Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

For n-butyl acrylate an extended one generation study according to OECD 443 and GLP was performed. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (Acrylate Reach TF, 2017). There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day.

A subchronic inhalation study in rats demonstrated no indication of a fertility impairing potential of n-butyl acrylate.

A two-generation reproduction toxicity study with the structural analogue methyl acrylate, which was considered as reliable surrogate for n-butyl acrylate, led to no effects on reproductive performance including fertility after whole body inhalative exposure. The NOAEC for reproductive function was 75 ppm (= ca. 0.269 mg/L), the highest concentration investigated.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
28 Jul 2011
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 70 days
- Basis for dose level selection: Dosage levels were selected based on the range-finding study. In that study, dosage levels of 40, 160, and 400 mg/kg/day were tested. Animals in the high-dosage group showed clinical signs (salivation and red material around the eyes, nose, and mouth), which were still observed to a lesser extent in the mid-dosage group, and the males were more affected than the females. Macroscopic examinations showed thickened and eroded stomach in the 400 mg/kg/day group males and thickened stomach was still observed in the 160 mg/kg/day group animals (predominantly in males). In the current study, the animals were dosed for a longer time period. Therefore, based on the results of the range-finding study, dosage levels of 20, 50, and 150 mg/kg/day were selected for the current study. The high-dosage level of 150 mg/kg/day was expected to show parental toxicity, whereas the low-dosage level of 20 mg/kg/day was not expected to show toxic effects.
- Inclusion/exclusion of extension of Cohort 1B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Termination time for F2: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not performed as P0 and F1 Cohort 1A did not reveal any results
- Route of administration: oral
Specific details on test material used for the study:
- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8 weeks
- Weight at study initiation: Male body weights ranged from 264 g to 378 g and female body weights ranged from 154 g to 228 g on the initial day of test substance administration
- Housing: F0 parental animals were housed 2–3 per cage by sex in clean, solid-bottom cages with heat-treated aspen bedding material (Aspen Bed™ 1, Northeastern Products Corporation, Warrensburg, NY). Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly
- Diet: the basal diet was provided ad libitum throughout the acclimation period and during the study. The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF, was a certified feed with appropriate analyses performed by the manufacturer.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system was provided ad libitum throughout the acclimation period and during the study.
- Acclimation period: 24 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 4, 10 and 30 mg/mL (corresponding to dosage levels of 0, 20 50 and 150 mg/kg/day)
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment.
- Length of cohabitation: 14 day mating period
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Following positive evidence of mating, the F0 males and females were individually housed in solid-bottom cages with bedding material until the scheduled necropsy. The dams were housed in these cages with their litters until weaning on Lactation Day 21. Following weaning of the F1 litters, the weaned F1 pups were housed together by litter for 1 week. Beginning on PND 28, the F1 offspring were housed 2–3 per cage by sex in clean, solid-bottom cages with bedding material until necropsy. Females for which there was no evidence of mating were placed in clean, solid-bottom cages with bedding material upon completion of a 14-day mating period with no further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 4, 10, and 30 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on Study Day 0, 7, 15, and 30 for the first month and once a month thereafter (total of 9 sets). Analysis was performed using a validated gas chromatography method using flame ionization detection. The analysed dosing formulations were, with minor exceptions, within the range for suspensions (85% to 115%) and were homogeneous. Analysed concentrations above the acceptance criteria had no impact on the study as the no-observed-adverse-effect level (NOAEL) was determined to be 150 mg/kg/day, the highest dose evaluated on this study.
Duration of treatment / exposure:
The vehicle and test substance formulations were administered to the F0 males and females for a minimum of 70 consecutive days prior to mating. Dose administration for the F0 males continued throughout mating and through the day prior to euthanasia, for a total of 129-131 doses. The F0 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia, for a total of 132-136 doses. Animals selected for the F1 generation were directly administered the vehicle or test substance following weaning (beginning on PND 21) and continuing through the day prior to euthanasia (PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]).
Frequency of treatment:
daily
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were selected based on the range-finding study (see any other information on materials and methods)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS
Detailed physical examinations were recorded weekly, beginning 1 week prior to the initiation of test substance administration, for all parental animals throughout the study period. In addition, detailed physical examinations were conducted on Gestation Days 0, 7, 14, and 20 for all females with evidence of mating and on Lactation Days 1, 7, 14, and 21. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration. Special attention was paid to the degree of salivation and lacrimation, presence or absence of urination and defecation (including polyuria and diarrhoea), pupil size, degree of palpebral closure, presence of convulsions, tremors, or abnormal movements, presence of posture and gait abnormalities, the presence of any unusual or abnormal behaviours, and any repetitive actions (stereotypies). Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal.

BODY WEIGHT
Individual F0 male body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, throughout the study and prior to the scheduled necropsy. Individual F0 female body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating treatment period (males and females) and for the entire F0 treatment period (males only). Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, 14, 17, and 21. After weaning (Lactation Day 21), weekly body weights were recorded for these females until the scheduled necropsy. For F0 females with no evidence of mating, weekly body weights were recorded until necropsy.

