of two males and three females, correlating to the gross finding “margo plicatus thickened”

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

·        No test substance-related adverse findings 

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Salivation after gavage dosing in many male and female animals during the entire study

·        Gross finding “margo plicatus thickened” in the forestomach of 6 males and 2 females

·        Hyperplasia of the squamous cell layer of the margo plicatus in the forestomach of 6 males and 3 females graded minimal, correlating to the gross finding “margo plicatus thickened”

·        Hyperplasia squamous cell diffuse in the forestomach of one male animal

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ PATHOLOGY

·        Salivation after gavage dosing in many male and female animals during the entire study

·        Gross finding “focus” in the forestomach of 1 female animal

·        Gross finding “margo plicatus thickened” in the forestomach of 2 females

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Salivation after gavage dosing in many male and female animals during the entire study

15 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Gross finding “focus” in the glandular stomach of 3 male and 14 female animals

·        Erosion/ulcer in the glandular stomach of two males and 13 females

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

·        No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related adverse findings 

Under the conditions of the present extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 50mg/kg bw/d for the F0 parental as well as F1 adolescent animals, based on changed clinical-pathological parameters, which wereo bserved 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 is150 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 is150 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.

 

OECD Guideline 422:

Hydroxypropyl acrylate was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 15, 50 and 150 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (drinking water). The duration of treatment covered a 2-week premating and a mating period for both sexes, approximately 2 days post-mating in males, as well as gestation and lactation in females.

The following adverse treatment-related findings were noted at 150 mg/kg bw/d. Decreased food consumption in the females during the entire premating period (up to 7% below control). Minimal to slight thickening of the mucosa of the duodenum correlating to the macroscopically observed dilation in all male and 9/10 females. Focal hyperplasia of the duodenal mucosa in 1/10 female animals. Diffuse squamous hyperplasia of the forestomach in all male animals (graded slight or moderate) and in 7/10 female animals (graded minimal or moderate). Erosion/ulcer in the cranial part of the forestomach in 6/10 male and 2/10 female animals. At 50 mg/kg bw/d minimal thickening of the wall of the duodenum correlating to the macroscopically observed dilation in 2/10 male animals. Minimal diffuse squamous hyperplasia of the forestomach in 6/10 male and female animals. At 15 mg/kg bw/d no test substance-related adverse findings were observed. No test substance-related adverse findings were observed for the F1 generation.

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Hydroxypropylacrylate was 150 mg/kg bw/d for male and female rats. Based on pathological findings characteristic of irritation in forestomach and duodenum in F0 parental rats of both sexes at 150 and 50 mg/kg as well as corresponding temporary reductions of food consumption in F0 females at 150 mg/kg bw/d a NOAEL of 15 mg/kg bw/d was determined for local effects in the gastrointestinal tract.

Data from the structurally analogous substances:

2 -Hydroxyethyl acrylate (CAS No. 818 -61 -1)

OECD TG 422:

In a study according OECD 422, HEA was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 12, 40 and 120 mg/kg body weight/day (mg/kg bw/day, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity (HARTF, 2020). The oral administration of HEA by gavage to male and female Wistar rats resulted in signs of parental toxicity at the mid- and high-dose of 40 and 120 mg/kg bw/day, such as a combination of clinical signs and gastrointestinal pathology, being the consequence of the irritating properties of the test item. Thus, the NOAEL for general systemic toxicity was 40 mg/kg bw/day for male and female Wistar rats based on the reduced food consuption (lactation phase) and the significant increased liver weights and the NOAEL for local effects (gastrointestinal pathology) was 12 mg/kg bw/day. The NOAEL for reproductive performance and fertility was set to 120 mg/kg bw/day for male and female Wistar rats. The NOAEL for developmental toxicity was 120 mg/kg bw/day.

Acrylic acid (CAS No. 79-10-7)

OECD Guideline 416 (2 -generation-studies):

Acrylic acid (CAS No. 79-10-7):

In a two-generation study according to OECD TG 416 acrylic acid was administered orally in the drinking water to male and female Wistar rats at doses of 0, 500, 2500, 5000 ppm (corresponding to approx. 53, 240, 460 mg/kg bw/day).At least 70 days after the beginning of treatment, F0 animals were mated to produce one litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dosing group as their parents. Groups of 25 males and 25 females selected from F1 pups as F1 parental generation were offered drinking water containing 0, 500, 2500 and 5000 ppm of the test substance post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals(BAMM, 1994; Hellwig et al., 1997).

The continuous administration of aqueous acrylic acid solutions to rats over two generations caused clear signs of toxicity in the highest dose group (5000 ppm = approx. 460 mg/kg body weight/day) in F0 and F1 parents. General toxicity was substantiated by e.g. reduced food and/or water consumption, impairment of body weights/body weight gains and gross and histopathological findings in the fore- and the glandular stomach (i.e. thickening of and minimal hyperkeratosis at the limiting ridge (margo plicatus), edema in the submucosa of the glandular stomach), which are a consequence of the administration of the acid solutions (indicative of the irritating properties of the test substance).

At 2500 ppm (= approx. 240 mg/kg body weight/day) the water consumption of the F1 parental animals was still clearly reduced, but no further substance-related adverse effects on the parental rats were seen.

Clear adverse substance-induced effects were also noted for the progeny of the high dose of the F0 and F1. Impaired body weight/body weight gain in the F1 and F2 pups and some indications for delays in the morphological development of the F2 pups were seen. The latter finding was likely associated with the decreased body weight/body weight gain. Similar, but much less pronounced effects were also observed for the F1 and/or F2 pups at 2500 ppm.

500 ppm (= approx. 53 mg/kg body weight/day) were tolerated by both parental generations and their offspring without any changes which could be causally related to test substance administration.

Acrylic acid had no adverse effects on reproductive parameters of the parental animals of either generation (F0 and F1) of all groups (500, 2500 and 5000 ppm). No adverse effects on fertility and pre-implantation development could be detected; no effects on reproductive organs have been observed. The mating index of males in both generations and in all dose groups was 100 %. The fertility rate in the F0 generation was between 92-96 %; in the F1 generation in all dose groups the fertility rate was 100 %. The rate of pregnancy in both generations was not reduced. In both generations there were no differences in numbers of pups born alive.

Therefore, the NOAEL (no observed adverse effect level) with respect to reproductive function was 5000 ppm (= approx. 460 mg/kg body weight/day). The NOAEL with respect to general toxicity of the test substance was 2500 ppm (= approx. 240 mg/kg body weight/day) for the F0 generation parental animals and 500 ppm (= approx. 53 mg/kg body weight/day) for the F1 males and females and the offspring (F1 and F2 pups).