FOOD CONSUMPTION
F0 male and female food consumption was measured weekly, beginning 1 week prior to the initiation of test substance administration and continuing until cohabitation. Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food intake was not recorded during the breeding period. Following the breeding period, individual food consumption for males and for females with no evidence of mating was measured on a weekly basis until the scheduled necropsy. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 14, 17, and 21. Food intake was reported for the corresponding intervals of gestation and lactation, and also for Gestation Days 0–20 and Lactation Days 1–21. Food efficiency (body weight gained as a percentage of food consumed) was also calculated

CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (Study Days 129-136 for the F0 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from 10 randomly selected F0 animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis parameters examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)

THYROID HORMONE ANALYSIS
Blood (approximately 1.5 mL) for thyroid hormone levels was collected via the retro-orbital sinus following isoflurane anaesthesia from F0 males and females (10/sex/group) following the completion of weaning of all F0 females
- Parameters evaluated: Thyroxine (T4) and Thyroid Stimulating Hormone (TSH)
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete oestrous cycles (i.e., the total number of returns to metoestrous [M] or dioestrous [D] from oestrus [E] or prooestrous [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the oestrous cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each F0 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F0 males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, 10 pups/litter, 5 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 10 pups. All selections were performed by computerized randomization. Following selection, culled pups were euthanized by decapitation (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital, and examined macroscopically; tissues with gross lesions were preserved in 10% neutral-buffered formalin.

PARAMETERS EXAMINED PRE-WEANING
- A daily record of litter size was maintained. Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.7 Pups found dead on PND 0 or 1 had the lungs removed and placed in a saline-filled jar. If the lungs sank to the bottom of the jar, the pup had no prior documentation of being viable, and there was no evidence of milk in the stomach, the pup was considered to be stillborn. If the lungs floated, the pup was considered to be found dead on the originally documented day (PND 0 or 1).
- Litters were examined daily for survival and any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1, 4, 7, 14, and 21.
- Pups were individually weighed on PND 1, 4 (before culling), 7, 14, and 21.
- Pups were individually sexed on PND 0, 4 (before culling), 7, 14, and 21.
- The anogenital distance of all F1 pups was measured on PND 1
- On PND 12, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae.

GROSS EXAMINATION OF DEAD PUPS
A detailed gross necropsy was performed on any pup found dead after PND 4 and prior to weaning. Tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings.

POST-WEANING DEVELOPMENTAL LANDMARKS
- Balanopreputial Separation: Each male was observed for balanopreputial separation beginning on PND 35. The age at which balanopreputial separation was first observed was recorded for each pup. Examination of the pups continued daily until balanopreputial separation was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a partial and complete balanopreputial separation or a persistent thread of tissue between the glans and prepuce was recorded.
- Vaginal Patency: Each female was observed for vaginal perforation beginning on PND 25. The age at which the vaginal lumen was first observed to open was recorded for each pup. Examination of the females was continued daily until vaginal patency was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a small “pin hole”, a vaginal thread, and complete vaginal opening were recorded.
- Oestrous Cyclicity (Cohort 1A): Beginning on the day vaginal opening was observed, vaginal lavages were performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female until the first sign of oestrus (cornified cells) was observed. The age of first vaginal oestrus after vaginal opening was recorded. Vaginal lavages were also performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each female for 2 weeks during PND 75–91 (the day of necropsy). The average cycle length was calculated and reported for complete oestrous cycles (i.e., the total number of returns to metaoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P]). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

POST WEANING OBSERVATIONS
- Clinical observations: Following weaning, all animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Detailed physical examinations were performed weekly until necropsy. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration.
- Body weights: F1 males and females were weighed weekly following weaning, and on the day of euthanasia.
- Food Consumption: F1 male and female food consumption was measured weekly, beginning on PND 28, until the day prior to euthanasia. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

CLINICAL PATHOLOGY (Cohort 1A)
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (PND 91 for the Cohort 1A F1 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from10 randomly selected F0 and F1 Cohort 1A animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)
Postmortem examinations (parental animals):
SACRIFICE
The surviving F0 males and females were euthanized on Study Days 129-136

GROSS NECROPSY
- A complete necropsy was conducted on all F0 animals that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO. For F0 females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females necropsied during gestation through Lactation Day 4. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
- The following F0 parental tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.

ORGAN WEIGHTS
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group, Liver, Ovaries, Pituitary gland, Prostate gland, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes [a], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix.
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.

HISTOPATHOLOGY
Microscopic examination was performed on all tissues from all F0 parental from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. In addition, reproductive organs of all F0 animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrous cyclicity or sperm number, motility, or morphology were affected) were subjected to histopathological examination. Additionally, all gross lesions were examined microscopically, irrespective of group.
Postmortem examinations (offspring):
SACRIFICE
- Euthanasia was scheduled PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]
- All nonselected F1 weanlings were euthanized on PND 21.
- These animals were subjected to post-mortem examinations (macroscopic and microscopic examination) as follows:

GROSS NECROPSY
- A complete necropsy was conducted on all F1 animals in Cohorts 1A and 1B that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO.
- The following F1 Cohorts 1A and 1B tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Both testes and epididymides from animals euthanized in extremis and from all males in Cohort 1B were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.
- Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on all nonselected F1 pups euthanized on PND 21.
-The following tissues/organs from 10 F1 nonselected pups/sex/group were retained in 10% neutral-buffered formalin for possible histopathologic examination: Brain, Mammary gland, Spleen, Thymus, Thyroids, All gross lesions

ORGAN WEIGHTS
- Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F1 animals in Cohort 1A at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a,c], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group[c], Liver, Ovaries [c], Pituitary gland [c], Prostate gland [c], Seminal vesicles with coagulating glands (with accessory fluids) [c], Spleen, Testes [a,c], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix [c]
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.
[c] Also weighed for F1 Cohort 1B animals.
- The brain, spleen and thymus were weighed from 10 nonselected F1 pups/sex/group on PND 21. In addition, the thyroids were weighed (after fixation) from the same 10 pups/sex/group that were used for blood collection

HISTOPATHOLOGY
- Microscopic examination was performed on all tissues from all F1 Cohort 1A animals from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. Additionally, all gross lesions were examined microscopically, irrespective of group.

SPERM PARAMETERS (Cohort 1A)
Immediately upon euthanasia, the reproductive tract of each F1 Cohort 1A male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F1 Cohort 1A males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium
Statistics:
see any other information on materials and methods
Reproductive indices:
- Male Mating Index (%) = No. of Males with Evidence of Mating / Total No. of Males Used for Mating x 100
- Female Mating Index (%) = No. of Females with Evidence of Mating or Females Confirmed Pregnant / Total No. of Females Used for Mating x 100
- Male Fertility Index (%) = No. of Males Siring a Litter/ Total No. of Males Used for Mating x 100
- Male Copulation Index (%) = No. of Males Siring a Litter/ No. of Males with Evidence of Mating (or Females Confirmed Pregnant) x 100
- Female Fertility Index (%) = No. of Females with Confirmed Pregnancy/ Total No. of Females used for Mating x 100
- Female Conception Index (%) = No. of Females with Confirmed Pregnancy/ No. of Females with Evidence of Mating (or Females Confirmed Pregnant) x 100
Offspring viability indices:
- Mean Live Litter Size = Total Viable Pups on PND 0/ No. Litters with Viable Pups on PND 0
- Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection)(% Per Litter) = Sum of (Viable Pups/Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born/Litter)/ No. of Litters/Group x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups/Litter at End of Interval N/Viable Pups/Litter at Start of Interval N)/ No. of Litters/Group x 100