Propylene glycol (CAS No. 57-55-6)

Continuous breeding study:

In addition, the possible metabolic cleavage product propylene glycol was tested in a continuous breeding study with mice, performed within the frame of the National Toxicology Program, was available for assessment (Morrissey, 1989; National Toxicology Program, 1985), comparable to OECD guidelines for multi-generation studies (i.e. OECD 416) with respect to the assessment of fertility parameters. Mice were exposed to the test substance for 7-day premating period and were then randomly grouped as mating pairs and cohabited and treated continuously for 98 days. Data were collected on all newborns during this period within 12 hours of birth, after which each litter was discarded. After the 98- day cohabitation, the pairs were separated but continued on treatments. During the next 21 days, any final litters were delivered and kept for at least 21 days (weaning). The mother was dosed through weaning and F1 mice were dosed until mated at 74 ± 10 days of age. For this, male offspring were mated to female off-spring from the same treatment group (n = 20/group/sex) and the F2 litters were examined for litter size, sex and pup weight. No differences were found between control and test P animals in the mean No. litters per pair, mean No. live pups per pair, mean No. live male pups per litter, mean No. live female pups per litter; proportion of pups born alive; sex of pups born alive; mean live pup weight per litter; mean live male pup weight per litter; mean live female pup weight per litter; adjusted mean live pup weight per litter; adjusted mean live male pup weight per litter; adjusted mean live female pup weight per litter. No differences were found between control and F1 animals in mean No. live pups per litter; mean No. liver male pups per litter; mean No. live female pups per litter; proportion of pups born alive and sex of pups born alive. The NOAEL for effects on fertility was established to be 10100 mg/kg bw/day (the highest dosetested).

Available data on reproductive toxicity of propylene glycol have been assessed and evaluated by the expert panel of the NTP CERHR (National Toxicology Program, 2004a). No human data were identified. Based on the data reported in the continuous breeding study by Morrissey (Morrissey, 1989; NTP, 1985) the Panel concluded that propylene glycol is not a reproductive toxicant in males or females or in their progeny under the conditions of this study. These data were assumed by the Panel to be relevant for assessing human hazard. Based on these findings, the Panel concluded that current estimated exposures topropylene glycol are of negligible concern for reproductive toxicity in humans.

Repeated dose toxicity: Oral route

In a GLP-compliant oral 90-day repeated dose toxicity study according to OECD Guideline 408 hydroxypropyl acrylate was administered daily by oral gavage to 10 male and 10 female Wistar Han rats at doses of 0 mg/kg bw/day (test group 1), 10 mg/kg bw/day (test group 2), 30 mg/kg bw/day (test group 3) and 100 mg/kg bw/day (test group 4) for at least 90 consecutive days. The dose selection was based on the results of an OECD TG 422 in Wistar rats, in which strong irritation including erosion and ulcer in the forestomach, as well as inflammatory changes in the duodenum were detected in the parental animals of the high dose (150 mg/kg). The effects in the forestomach and duodenum were still observed in the mid dose (50 mg/kg), but less pronounced.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, functional observational battery, motor activity, ophthalmology, thyroid hormone assessment, clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis), spermatogenesis evaluation, gross necropsy findings, organ weights, and histopathologic examinations.

All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on body weight, hematology, coagulation, or urinalysis. There were no test substance-related ophthalmic, macroscopic, microscopic findings, or effects on ovarian follicle counts, thyriod hormones (T3, T4 or TSH levels), sperm morphology or differential counts.

Test substance-related higher food consumption was noted in the 100 mg/kg/day group males and females (Days 1–90), and in the 30 mg/kg/day group males (Days 29–90), compared to the control group.

Test substance-related higher cholesterol and potassium, and lower mean chloride levels were noted in the 100 mg/kg/day group males on Week 13, compared to the control group.

Test substance-related higher mean organ weights were noted in the liver and thyroid/parathyroid gland of the100 mg/kg/day group males. Higher mean kidney weights were present in the 100 mg/kg/day group males, and lower mean thymus weights were seen in the >10 mg/kg/day group females, but relationship of these to the test substance was considered uncertain.

Based on the results of this study, oral administration of the test substance to Wistar Han rats at dosage levels of 10, 30, and 100 mg/kg/day for a minimum of 90 days was well tolerated at all dosages. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.

Conclusion:

The results of the OECD 443 and OECD 422 study with hydroxypropyl acrylate and a 2-generation-study (OECD Guideline 416) with the structural analogues acrylic acid (CAS No. 79-10-7) in rats by the oral route gave no indication of a fertility impairing effect caused by hydroxypropyl acrylate. In addition, no indications of a possible reproductive toxicity were determined in a continuous breeding study with the metabolic cleavage product propylene glycol.

Effects on developmental toxicity

Description of key information

There is sufficient data available to assess the endpoint developmental toxicity.

No indications of a developmental toxic or teratogenic effect were seen in a rat study with hydroxypropyl acrylate, which is supported by developmental toxicity studies in rabbits as a second species, conducted with the structural analogues 2 -hydroxyethyl acrylate (CAS No. 818 -61 -1) and acrylic acid (CAS No. 79-10-7) and the data in rats, mice, rabbits and hamsters with propylene glycol (CAS No. 57-55-6).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication, comparable to current guidelines

Principles of method if other than guideline:

Groups of 20-29 bred female rats (17-25 pregnant) were exposed to the compound 6h/day on days 6 through 20 of gestation by inhalation. Control animals were exposed concurrently to filtered room air. The test concentrations were 1, 5, and 10 ppm (corresponding to approx. 5.4, 27.0, and 54.0 µg/L)*.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l' Arbresle, France)
- Age at study initiation: Young, nulliparous females
- Weight at study initiation: 200-220 g
- Housing: Single in clear polycarbonate cages with stainless-steel wire lids and hardwood shaving as bedding.
- Diet: Food pellets (UAR Alimentation Villemoisson, France), ad libitum
- Water: Filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

EXPOSURE
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6-20 m3/h). The chamber temperature was set at 23 ± 2°C, and the relative humidity at 50 ± 5 %. The exposure system delivered, with an infusion pump, a constant rate of liquid chemical from the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a countercurrent fashion to the liquid flow. The vapourized compound was introduced into the main air inlet pipe of the exposure chambers.
Concentrations of acrylate ester were monitored continuously with a gas-chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with dichloromethane. The resulting samples were analyzed by gas chromatography using appropriate internal standards. Since 2-HPA has a rather low vapour pressure (0.07 mm Hg at 20 °C), the presence of liquid particles was evaluated at the highest concentration generated (i.e. 25 ppm). Airborn particles were measured with an Aerodynamic Particle Sizer.
No differences in particle counts were observed between the clean filtered air (control) and the vapour-laden air in the exposure chambers.


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass/stainless-steel inhalation chambers
- Source and rate of air: Test atmospheres were generated through an additional air-flow  rate passed through the fritted disk of a heated bubbler containing 2-hydroxypropyl acrylate.  The vapourized compound was introduced into the main air inlet pipe of the exposure chamber.
- Air flow rate: 6-20 m3/h


TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical concentrations (mean ± SD):
1.0 ± 0.1 ppm (nominal: 1 ppm)
5.1 ± 0.3 ppm (nominal: 5 ppm)
10.3 ± 0.8 ppm (nominal: 10 ppm)

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2-3
- Length of cohabitation: Overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 to 20 of gestation

Frequency of treatment:

daily, 6h/d

Duration of test:

until gestation day 21

Remarks:

Doses / Concentrations:
1, 5, and 10 ppm (corresponding to approx. 5.4, 27.0, and 54.0 µg/L)
Basis:
nominal conc.

No. of animals per sex per dose:

20

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
Exposure concentrations were based on preliminary studies in which severe maternal toxicity (i.e. weight loss during GD 6-13 and pronounced reduction in weight gain during GD 13-21) was observed at 25 ppm hydroxypropyl acrylate.

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day (GD) 0, 6, 13 and 21.