Where N = PND 0–1, 1–4 (Pre-Selection), 4 (Post-Selection)–7, 7–14, 14–21, Birth to PND 4 (Pre-Selection), or 4 (Post-Selection)–21
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related clinical observations were noted during the F0 generation at the daily examinations, weekly detailed physical examinations, and/or approximately 1 hour following dose administration. Clonic convulsions were noted for 1 male in the 20 mg/kg/day group, 1 female in the 50 mg/kg/day group, and 1 male and 1 female in the 150 mg/kg/day group sporadically throughout the treatment period; this finding was noted at the daily examinations, weekly detailed physical examinations, at the time of dose administration, and/or approximately 1 hour following dose administration. This finding was transient and was not observed in the F1 generation, and therefore was not considered test substance-related. Other observations noted in the test substance-treated groups, including red and/or yellow material, scabbing, and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test substance-related effects on survival were noted for F0 males and females at any dosage level. One female (in the 50 mg/kg/day group) was euthanized in extremis on Gestation Day 26 due to poor general health. The cause of morbidity was the acute inflammation of the uterus observed microscopically and the partially decomposed fetuses observed grossly. Because all animals in the high-dose group (150 mg/kg/day) survived to the scheduled necropsy, the moribundity in the 50 mg/kg/day group was not considered test substance-related. All other F0 parental animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related effects on F0 mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day groups. Differences from the control group were not statistically significant, with the following exceptions. For F0 males, significantly (p < 0.01) lower mean body weight gains were noted in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91–98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect the entire generation interval (Study Days 0–126) or mean body weights in these groups, and therefore were not considered test substance-related. A significantly (p < 0.05) higher mean body weight was noted for the 150 mg/kg/day group F0 females compared to the control group on Study Day 14; this difference was transient and no corresponding effect on mean body weight gain was noted in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean body weight gain was noted in the 50 mg/kg/day group F0 females compared to the control group during Gestation Days 17–20; this difference was transient, did not occur in a dose-related manner, was not of sufficient magnitude to affect the overall gestation treatment interval (Gestation Days 0–20) or mean body weights in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F0 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Differences from the control group were not statistically significant, with the following exceptions. In F0 females, significantly (p < 0.05 or p < 0.01) higher mean food consumption was noted in the 20 mg/kg/day group during Study Days 7–14 and in the 150 mg/kg/day group during Study Days 0–7 and 14–21; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.
- Mean F0 maternal food consumption was unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
- Mean F0 maternal food consumption was unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F0 food efficiency in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration.
- Differences from the control group were not statistically significant, with the following exceptions. Significantly (p < 0.01) lower mean food efficiency was noted for F0 males in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91-98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or a corresponding effect on mean food consumption was not noted at these dosage levels, and therefore they were not considered test substance-related.
- Mean F0 maternal food efficiency was unaffected by test substance administration during gestation.
- Mean F0 maternal food efficiency was unaffected by test substanceadministration during lactation.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related alterations in serum chemistry parameters. Significantly (p < 0.05) higher mean serum albumin was observed in the 150 mg/kg/day group F0 females when compared to the concurrent control group. The mean and individual animal values were within the Charles River Ashland historical control ranges for female rats. The finding was considered a result of individual animal variation rather than a true test substance-related change.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were noted in the nonglandular stomach and liver of the 150 mg/kg/day group F0 males and females and the kidneys in the 150 mg/kg/day group F0 females.
- Nonglandular stomach findings were limited to minimal to moderate epithelial hyperplasia and hyperkeratosis. The hyperplasia was characterized by increased thickness (increased number of cell layers) of the squamous epithelium with an increased thickness of the keratinized oute layers of the epithelium (hyperkeratosis). In one 150 mg/kg/day group F0 female, minimal edema and congestion were observed in the submucosa adjacent to the hyperplasia and hyperkeratosis. These changes were not associated with any clinical pathology changes. Although considered an adaptive change, the mild to moderate severity of the hyperplasia and/or hyperkeratosis in the F0 generation were considered adverse in the 150 mg/kg/day group males and females.
- Liver changes observed microscopically included increased incidence of minimal to mild biliary hyperplasia in the 150 mg/kg/day group F0 males and females and minimal to mild randomly scattered hepatocellular necrosis in the 150 mg/kg/day group F0 males. Biliary hyperplasia was characterized by increased numbers of small bile duct profiles in the portal triads. Biliary hyperplasia can occur as a normal age related finding in rats and was observed in the concurrent control group in this study. The severity was minimal to mild and a clinical pathology correlate for the finding was not observed. Similarly, randomly distributed hepatocellular necrosis can be observed spontaneously in rats and was observed in the control group in this study. The severity of the necrosis was minimal to mild and the change lacked clinical pathology correlates. There was no association of the biliary hyperplasia to the necrosis. Changes observed in the liver were considered nonadverse.
- Increased severity of mineralization at the corticomedullary junction of the kidneys was observed in the 150 mg/kg/day group F0 females. This increase in severity (minimal to mild) did not result in alterations to the clinical pathology parameters related to renal function and were considered nonadverse.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) in F0 males or females. Differences from the control group were slight, did not occur in a dose-related manner, and/or were not statistically significant.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean lengths of oestrous cycles in the test groups were also similar to the control group value. None of these differences were statistically significant. All females were noted to be cycling, and the percentage of females cycling regularly was similar across all groups.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were slight and were not statistically significant, with the following exceptions. Left cauda epididymal sperm concentration was significantly (p < 0.05) lower in the 150 mg/kg/day group compared to the control group. However, there were no effects on reproductive performance (mating and fertility) and no other effects on spermatogenic endpoints at this dosage level; therefore, the lower epididymal sperm concentration noted at 150 mg/kg/day was not considered test substance-related. Significantly (p < 0.05) lower mean motility and progressive motility were noted in the 20 mg/kg/day group compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on F0 reproductive performance were observed at any dosage level. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.
No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive effects observed
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects (nonglandular stomach)
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on survival were noted for F1 animals at any dosage level. In the 50 mg/kg/day group, 1 female was found dead on PND 82 following clinical observations of gasping prior to and following dose administration on the day of death. Based on the findings noted for this female at necropsy (esophageal perforation and clear fluid contents in the thoracic cavity [approximately 8.0 mL]), the cause of death was determined to be intubation error, and therefore was not considered test substance-related. In the control group, 1 male was euthanized in extremis on PND 28 due to clinical observations of head tilt and circling on the day of euthanasia; at necropsy, this male was noted with dilated lateral ventricles in the brain. This correlated to the marked chronic active inflammation in the brain. All other F1 animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F1 male and female pup body weights and body weight changes in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period.
- No test substance-related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day group F1 males and females. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.01) lower mean body weight gain was noted in the 20 mg/kg/day group F1 males compared to the control group during PND 42-49; this difference was transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weights at this dosage level, and therefore was not considered test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean F1 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p < 0.05 or p < 0.01) lower mean food consumption was noted in the 20 mg/kg/day group F1 males during PND 42–49 and 63–70, in the 50 mg/kg/day group F1 males during PND 70–77, and in the 50 mg/kg/day group F1 females during PND 49–63 and 70–77 compared to the control group; these differences were transient, did not occur in a dose-related manner, and were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.
Food efficiency:
no effects observed
Description (incidence and severity):
Mean F1 food efficiency in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- There were no test substance-related effects on serum chemistry parameters.
Urinalysis findings:
no effects observed
Sexual maturation:
no effects observed
Description (incidence and severity):
- Mean estrous cycle lengths in the test substance-treated groups were similar to the control group.
- No test substance-related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level.
- There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.
- Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration.
- Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for PND 21 pups at any dosage level.
-There were no test substance-related changes in organ weights for F1 males and females in Cohort 1A. The absolute and relative pituitary weights were significantly (p < 0.05 or p < 0.01) higher in the 50 and 150 mg/kg/day group F1 females when compared to the concurrent control. These values fell within the historical control reference ranges, were not associated with any microscopic findings, and/or were considered a result of individual animal variation rather than a true test substance-related effect.

-No test substance-related effects on organ weights (absolute and relative to final body weight) were observed at any dosage level for F1 animals in Cohort 1B (necropsied on PND 103–110). Differences from the control group were slight and not statistically significant, with the following exceptions. A higher mean absolute left testis weight was noted in 50 mg/kg/day group F1 males and higher mean relative (to final body weight) left and right testis weights were noted in the 20 and 50 mg/kg/day group F1 males compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- The numbers of pups (litters) found dead from PND 0 through the selection of the F1 generation were 50(13), 46(14), 24(5), and 31(11) in the control, 20, 50, and 150 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration of the test substance were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach and lungs that did or did not float, internal findings included renal papilla(e) not fully developed (Woo and Hoar Grade 1) for 2 pups in the 50 mg/kg/day group and red fluid contents in the bladder for 1 pup in the 20 mg/kg/day group. Because the findings in the 20 and 50 mg/kg/day groups were noted infrequently and in a non-dose-related manner, they were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of culled pups euthanized on PND 4. Internal findings included the developmental variations of distended ureters noted for one pup in the 50 mg/kg/day group and hemorrhagic ring around the iris noted for 1 pup each in the control and 20 mg/kg/day groups. These findings occurred in single pups and in a non-dose-related manner, and therefore were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of pups euthanized on PND 21. Dilated renal pelvis was noted for 1, 2 , and 1 pups in the 20, 50, and 150 mg/kg/day groups, respectively; 1 of these same pups in the 50 mg/kg/day group was also noted with distended ureters. In the 150 mg/kg/day group, 1 pup was noted with opacity of the eyes. Foamy contents in the trachea was noted for groups, respectively; this finding was also noted for 1 pup in the control group. A short tail was noted for 1 pup each in the control and 50 mg/kg/day groups. In the 50 mg/kg/day group, 1 up was noted with yellow areas on the liver and 1 Pup was noted with a fractured tail. Dark red discoloration of the eyes was noted for 1 pup each in the control and 20 mg/kg/day groups; 11 pup was also noted with a small left eye. The aforementioned findings were noted insingle pups or litters, occurred infrequently or similarly in the control group, and/or did not occur in a dose-related manner, and therefore were not considered test substance-related.
- At the PND 21 necropsy of F1 weanlings selected for hormone analysis, no internal findings that could be attributed to parental administration of the test substance were noted at any dosage level. Internal findings were limited to dark red discoloration on the thyroid glands for a Male Pup in the 20 mg/kg/day group and an accessory spleen for a Female Pup in the 150 mg/kg/day group. Because these findings were limited to a single fetus and/or did not occur in a dose-related manner, they were not considered test substance-related.
- Thickened stomachs were observed in the 20, 50, and 150 mg/kg/day group F1 males and the 150 mg/kg/day group F1 females in Cohort 1A and were considered test substance-related. At the scheduled F1 male and female necropsies for Cohort 1B, thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females and were considered test substance-related; given the lack of systemic toxicity
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic changes were observed in the nonglandular stomach for F1 males and females in Cohort 1A at all dosage levels. Hyperkeratosis was observed in all test substance-administered groups while epithelial hyperplasia was observed in the 50 and 150 mg/kg/day groups. The findings were not associated with clinical pathology changes but were of similar, although slightly less, severity when compared to the F0 generation and were considered adverse in the 150 mg/kg/day group males and females.
There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.
Other effects:
no effects observed
Description (incidence and severity):
- No test substance-related effects on anogenital distance were noted for F1 pups in the 20, 50, and 150 mg/kg/day groups. Longer anogenital distances (absolute and relative to the cube root of pup body weight) were noted for F1 male pups in the test substance-treated groups compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). However, the aforementioned differe ces did not occur in a dose-related manner and the mean absolute values in the 20, 50, and 150 mg/kg/day groups (4.07 mm, 4.19 mm, and 4.17 mm, respectively) were within the range of values in the Charles River Ashland historical.
- Areolae/nipple anlagen in the F1 male pups was not affected by test substance administration. There was no retention of nipples noted in any male pup on study on PND 12.
- There were no test substance-related changes in total T4 or TSH in pups that were culled on PND 4.
- There were no test substance-related changes in serum T4 or TSH in F1 weanlings on PND 21.
- There were no test substance-related changes in serum T4 or TSH in F1 animals assigned to Cohort 1A.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: local effects (nonglandular stomach)
Critical effects observed:
no
Reproductive effects observed:
no
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 416.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Anogenital distance, a triggered end point as per test guidelines, was not measured in the F2 pups because there were no significant effects observed on F1 sex ratio or age at vaginal opening or preputial separation.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
See above
Qualifier:
according to
Guideline:
other: JMAFF, Guideline 2-1-17, Reproduction Study (2000)
Deviations:
yes
Remarks:
See above
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River Laboratories Inc. (Portage, Michigan)
Age: Approximately six weeks at initiation of treatment.