FOOD CONSUMPTION: YES
Food consumption was measured for the intervals GD 6-13 and 13-21.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10 %) to detect very early resorptions.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin's solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70 %), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

Data were presented as mean ± SD. The number of  implantation sites and live fetuses and the various body weights were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's test if differences were found. The percentages of non-live  implants and  resorptions and the proportions of fetuses with alterations in each litter were evaluated by using the Kruskal-Wallis test, followed by the Dixon-Massey test where appropriate. Rates of pregnancy, fetal sex ratio, and percentage of litters with malformations or external, visceral, or skeletal variations were analyzed by using Fisher's test. Where applicable, least-squares analysis was carried out. For all statistical tests, the level of significance was set a priori at alpha = 0.05.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal deaths were observed. Maternal weight gain was significantly less than control during the first half of exposure at 10 ppm and absolute weight gain at 5 and 10 ppm. Food consumption was slightly reduced during treatment at 10 ppm.

Dose descriptor:

NOAEC

Effect level:

ca. 0.005 mg/L air (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of implantation sites and live fetuses, and the incidence of non-live implants and resorptions were comparable among groups. There was no effect on fetal weights at any exposure level. Visceral malformations were observed in single fetuses in the control and the 5 ppm groups. Several visceral and skeletal variations were observed, with no significant differences between treated and control groups.

Dose descriptor:

NOAEC

Effect level:

ca. 0.054 mg/L air (nominal)

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Abnormalities:

no effects observed

Developmental effects observed:

no

Maternal body weights:

 

Concentration [ppm/6h/d]	Maternal body weight GD 6 [g]	Absolute weight gain [g]
0	266 ± 16	27 ± 13
1	272 ± 15	27 ± 9
5	271 ± 13	19 ± 11*
10	272 ± 15	16 ± 12**

 

 

Reproductive parameters:

 

Conc. [ppm/6h/d]	No. of litters	No. of implantation sites/litter	% of non-live implants/litter	% of resorption sites/litter	No. of live fetuses/litter	Average fetal body weight/litter [g]
0	21	14.48 ± 3.06	6.10 ± 8.37	6.10 ± 8.37	13.62 ± 3.29	5.48 ± 0.21
1	20	14.40 ± 2.23	9.76 ± 15.58	9.76 ± 15.58	13.00 ± 2.90	5.61 ± 0.29
5	22	14.27 ± 4.09	10.83 ± 21.79	10.83 ± 21.79	14.00 ± 3.22	5.58 ± 0.33
10	21	14.52 ± 3.74	6.70 ± 5.90	6.70 ± 5.90	13.57 ± 3.70	5.48 ± 0.27

 

 

Concentration [ppm/6h/d]	0	1	5	10
Mean % of fetuses with:
- any malformations/litter	0.43 ± 1.98	0	0.26 ± 1.21	0
- external variations/litter	0	0	0	0
- visceral variations/litter	5.05 ± 12.39	7.45 ± 13.11	7.66 ± 13.08	4.65 ± 6.82
- skeletal variations/litter	10.40 ± 11.37	11.70 ± 19.45	23.76 ± 28.69	7.37 ± 9.35
- any variations/litter	7.68 ± 8.51	9.56 ± 10.87	15.67 ± 18.03	5.92 ± 5.70

 

* ,** Significant differences from the control (0 ppm) value, p< 0.05, and p< 0.01, respectively.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-05-2020 - 03-02-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species and strain
Sexually mature, virgin New Zealand White rabbits (Crl:KBL(NZW)) were supplied at 15-18 weeks of age by Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France). Only animals free from clinical signs of disease were used for the investigations.

Animal identification
The breeder produced unique identification of the rabbits by ear tattoo.

Reason for species selection
The strain was selected since historical control data is available from the test facility for New Zealand White rabbits. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

HOUSING AND DIET
During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm).

For enrichment, wooden gnawing blocks were added (Typ SAFE® gnawing block), supplied by
J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany).

The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a range of temperature of 17-21°C and a range of relative humidity of 45-65%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.

The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).

Before the study started, the animal room was completely disinfected using a disinfector (Geschko MLT 17 hydrogen peroxide gas generator (PEA; Germany)). The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.

The food used was pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles) were available ad libitum throughout the study (from the day of arrival to the day of necropsy).

Based on the pregnant animals the body weight on GD 0 varied between 3251 – 4432 g.

IN-LIFE DATES: From: 29 May 2020 To: 14 Jul 2020

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water over a period of a maximum of 7 days at room temperature had been verified prior to the start of the study with the same batch.

Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations.

All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director.

The results of the analyses of the test substance preparations in drinking water confirmed the correctness of the prepared concentrations. The measured concentrations of the samples of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.

A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.

Duration of treatment / exposure:

The test substance was administered to the animal orally by gavage from implantation to one day prior to the expected day of parturition (GD 6-28) always at approximately the same time of day. The animals of the control group were treated in the same way with the vehicle (drinking water). 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.

Frequency of treatment:

once daily

Dose / conc.:

6 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

On GD 29 blood samples were obtained in randomized order from all surviving animals from the ear veins. After blood sampling, the females were euthanized and examined. The fetuses were removed from the uterus and investigated.

Dose selection rationale
The high dose was selected based on signs of toxicity noted at dose levels of 90 and 180 mg/kg bw/d in a previously conducted maternal toxicity range-finding study (BASF project 20R0173/16R191, non-GLP) which preceded this definitive prenatal developmental toxicity study.
In this maternal toxicity range‐finding study, 5 presumed pregnant NZW rabbits were adminis- tered the test substance via oral gavage from gestational day (GD) 6 through GD 28, at doses of 90 and 180 mg/kg bw/d.
The 180 mg/kg bw/d dose caused in the pregnant rabbits an immediate cessation of food consumption after the first dosing. One animal was found dead and all remaining animals were killed for humane reasons after the second dosing.
The 90 mg/kg bw/d dose caused a significant reduction of food consumption (up to 61% below control) during the first two treatment weeks (GD 6-19). During the last treatment week recovery was noted, however, mean food consumption stayed about 14 % below control during the treatment period. In addition, clinical signs like poor general condition, high-stepping gait, blood in bedding and no or reduced defecation were observed in individual rabbits. One animal aborted.
Clinical pathology revealed no relevant findings.
At macroscopic pathology discolorations and foci were detected in the stomach of 3 rabbits at 180 mg/kg bw/d and one rabbit at 90 mg/kg bw/d. Histopathologically erosions/ulcers were detected in all 180 mg/kg bw/d rabbits and one 90 mg/kg bw/d rabbit. At 180 mg/kg bw/d these were accompanied by signs of minimal or mild to moderate hemorrhages and inflammation.
Thus, the selected high dose for the present definitive study (60 mg/kg bw/d) represented a dose which was supposed to cause a moderate reduction of food consumption and/or body weight/body weight gain in pregnant female rabbits. Considering the specific characteristic of rabbit gastrointestinal physiology and the consequences of their disruption for the maintenance of pregnancy in rabbits this procedure meets the principles of guidelines OECD 414 (adopted 2018) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in compliance with EU Directive 2010/63/EU on animal protection.

Maternal examinations:

Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).


Clinical symptoms
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.


Food consumption
The consumption of food was recorded daily during GD 0-29.


Body weight data
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.


Corrected (net) body weight gain
The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).