Physical and Acclimation
Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed two-three per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

Housing
After assignment to study, animals were housed singly in stainless steel cages, except during breeding (one male and one female) and during the littering phases of the study. During littering, dams (and their litters) were housed in plastic cages provided with ground corncob nesting material from approximately GD 19 until completion of lactation. Cages had wire mesh floors and were suspended above catch pans. Non-woven gauze were placed in the cages to provide a cushion from the flooring for rodent feet and also provided environmental enrichment. In order to better visualize copulation and plugs, gauze was not placed in cages during the breeding phase. Cages contained a feed crock and a pressure activated lixit valve-type watering system. Environmental conditions were maintained as follows:
Temperature: 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 12-15 times/hour
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water was provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or the water that would have impacted the results of this study. Copies of these analyses are maintained at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Animal Welfare
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study were DART 01 and Animal ID 01.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
Vapor Generating System
Chambers
The animals were exposed to filtered air or methyl acrylate vapors in 14.5 m3 inhalation exposure chambers under dynamic airflow conditions. Chamber airflow was maintained at approximately 2900 liters per minute, a rate that is sufficient to provide 12 air changes per hour and thus maintain normal concentrations of oxygen. The chambers were operated at a slight negative pressure relative to the surrounding area. All test chamber exhaust was passed through an activated charcoal bed to remove test material from the exhaust stream.

Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of methyl acrylate vapor was present throughout the breathing zone of the animals.

Generation System
The various concentrations of methyl acrylate were generated using the glass J-tube method (Miller et al., 1980). Liquid test material was pumped into the glass J-tube assemblies (1 per exposure chamber) and vaporized by the flow of nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated as needed with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. All chambers, including the 0 ppm (control) chamber received the same amount (20 liters per minute) of supplemental nitrogen (carrier gas). The minimum amount of nitrogen necessary to reach the desired chamber concentrations was used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas were mixed and diluted with supply air to achieve a total flow of approximately 2900 liters per minute at the desired test chamber concentration.

Exposure Environmental Conditions
Airflow through the chambers was determined with a manometer, which measures the pressure drop across a calibrated orifice plate, and was maintained at approximately 450 liters per minute. Chamber airflow data were collected using Setra Differential Pressure Transducers (Setra Systems, Inc., Boxborough, Massachusetts). The signal from the pressure transducer was sent to the CAMILE TG 4 Acquisition and Control System and recorded in liters per minute. The differential pressure transducer was calibrated with a gas meter (Singer Aluminum Diaphragm Meter, Model AL-2300, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. Chamber temperature and relative humidity data were collected using Omega HX94C Probes (Omega Engineering Inc., Stamford, Connecticut) coupled to the CAMILE TG 4 Data Acquisition and Control System. The chamber temperature and relative humidity were controlled by a system designed to maintain values of approximately 22 ± 2°C and 40 to 60%, respectively. Chamber temperature, relative humidity, and airflow data were manually recorded from the CAMILE TG 4 Data Acquisition and Control System once per hour.