Clinical Pathology
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.


The following examinations were carried out in all animals per test group:

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):

Parameters and methods:

Parameter Unit Method
Leukocyte count (WBC) giga/L cytochemistry coupled with flow cytometry
Erythrocyte count (RBC) tera/L flow cytometric laserlight scattering
Hemoglobin (HGB) mmol/L cyanmethemoglobin method; according to ICSH
Hematocrit (HCT) L/L calculation: MCV x erythrocytes
Mean corpuscular volume
(MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Mean corpuscular
hemoglobin (MCH) fmol calculation: hemoglobin/erythrocytes
Mean corpuscular
hemoglobin concentration
(MCHC) mmol/L calculation: hemoglobin/hematocrit
Platelet count (PLT) giga/L flow cytometric laserlight scattering
Differential blood count % & giga/L cytochemistry coupled with flow cytometry
Reticulocytes (RETA) giga/L cytochemistry coupled with flow cytometry


Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters
Parameters and methods:
See table below in any other information


Necropsy
On GD29 all surviving animals were sacrificed by an intravenous injection of pentobarbital (dose: 2ml/animal) in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
All animals which died intercurrently (animal No. 31) or were sacrificed in a moribund state (including animals with abortion; animal No. 91) were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights were determined in all does sacrificed on schedule:
1. Adrenal glands
2. Kidneys
3. Liver
4. Spleen
All paired organs were weighed together (left and right).

The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights. Organ weights of animals, that were not pregnant, were excluded from the calculation of mean values.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Duodenum
4. Kidneys
5. Liver
6. Spleen
7. Stomach (forestomach and glandular stomach)

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the following table:

Organs Test groups
0 1 2 3
1. Stomach (forestomach and glandular stomach) A1 A1 A1 A1
2. Duodenum A1 A1 A1 A1
A = hematoxylin and eosin (H&E) stain 1 = all animals/test group

A correlation between gross lesions and histopathological findings was attempted.

Ovaries and uterine content:

Cesarean section

On GD 29, the surviving does were euthanized in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal). After exsanguination, the animals were necropsied and assessed by gross pathology.

The uteri and the ovaries were removed and the following data were recorded:

- Weight of the unopened uterus

- Number of corpora lutea

- Number and distribution of implantation sites classified as:

• Live fetuses

• Dead implantations:

a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)

b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)

c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the does (except of gross pathology including organ sampling and weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.

Blood sampling:

In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.


Additional blood samples for non-GLP research purposes

Additional samples during venipuncture were collected for external research projects beyond the scope of this study and without GLP status. The results of the study were not influenced by this procedure because blood sampling occurred just prior to sacrifice/at necropsy. The last aliquot was taken for these projects, thus blood sampling for the regulatory investigations was not affected. Thus, the sampling procedures did not affect the outcome and compliance of the GLP study. The data from these research projects do not affect the outcome, assessment and compliance of this GLP study.
From each animal 1 mL of blood was collected with EDTA as anticoagulant (10 µL of a 10% solution). The samples were centrifuged. The plasma was separated. The blood was sampled and prepared in original Eppendorf tubes. The preparation of the samples was done under cooling. All samples were stored covered with a N2 atmosphere and then stored at –80°C for research.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.
Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital (Narcoren®; dose: 0.2 mL/fetus; one part Narcoren® diluted with one part physiological saline).

Soft tissue examination of the fetuses
After the fetuses had been sacrificed, the abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure.
The sex of the fetuses was determined by examination of the gonads in situ.

After these examinations, the heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head. After the examination the heads were discarded.

All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.

Skeletal examination of the fetuses
After fixation in ethyl alcohol, the skeletons (with and without skulls) were stained according to a modified method of KIMMEL and TRAMMELL. The stained skeletons were placed on an illuminated plate, investigated and archived individually.

Evaluation criteria for assessing the fetuses
Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BELTRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the nomenclature used to describe observations of fetal morphology, the terms still vary considerably between laboratories, investigators and textbooks in the fields of teratology and developmental toxicity.
In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:

Malformation
A permanent structural change that is likely to adversely affect survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

All fetal findings were listed in tables according to these classifications.

Statistics:

see any other information below

Indices:

The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals / number of fertilized animals) x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of corpora lutea – number of implantations / number of corpora lutea) x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of implantations – number of live fetuses) / number of implantations) x 100

Historical control data:

see attached file

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No defecation was observed in three control females, one low-dose, two mid-dose and six high-dose females. Reduced defecation was observed in eight control, four low-dose, four mid- dose and seven high-dose females (0, 6, 20 and 60 mg/kg bw/d). Incidence and distribution of these findings do not indicate a relationship to the test substance.

There were no further clinical findings in the other does in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One low-dose female (No. 31 - 6 mg/kg bw/d) was found dead before treatment on GD 26, after showing no or reduced defecation during several days before. The cause of this preterminal death remains unknown, because of the lacking dose-response it is, however, considered not to be related to the test substance.

One high-dose female (No. 91 - 60 mg/kg bw/d) was sacrificed after abortion ahead of schedule (GD 25). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant difference was observed for the mean body weights (BW) of the high- dose females (60 mg/kg bw/d) when compared to the control group.

Collectively, the mean body weight gain during the treatment period was comparable between all test groups (6, 20 and 60 mg/kg bw/d) and the control group.

Notably, there were episodes of significant differences between control and high-dose group such as GD 19-21 where the high-dose does lost less weight (-3.9 g vs. -55.4 g in control), or GD 23-25 where they gained more weight than the control does (47.2 g vs. 6.8 g in control). These episodes compensated for a longer time period (GD 6-19) where the high-dose rabbit gained consistently (but not statistically significantly) less body weight than the controls.

Corrected (net) body weight gain
Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 6, 20 or 60 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group the mean food consumption of the does in test group 3 (60 mg/kg bw/d) was reduced from GD 12 onwards, attaining statistical significance on GD 12-14 and GD 15-18 (up to 40% below control). Afterwards it recovered, being comparable to or even exceeding the control values (attaining statistical significance on GD 25-27). Overall, the high- dose does consumed about 9% less food than the concurrent control does during the treatment period (GD 6-28).

The food consumption of the low- and mid-dose rabbits (6 and 20 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes of hematological parameters were observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

At gestation day 29, in does of test group 3 (60 mg/kg bw/d) cholesterol values were significantly increased, but the mean was within the historical control range (cholesterol 0.18-0.53 mmol/L). Therefore, this change was regarded as incidental and not treatment related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative kidney weights were statistically significantly increased in test group 3 :

Relative weights
Test group (mg/kg bw/d) 1 (6) 2 (20) 3 (60)
Kidneys 96% 104% 110%**
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Since only the relative kidney weights in test group 3 were significantly increased and since relative kidney weights lay within the range of historical control values, weight changes were regarded as incidental and not treatment related.


Uterus weights
The mean gravid uterus weight of the rabbits of test groups 1-3 (6, 20 or 60 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All 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.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All 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.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):
• females Nos. 14, 18, 22, 23 - not pregnant

Test group 1 (6 mg/kg bw/d):
• females Nos. 43, 48 - not pregnant
• female No. 31 - died intercurrently

Test group 2 (20 mg/kg bw/d):
• females Nos. 53, 60, 65, 75 - not pregnant

Test group 3 (60 mg/kg bw/d):
• females Nos. 77, 80, 88 - not pregnant
• female No. 91 - sacrificed after abortion


Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

Number of abortions:

effects observed, non-treatment-related

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Early or late resorptions:

effects observed, non-treatment-related

Dead fetuses:

effects observed, non-treatment-related

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Female rabbits were placed into the study in four cohorts. Each dose group was represented in each cohort. The conception rate was 84% in the control and mid-dose groups (0 and 20 mg/kg bw/d), 88% in the high-dose group (60 mg/kg bw/d) and 92% in the low-dose group (6 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study (according to the test guidelines).

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for pre- and post-implantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

In high-dose doe No. 98 (60 mg/kg bw/d) 2 early resorptions were recorded after staining the apparently non-pregnant uterus at C-section (i.e. pregnant by stain), resulting in a 100% post- implantation loss for this animal. This isolated finding did not exert a significant effect on the group mean and is regarded as incidental and not treatment related.

Two dead fetuses were found at C-section of high-dose doe No. 93 which is a rare finding but may occur spontaneously in this rabbit strain. As there was no evidence whatsoever for any potential developmental toxicity of the test item in this study, this is considered to be an incidental finding.

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (6, 20 and 60 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal external malformations
One external malformation was recorded for two fetuses, each, in test groups 0 and 1 (0 and 6 mg/kg bw/d) as listed in the table below. In one control fetus it was associated with an additional visceral malformation. However, this finding was an isolated event in single fetuses, thus, it is considered to be incidental. No statistically significant differences of overall incidences were noted between the groups

Individual fetal external malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 3-02 M umbilical hernia
21-09 F a) umbilical hernia
1 (6 mg/kg bw/d) 30-06 F umbilical hernia
35-02 M umbilical hernia
2 (20 mg/kg bw/d) none
3 (60 mg/kg bw/d) none
a) fetus with additional visceral malformations

Total external malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N(%) 2 (1.1) 2 (1.2) 0.0 0.0
Litter
incidence N(%) 2 (9.5) 2 (9.1) 0.0 0.0
Affected
fetuses/litter Mean% 1.3 1.1 0.0 0.0



Fetal external variations
One external variation (paw hyperflexion) was recorded in two control fetuses, one mid-dose fetus and two high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, thus it is considered to be incidental.

Total external variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 2 (1.1) 0.0 1 (0.5) 2 (1.2)
Litter
incidence N (%) 2 (9.5) 0.0 1 (4.8) 2 (10)
Affected
fetuses/litter Mean% 1.1 0.0 0.4 1.3


Fetal external unclassified observations
One unclassified observation, i.e. placentae necrobiotic, was recorded in one fetus of test group 3 (60 mg/kg bw/d). This finding is considered not to be related to treatment.

Total external unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 0.0 0.0 0.0 1 (0.6)
Litter
incidence N (%) 0.0 0.0 0.0 1 (5.0)
Affected
fetuses/litter Mean% 0.0 0.0 0.0 0.6

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in single fetuses of all test groups including the control (0, 6, 20 and 60 mg/kg bw/d), as shown in the table below. Two fetuses had associated soft tissue malformations. All findings were considered to be spontaneous in origin and not treatment-related.

No statistically significant differences between the groups were noted. The overall incidences were well within the historical control range of the test facility.

Individual fetal skeletal malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 19-04 M fused thoracic arch, fused rib
1 (6 mg/kg bw/d) 41-01 M additional vertebral arch and corresponding rib, intercostal rib
41-03 M a) cleft sternum
2 (20 mg/kg bw/d) 73-10 F absent lumbar vertebra
3 (60 mg/kg bw/d) 82-07 M severely malformed vertebral column and/or ribs
87-07 M a) severely malformed vertebral column and/or ribs
a) fetus with additional soft tissue malformations


Total skeletal malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 1 (0.5) 2 (1.2) 1 (0.5) 2 (1.2)
Litter
incidence N (%) 1 (4.8) 1 (4.5) 1 (4.8) 2 (10)
Affected
fetuses/litter Mean% 0.4 4.5 0.5 1.1


Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.

Total fetal skeletal variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 175 (93) 143 (88) 165 (87) 161 (93)
Litter
incidence N (%) 21 (100) 22 (100) 21 (100) 20 (100)
Affected
fetuses/litter Mean% 93.5 86.4 86.2 92.7


For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table below. All incidences were expressed on a fetus per litter basis.

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding Test group 0 Test group 1 Test group 2 Test group 3 HCD
Mean % (range)

Misshapen
sacral vertebra 2.1 2.3 5.3 4.8* 5.0 (1.9 - 8.6)

Incomplete
ossification
of talus;
cartilage present 2.6 2.9 2.3 6.7* 2.3 (0.0 - 4.5)

Unossified talus;
cartilage present 0.5 1.2 3.1 5.8* 1.3(0.0 - 2.6)

HCD = Historical control data
* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])


The findings ‘incomplete ossification of talus’ and ‘unossified talus’ (with present cartilage, respectively) were statistically significantly increased and outside the historical control range in test group 3 (60 mg/kg bw/d). These findings may represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases.

The increased incidence of ‘misshapen sacral vertebra’ in test group 3 was not related to dose and clearly inside the historical control range. Thus, this finding is considered not to be associated with treatment.


Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.

Total unclassified cartilage observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 20 (11) 12 (7.4) 30 (16) 13 (7.5)
Litter
incidence N (%) 11 (52) 8 (36) 12 (57) 5 (25)
Affected
fetuses/litter Mean% 10.7 6.0 14.3 8.5

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in the test groups 0, 1 or 3 (0, 6 and 60 mg/kg bw/d), as listed in the table below. Three fetuses in different test groups had additional external or skeletal malformations.

The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Individual fetal soft tissue malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 15-10 F small thymus
21-09 F a) small spleen
24-05 M, 24-06 F, 24-08 M small spleen
1 (6 mg/kg bw/d) 39-04 F small spleen
41-03 M b) cor triloculare
2 (20 mg/kg bw/d) none
3 (60 mg/kg bw/d) 87-07 M b), 87-08 F small spleen
97-05 F small spleen
a) fetus with additional external malformations
b) fetus with additional skeletal malformations

Total soft tissue malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 5 (2.6) 2 (1.2) 0.0 3 (1.7)
Litter
incidence N (%) 3 (14) 2 (9.1) 0.0 2 (10)
Affected
fetuses/litter Mean% 2.0 2.8 0.0 1.8


Fetal soft tissue variations
The examinations of the soft tissues revealed malpositioned carotid branches and an absent lung lobe (Lobus inferior medialis) in all test groups including the control (0, 6, 20 and 60 mg/kg bw/d). Other variations, such as cystic dilatation of the brain and supernumerary branch from aortic arch (test group 2, respectively), dilated cerebral ventricle (test groups 1 and 2), malpositioned carotid origin (test group 0), dilated aortic arch and narrowed pulmonary trunk (test group 1, respectively) and dilated renal pelvis (test group 3) occurred in individual fetuses of the different test groups.

No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range.