Chamber Environmental Conditions
Chamber temperatures values for all chambers were maintained between 20.3 and 23.3°C. Chamber relative humidity was maintained in the range of 35.9 and 64.5% for all exposure chambers. Minor excursions of daily values from the protocol-specified relative humidity range (40-60%) for the chambers were noted. These minor excursions did not affect the integrity of the study. Chamber airflow in all four chambers was maintained throughout
the study duration, ensuring 12-15 calculated air changes per hour at approximately 2900 liters per minute of total airflow.
Details on mating procedure:
Breeding for the P1 and P2 adults commenced after approximately ten weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating occurred or two weeks elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then removed from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. If available, one rat/sex/litter was randomly selected for the P2 mating to produce the F2 generation. More than one weanling may have been selected from the litters, if necessary, to achieve 27 breeding pairs/dose level for the second generation. Cohabitation of P2 male and female littermates was avoided. In cases where a mating partner had died or was otherwise not available, the animal was paired with the next available partner. A second breeding of the first or second generation adults was not conducted.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations of methyl acrylate, measured approximately in the center of the breathing zone of the animals, were determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut) and reported by a strip chart recorder. The IR spectrophotometer was calibrated and a standard curve was compiled prior to and at the midpoint of the study, using air standards prepared by vaporizing measured volumes of methyl acrylate into Tedlar sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with the metered volumes of dry, compressed air. Chamber concentrations during the exposures were interpolated using the standard curve. The analytical system was checked prior to each exposure with a methyl acrylate standard gasbag of known concentration. The CAMILE TG 4 Data Acquisition and Control System toggled the IR spectrophotometer between the chambers for concentration sampling. The nominal concentration of the test material in each chamber was estimated based on the amount of test material used and the total airflow through the chamber. Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of vapor was present throughout the breathing zone of the animals.
Duration of treatment / exposure:
Six hours/day, seven days/week for approximately 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (four weeks) for each of two generations
Frequency of treatment:
Daily. Maternal rats were not exposed to methyl acrylate after GD 20 through LD 4 in order to allow for parturition and initiation of lactation.
Details on study schedule:
Groups of 27 male and 27 female Crl:CD(SD) (Sprague-Dawley derived) were exposed to targeted concentrations of 0, 5, 25, or 75 ppm methyl acrylate for six hours/day, seven days/week for approximately 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (four weeks) for each of two generations. Maternal rats were not exposed to methyl acrylate after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Females with no evidence of mating were exposed until termination. Maternal rats were exposed from LD 5-LD 28. During the lactation period, pups were not placed in the exposure chambers, but remained in nesting boxes separated from the dam for approximately six hours /day on PND 5-28. Due to the daily exposures of the dams and separation from the pups, weaning occurred on PND 28 to allow the pups extra time to grow prior to single housing. Weanlings selected for the second generation began exposure on PND 28. Weanlings that were not selected for the second generation were not placed in the exposure chambers on PND 28, but were necropsied on PND 29. A comprehensive evaluation of male and female reproductive systems was conducted, and included an evaluation of gonadal function, the estrous cycle, mating performance, conception, gestation, parturition and lactation, as well as survival, growth and development of the offspring. In-life observations, body weights, feed consumption and litter data were evaluated. In addition, a gross necropsy of the P1 and P2 adults was conducted with extensive histopathologic examination of reproductive organs and target tissues. The material administration began on April 18, 2008 and was continued up until the day prior to necropsy. The F1weanlings were necropsied from August 18-31, 2008. The P1 adults were necropsied from August 11-14, 2008 (males) and September 3-4, 2008 (females). The F2 weanlings were necropsied from December 29, 2008 to January 11, 2009. The P2 adults were necropsied from January 5-8, 2009 (males) and January 12-13, 2009 (females).
Remarks:
Doses / Concentrations:
0, 5, 25, and 75 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 5.3 ± 0.2, 24.9 ± 0.4, and 73.4 ± 1.8 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm (corresponding to approx. 0, 0.019, 0.092, and 0.269 mg/L)
Basis:
analytical conc.
Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (86.09 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG, 2005]
No. of animals per sex per dose:
27/sex/dose
Control animals:
yes, sham-exposed
Details on study design:
In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.
Positive control:
None
Parental animals: Observations and examinations:
Daily Observations
A cage-side examination was conducted twice daily, approximately the same time eachday. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could be observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Any animals found dead were necropsied as soon as practical. In addition, all animals and litters if available were
observed for morbidity, mortality, and the availability of feed and water at least twice daily. Moribund animals not expected to survive until the next observation period were humanely euthanized that day. In addition, females were observed for signs of parturition beginning on or about GD 20.

Clinical Observations
Clinical observations were performed immediately after exposure, with the exception of day 1, which was conducted pre-exposure. Clinical examinations were conducted weekly on all males throughout the study, and weekly on all females throughout the pre-breeding period. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14 and 21. Females were observed for signs of parturition beginning on or about GD 20. Dams unable to deliver or exhibiting signs of severe dystocia were humanely euthanized and necropsied. Females that delivered litters were subsequently evaluated on LD 0, 1, 4, 7, 14, 21 and 28. Clinical observations were not conducted on females that failed to mate or deliver a litter for the remainder of the study until the week prior to the scheduled necropsy, unless deemed appropriate based on cageside observations. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Body Weights/Body Weight Gains
All rats were weighed during the pre-exposure period and weekly during the 10-week prebreeding periods. Males continued to be weighed weekly after the pre-breeding period until termination. Mated females were weighed on GD 0, 7, 14, and 21. Lactating females were weighed on LD 1, 4, 7, 14, 21 and 28. Females that failed to mate and/or deliver a litter were weighed during the subsequent gestation and/or lactation segments of the study. Body weight gains of females were calculated for the following intervals in both generations: GD 0-7, 7-14, 14-21, 0-21 and LD 1-4, 4-7, 7-14, 14-21, 21-28 and 1-28.