Total soft tissue variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 4 (2.1) 7 (4.3) 7 (3.7) 7 (4.0)
Litter
incidence N (%) 4 (19) 6 (27) 6 (29) 4 (20)
Affected
fetuses/litter Mean% 1.7 5.2 4.3 4.1

Fetal soft tissue unclassified observations


Three unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in two control, one low-dose, two mid-dose and one high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, therefore, it was neither assessed as treatment-related nor as adverse. Furthermore, a discolored spleen was seen in three fetuses of the same high-dose litter, and an infarct of liver was recorded in one fetus of test group 3. These findings are considered not to be treatment-related.

Total soft tissue unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 2 (1.1) 1 (0.6) 2 (1.1) 5 (2.9)
Litter
incidence N (%) 1 (4.8) 1 (4.5) 2 (9.5) 3 (15)
Affected
fetuses/litter Mean% 1.6 0.5 1.0 2.5

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were comparable to the control value.

Details on embryotoxic / teratogenic effects:

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all test groups (0, 6, 20 or 60 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.

Six fetuses had more than one malformation. Male control fetus No. 19-04 had a fused thoracic arch and a fused rib, while female control fetus No. 21-09 showed an umbilical hernia and a small spleen. Furthermore, for male low-dose fetus No. 41-03 (6 mg/kg bw/d) a cor triloculare combined with a cleft sternum was recorded. Male low-dose fetus No. 41-01 had malformations affecting the rib cage, i.e. additional vertebral arch and corresponding rib and an intercostal rib. Male high-dose fetus No. 87-07 (60 mg/kg bw/d) showed a small spleen and a severely malformed vertebral column and/or ribs. Lastly, male high-dose fetus No. 82-07 had also a severely malformed vertebral column and/or ribs. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups.

The findings ‘umbilical hernia’ and ‘small spleen’ which were seen in the multiple malformed fetuses, occurred also in further individual fetuses of test groups 0, 1 and 3. Other malformations, such as small thymus (test group 0) and absent lumbar vertebra (test group 2) were scattered observations in individual fetuses. They all were not dose-related and most of them can be found in the historical control data at comparable frequency. An association of these findings to the treatment is not assumed.

Total fetal malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 7 (3.7) 5 (3.1) 1 (0.5) 4 (2.3)
Litter
incidence N (%) 5 (24) 4 (18) 1 (4.8) 3 (15)
Affected
fetuses/litter Mean% 3.2 6.2 0.5 2.3



A spontaneous origin is assumed for the external variations, soft tissue variations and the broad range of skeletal variations which were noted in fetuses of all test groups including controls.

If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at comparable frequency.

The only exception: statistically significantly increased findings ‘incomplete ossification of talus’ and ‘unossified talus’ represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. As these non or incomplete ossifications were solely found in this single location, and no other signs of a delay of skeletal development were noted, a spontaneous origin of these findings is assumed. In addition, such minor developmental delays are considered not to be adverse events.

Total fetal variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 175 (93) 143 (88) 165 (87) 161 (93)
Litter
incidence N (%) 21 (100) 22 (100) 21 (100) 20 (100)
Affected
fetuses/litter Mean% 93.5 86.4 86.2 92.7


A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups. The distribution and type of these findings do not suggest any relation to treatment.

Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (60 mg/kg bw/d).

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Hydroxyethyl acrylate to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused beginning evidence of maternal toxicity at a dose of 60 mg/kg bw/d, such as slightly but consistently reduced food consumption as well as reduced body weight/weight gain during the first two treatment weeks.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is
20 mg/kg bw/d.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is
60 mg/kg bw/d, the highest dose tested.

The test substance is not teratogenic in rabbits at the tested dose levels.

Executive summary:

In a prenatal developmental toxicity study, Hydroxyethyl acrylate was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28).

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Clinical examinations revealed no toxicologically relevant difference between the animals receiving 6 or 20 mg/kg bw/d Hydroxyethyl acrylate and the controls.

At the high-dose level of 60 mg/kg bw/d slightly, but consistently lower food consumption as well as lower body weight gain during the first two treatment weeks may represent beginning signs of maternal toxicity.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 60 mg/kg bw/d.

Regarding pathology, all 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.

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc. (Denver, PA)
- Age at study initiation: 5.5-6 months old
- Weight at study initiation: 2.8-4.0 kg
- Housing: Single in in stainless steel, wire-mesh cages (61 x 61 x 41 cm)
- Diet (ad libitum): AGWAY® PROLAB® Animal Diet (Agway Inc.) Except during exposures
- Water (ad libitum): Tap water (except during exposures)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-21
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Females assigned to the study were exposed to acrylic acid vapour or filtered air for 6 hours/day during the period of major organogenesis (gestation day -gd- 6 through 18). Filtered air was bubbled through a glass reservoir containing liquid acrylic acid. For all vapour concentrations, a Dwyer Flowmeter was used to measure airflow prior to passing the air through the acrylic acid. The vapour, was introduced into the exposure chambers through 1 inch glass tubing containing stainless steel wool.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of acrylic acid vapour in each exposure chamber was monitored throughout the 15 days of exposure by sampling with XAD-8 sorbent tubes and subsequent analysis using high performance liquid chromatography (HPLC) analysis. The HPLC system was composed of a Model 981 Lambda Max LC Spectrophotometer, a Programmable Systems 680 Gradient Controller, a 712 WISP, and a Model 501 Solvent Delivery System. A Spectra Physics SP4270 computing Integrator provided a record of the chromatograms, chromatographic analyses, and peak height measurement. The concentration in each exposure chamber atmosphere was determined approximately 3 times during each 6-hour exposure. The control chamber was sampled once daily. The nominal concentration was calculated by dividing the total quantity of acrylic acid delivered to the chamber by the chamber airflow rate.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1

Duration of treatment / exposure:

From day 6 to day 18 of gestation

Frequency of treatment:

6 hours/day

Duration of test:

29 days

Dose / conc.:

25 ppm

Remarks:

corresponding to approx. 0.075 mg/L). Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]

Dose / conc.:

75 ppm

Remarks:

corresponding to approx. 0.224 mg/L). Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]

Dose / conc.:

225 ppm

Remarks:

corresponding to approx. 0.673 mg/L). Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]

No. of animals per sex per dose:

16

Control animals:

yes, sham-exposed

Maternal examinations:

CAGE SIDE OBSERVATIONS: not specified

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the exposure period, animals were observed for clinical signs once daily. Preceding and following each exposure, individual does were observed for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were measured on gd 0, 3, 6, 12, 18, 24, and 29.

FOOD CONSUMPTION: Yes
Food consumption was measured daily throughout the study, beginning on gd 3.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic cavities were examined grossly. The right nasal turbinates were examined. Maternal liver and kidney weights were determined.

Ovaries and uterine content:

Each uterus was removed from the peritoneal cavity, weighed, and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites (early and late) were recorded. Ovaries were removed from the peritoneal cavity and ovarian corpora lutea of pregnancy were counted. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for confirmation of pregnancy status.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

After removal from the uterus, all live fetuses received a single lethal intraperitoneal injection of sodium pentobarbital. All live and dead fetuses were weighed and examined externally for variations and malformations including cleft palate. All live fetuses in each litter were examined for thoracic and abdominal visceral abnormalities. The sex of each fetus was determined during dissection by examination of the reproductive organs. One-half of the live fetuses in each litter were decapitated. The heads were fixed in Bouin's solution for subsequent examination of craniofacial structures. All fetuses (50% intact, 50% decapitated) in each litter were eviscerated, air-dried, processed for skeletal staining with alizarin red S and examined for skeletal malformations and variations. All fetal skeletal preparations were retained.


- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

The unit of comparison was the pregnant doe or the litter. The data for quantitative continuous variables were intercompared for the 3 exposure groups and the control group by use of Levene's test for equality of variances; analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, pooled t-tests were used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by separate variance t-tests for pairwise comparisons.
Nonparametric data were statistically evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U test when appropriate. Frequency data were compared using Fisher's Exact Test. With the exception of the data analysis for fetal malformations and variations, all statistical analyses were performed using BMDP Statistical Software (Dixon, 1990). For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No animal died in any group. In the 225 ppm group, individual clinical signs observed included: perinasal wetness beginning as early as the first day of exposure and ending by the second day after the last exposure; perinasal encrustation (from days 14 through 22); and nasal congestion observed during and subsequent to exposures (through Day 22). Nasal congestion was observed in a single dose from the 75 ppm group on day 12 only. There were no clinical signs observed during or subsequent to exposures to 25 ppm acrylic acid vapour.

Dose descriptor:

NOAEC

Effect level:

0.075 mg/L air (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no evidence of developmental toxicity including teratogenicity at any exposure concentration.

Dose descriptor:

NOAEC

Effect level:

0.673 mg/L air (nominal)

Basis for effect level:

other: fetotoxicity

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Dose descriptor:

NOAEC

Effect level:

0.673 mg/L air (nominal)

Basis for effect level:

other: teratogenicity

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Abnormalities:

no effects observed

Developmental effects observed:

no

Maternal food consumption:

Exposure-related decreases in food consumption were observed in the 75 and 225 ppm groups during the first 5 days of the exposure period. Throughout the remainder of the exposure period, daily food consumption was consistently reduced in the 225 ppm group and the decreases occasionally reached statistical significance. Occasional reductions in daily food consumption were also observed during the exposure period subsequent to Day 11 (Days 17 to 19) in the 75 ppm group. Average food consumption/day calculated for the entire exposure period was reduced in the 225, but not 75, ppm group. Statistically significant increases in food consumption were observed subsequent to the exposure period in the 225 ppm group for Days 23 to 24 and 28 to 29 and in the 75 ppm group for Day 23 to 24. Mean food consumption values for Days 24 to 27 suggested a trend toward increased food consumption throughout the post exposure period for the 225 ppm group. Mean values for the 75 ppm group suggest a trend toward increased food consumption through Day 26. The statistically significant reduction (of approximately 16%) in food consumption for Day 8 to 9 in the 25 ppm group was not considered to be biologically significant based on slightly greater food consumption values (including increases of up to 14%) in the low concentration group prior to exposures. Occasional increases or decreases in food consumption values for the 25 ppm group subsequent to Day 9 were not considered to be exposure related due to the lack of a dose-response pattern of effects.

Gestation day	Group mean food consumption (g)
	0 ppm	25 ppm	75 ppm	225 ppm
6 – 7	191.42	166.96	147.31^**	101.55^**
7 – 8	193.42	169.23	149.51^**	110.35^**
8 – 9	195.13	163.75^*	150.69^**	121.66^**
9 – 10	185.38	166.73	153.59^*	132.49^**
10 – 11	184.98	200.79	154.79^*	134.49^**
12 – 13	180.85	123.21^*	203.10	136.18^**
16 – 17	187.39	186.42	161.23	152.77^*
18 – 19	208.56	178.61	167.23^*	147.33^**
23 - 24	158.19	176.43	204.25^**	199.75^*
28 - 29	134.35	167.32^*	160.41	188.19^**

^* Significantly different from control group (p<0.05.)

^**Significantly different from control group (p<0.01.)

Maternal body weights and weight changes:

Mean gestational body weights were equivalent across groups throughout gestation. There were no statistically significant exposure-related reductions in weight gain. However, mean body weight losses were observed in all acrylic acid-exposed groups for Days 6 to 12. Losses in the 25 ppm group were not considered to be biologically significant, since body weight gains were greater than control values (by approximately 60 g) during the preexposure period and the reductions for Days 6 to 12 were not associated with consistent reductions in food consumption for the first half of the exposure period. Reduced body weight gain values in the 75 and 225 ppm groups for Days 6 to 12 were considered to be an exposure-related effect since the reductions were coincident with consistent reductions in food consumption for the first five days of the exposure period. Likewise, increased body weight gains in the 75 and 225 ppm groups for Days 18 to 29 were associated with increases in food consumption during the postexposure period.

Gestation day	Gestational mean body weight changes (g)
	0 ppm	25 ppm	75 ppm	225 ppm
0 - 3	-34.08 (150.76)	-7.47 (152.69)	-6.83 (150.42)	-43.38 (214.46)
3 - 6	203.77 (96.19)	240.45 (76.84)	206.55 (96.92)	163.45 (251.41)
6 - 12	68.43 (65.47)	-18.87 (93.75)	-37.67 (93.47)	-41.06 (201.25)
12 - 18	146.67 (69.62)	160.54 (72.07)	123.51 (63.56)	148.45 (65.59)
18 - 24	112.36 (73.58)	150.13 (109.11)	178.41 (73.84)	176.15 (86.22)
24 - 29	26.65 (89.84)	51.55 (125.51)	64.32 (87.17)	143.49^**(96.91)

^**Significantly different from control group (p<0.01.)

Numbers in parentheses indicate standard deviation

Maternal Necropsy:

Upon necropsy on Day 29, pertinent findings included ulcerations in the nasal turbinates of 1 female in the 225 ppm group. There were no exposure related changes in mean body weight at sacrifice, gravid uterine weight, corrected body weight, or corrected weight change. There were no significant effects of exposure on relative and absolute kidney or liver weights. (Apparently slight increases in absolute and relative liver weights in the 75 and 225 ppm groups were due to single animals in each group which had abnormally large livers). There were no effects of exposure on the number of ovarian corpora lutea, the number of total, viable, or nonviable (early and late resorptions and dead fetuses) implantations/litter. Although percent preimplantation loss was statistically significantly increased in the mid and high concentration groups, the increases were not concentration dependent. Percent live fetuses and sex ratio were equivalent across groups.

Gestational parametres:

Fetal body weights were unaffected by test substance exposure. There were no increases in the incidences of individual external, visceral or skeletal malformations by category, or of total malformations among all groups. There were no increases in the incidences of individual fetal external, visceral, or skeletal variations, of variations by category, or of total variations among all groups.

Concentration (ppm)	0	25	75	225
Corpora lutea (mean)	8.4	9.1	9.5	8.8
Implantations (mean)	8.6	8.8	8.2	8.5
Viable implants (mean)	8.4	8.6	7.5	7.9
Percent live fetuses (mean)	97.8	98.4	91.3	95.0
Dead implantations (mean)	0.1	0.1	0.0	0.1
Mean weight of live fetuses (g)	43.99	42.44	42.44	42.93

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity:

In a study where the developmental toxicity of seven acrylates was investigated (Saillenfait 1999), groups of 25 pregnant rats were exposed to 0, 1, 5 or 10 ppm hydroxypropyl acrylate vapour (corresponding to approx. 0.0054, 0.027, and 0.054 mg/L) for 6 hrs/day from days 6 through 20 of gestation. Maternal body weights were lower than control for the 10 ppm group and weight gain was reduced for the 5 and 10 ppm groups.

Uteri were removed and weighed, and the number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide to detect very early resorptions. Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin’s solution and examined for internal soft tissue changes. The other half were fixed in ethanol, eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.

There was no significant difference in the numbers of implantation sites and live fetuses, in the incidence of non-live implants and resorptions, or in the fetal sex ratio, or fetal body weight between control and treated animals.

No treatment related increase in embryo/foetal lethality or fetal malformations was observed at any dose level. The incidence of external, visceral, and skeletal variations was similar to controls.

The NOAECs were 1 ppm (0.0054 mg/L) for maternal toxicity and 10 ppm (0.054 mg/L) for embryo-foetal toxicity and teratogenicity.

 

In addition, developmental studies in rabbits are available for the structural analogues 2-hydroxyethyl acrylate (CAS 818-61-1) and acrylic acid (CAS No. 79-10-7): 

Data from the structural analogue 2-hydroxyethyl acrylate (CAS No. 818-61-1):

In a prenatal developmental toxicity study (BASF 2021), 2-hydroxyethylacrylate was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28). 

Clinical examinations revealed no toxicologically relevant difference between the animals receiving 6 or 20 mg/kg bw/d Hydroxyethyl acrylate and the controls. At the high-dose level of 60 mg/kg bw/d slightly, but consistently lower food consumption as well as lower body weight gain during the first two treatment weeks may represent beginning signs of maternal toxicity. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 60 mg/kg bw/d. Regarding pathology, all 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. There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

 

Data from the structural analogue acrylic acid (CAS No. 79-10-7):

Groups of 16 pregnantrabbits were exposed (6 h/d, whole-body) to atmospheres containing acrylic acid at 0, 25, 75, and 225 ppm (corresponding to approx. 0.075, 0.224, 0.673 mg/L) during days 6-18 of gestation (BAMM, 1993; Neeper-Bradley et al., 1997). All dose groups were observed daily for morbidity and mortality. During the exposure period, animals were observed for clinical signs preceding and subsequent to daily exposures and from outside during actual exposures. Maternal body weights were measured on gestation day 0, 3, 6, 12, 24, and 29. Food consumption was measured daily throughout the study beginning on gestation day 3. After sacrifice on gestation day 29, maternal liver and kidney weights were determined. All foetuses were weighed and examined for external malformations and variations, for thoracic and abdominal visceral abnormalities including internal sex organs, for craniofacial abnormalities and for skeletal malformations and variations. Dose-related clinical signs (as perinasal/perioral wetness and nasal congestion, as well as reduced body weight gain and food consumption) were observed in the 75 and 225 ppm groups. The overall pregnancy rate was equivalent for all groups (94-100 %). No dose-related effects were observed in the reproduction function of the dams. There were no effects on the number of ovarian corpora lutea, the number of total viable or non-viable (early and late resorptions and dead foetuses) implantations/litter. Percentage live foetuses and sex ratio were equivalent across groups. Foetal body weights were unaffected by test substance exposure. There were no exposure-related increases in the incidences of external, visceral or skeletal malformations or variations.

NOAEC for maternal toxicity was 25 ppm (= approx. 0.075 mg/L).

NOAEC for developmental toxicity: 225 ppm = 0.673 mg/L.

 

Data form the metabolic cleavage product propylene glycol (CAS No. 57-55-6)

In the developmental toxicity study with mice (Bushy Run Research Center, 1993), propylene glycol was administered to pregnant mice at dose levels of 0, 0.5, 5.0 and 10.0 ml/kg bw/day (0, 520, 5200 and 10400 mg/kg bw/day) on gestation days 6 through 15. Mice were sacrificed on gestation day 18 and evaluation of fetuses, uterine weight, number of corpora lutea and implantation sites was performed. Increased water consumption was observed in the 10.0 ml/kg bw/day group and, although not statistically significant, in the middle dose group. No further treatment-related clinical signs, effects on maternal body weights and body weight gains, food consumption were observed at any dose level and no treatment-related necropsy findings of the dams at the scheduled sacrifice on gestation day 18 were found. Therefore, the increase in water consumption is likely to be a normal physiological reaction to the administration of a high quantity of substance by gavage, which may cause a surge in the osmolarity of body fluids. Consequently, this effect is not considered to be of toxicological significance. There were no effects of treatment on gravid uterine weight, the number of ovarian corpora lutea, the number of total, viable or nonviable implantations/litter or on sex ratio. Also, no effects on fetal body weights/litter were observed which were attributed to treatment. There were no treatment related increases in the incidences of individual fetal external or visceral variations. Based on the results of the study, the NOAEL for developmental toxicity was established to correspond to 10400 mg/kg bw/day.

 

Additional studies with propylene glycol on developmental toxicity in rats, mice, hamsters and rabbits, conducted under the contract for the FDA (FDRL. Teratologic evaluation of FDA 71-56 (propylene glycol) are available. (Food and Drug Research Laboratories, Inc., 1973), These studies showed no signs of maternal, fetal toxicity or teratogenicity at the maximum tested doses (1600 mg/kg bw/day for rats, and mice, 1550 mg/kg bw/day for hamsters and 1230 mg/kg bw/day for rabbits). The study reports had some shortcomings in that no detailed information on study design and no statistical information was reported. Propylene glycol was also tested in a mouse screening assay (Kavlock et al., Teratog. Carcinog. Mutagen. 1987, 7, 7-16); however, the endpoints evaluated (number of dams pregnant, mortality, the number of dams with resorptions, the number of live pups and their weights on postnatal days 1 and 3) and the dosing period (gestation days 8-12) were not adequate for a comprehensive developmental toxicity study. Nevertheless, against the criteria for such a screening study, the outcome was negative. Available data on developmental toxicity of propylene glycol were evaluated and assessed by the expert panel of the CERHR In summary, the Panel concluded that the available data are sufficient to evaluate the developmental toxicity of monopropylene glycol. The panel concluded that data from the Bushy Run Research Center (1993) indicated that monopropylene glycol is not a developmental toxicant in mice. The maternal NOEL was 5000 mg/kg bw/day based on increases in water consumption observed at 5000 and 10000 mg/kg bw/day. The panel concluded that it was reasonable to speculate that this effect was a physiological response to the high doses of propylene glycol administered. Data in other species, although inadequately presented, are consistent with the findings in mice. Despite the limitations of each study, no adverse developmental or maternal effects were noted in any species at the highest tested dose (10000 mg/kg bw/day in mice in the screening study; 1600 mg/kg bw/day in rats, 1550 mg/kg bw/day in hamsters and 1230 mg/kg bw/day in rabbits). The overall data were judged to be adequate for human risk assessment. Based on these findings, the Panel concluded that current estimated exposures to monopropylene glycol are of negligible concern for developmental toxicity in humans. From a REACH perspective, the Bushy Run study provides unequivocal and reliable evidence that this substance does not cause developmental toxicity in mice. There are other supporting studies that confirm this. There is no single study that is considered sufficiently reliable on its own to meet the requirement for a developmental toxicity study in a second species. However, the FDA studies in three further species (rat, hamster and rabbit) do provide what is regarded as sufficient weight of evidence that the result in mice is not anomalous and that propylene glycol does not cause developmental effects

Justification for classification or non-classification

Based on the available data, classification as a reprotoxic substance is not triggered according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.