Feed Consumption
Feed consumption was determined weekly during the 10-week pre-breeding period for all animals by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was measured weekly in males and dietary concentrations were adjusted accordingly. During gestation, feed consumption was measured at weekly intervals of sperm-/or plug- positive females on GD 0, 7, 14, and 21. Feed consumption was not recorded for non-confirmed mated females. During lactation, feed consumption was measured on LD 1, 4, 7, 11, 14, 17, 19, 21, 23, 26, and 28. Feed consumption was not measured in females that failed to mate or deliver a litter.
Oestrous cyclicity (parental animals):
Vaginal lavage samples were collected daily for all P1 and P2 females for three weeks prior to mating and during cohabitation until each female was sperm or plug positive or until the two-week mating period elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring loosely adherent vaginal cells to a slide with a pipette. Vaginal lavage slides were examined to determine estrous cycle length and pattern. Additionally, on the day of scheduled necropsy, the stage within the estrous cycle was determined for all P1 and P2 female rats.
Sperm parameters (parental animals):
Sperm parameters were evaluated in all P1 and P1 males at termination. Unless circumstances dictated otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis – counts; right testis – histopathology; left testis – counts.

Motility
Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing SpermPrep Medium (ZDL, Inc., Lexington, Kentucky) and was incubated at room temperature for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed in a chamber of the HTM Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. Images from the motility analyses were recorded on CD-R and archived with the study file. After sperm were released, the epididymis was placed in Bouin’s fixative and saved for histological examination.

Counts
The left testis and cauda epididymis were weighed and frozen at -20°C for subsequent determination of the number of homogenization-resistant spermatids and sperm per testis/cauda epididymis and per gram of testicular/epididymal tissue. The thawed testis or epididymis were minced, diluted and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and the spermatid or sperm count was determined from an aliquot loaded into the IVOS analyzer as described by Stradler et al. (1996). Because there were no treatment-related differences in testicular/epididymal sperm counts, only samples from the high-dose and control animals were evaluated.

Morphology
An aliquot of sperm suspension was placed on a slide, and a smear was prepared and then air dried for subsequent evaluation of sperm morphology. At least 200 sperm from each control and high-dose group male were evaluated and classified as normal or abnormal as described by Filler (1993). Sperm morphology was scored blind with respect to treatment group.
Litter observations:
Litter Observations
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. The following information was recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, 21 and 28, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, 21 and 28. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. In addition, pup clinical observations were recorded on each litter on PND 0, 1, 4, 7, 14, 21 and 28. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly if possible for external and visceral defects. These pups were preserved in neutral, phosphate-buffered 10% formalin.

Culling
To minimize variation in pup growth due to differences in litter size, F1 and F2 litters were standardized to eight/litter on PND 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated selection procedure, so that four males and four females (whenever possible) remained in each litter. Litters with eight or fewer pups were not culled. Culled pups were euthanized by administration of Socumb euthanasia solution (Veterinary Laboratories, Inc., Lenexa, Kansas) into the buccal cavity, and then discarded.
Postmortem examinations (parental animals):
Necropsy
Adult males (fasted) were submitted for necropsy after completion of their respective mating period when it was deemed that they were no longer needed for assessment of reproductive effects. Adult females (fasted) were terminated after weaning of their litters, or at least 24 days after the end of the mating period for females not producing a litter. On the morning of the scheduled necropsy, all surviving P1 and P2 males and females were weighed. Vaginal lavage smears were prepared from all surviving P1 and P2 females for later determination of estrous cycle stage. The animals were anesthetized by the inhalation of CO2, the tracheas were exposed and clamped, and the animals were euthanized by decapitation.

A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.

The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately two minutes and were examined for the presence and number of implantation sites. After evaluation, uteri was gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.

Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, adrenal glands, spleen, thyroid with parathyroids (weighed after fixation) were recorded, and the organ-to-body weight ratios calculated. In addition, weights of the left testis and left cauda epididymis were collected for use in calculating sperm count parameters.

Representative samples of tissues listed in Table 3 were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the right testis, right epididymis, and ovaries (P2 females only) was preserved in Bouin’s or another appropriate fixative. Transponders were removed and placed in jars with the tissues. During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for these animals except that terminal body and organ weights were not recorded and the testes, epididymides and ovaries were preserved in neutral, phosphate-buffered 10% formalin.

Histopathology
Histologic examination of the tissues was conducted on all control and high-dose adult rats, and on the reproductive organs of any study rats with
signs of reduced fertility. The thyroid glands of all P2 adult male and female control and high-dose rats were examined microscopically because the absolute thyroid gland weights of P2 high-dose males and females were statistically identified as lower than controls. Examination of additional tissues from the low- and mid-dose groups was limited to the nasal tissues/pharynx and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.

Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Examination of the ovaries included enumeration of primordial follicles using a method similar to Bucci et al. (1997). From among the surviving post-lactational P2 females in the control and high-dose groups, 15 per group were randomly selected for this examination.

Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to
significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue as adversely affected, but not to the point of organ failure. The health status of the animal may or may not be affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A grade of severe was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may be life threatening.

A complete set of tissues was examined from rats found dead, moribund, or euthanized due to accidental trauma. Histological examination was
conducted in a similar manner as described above, except that the testes were stained with hematoxylin and eosin.
Postmortem examinations (offspring):
Necropsy
Three pups/sex/litter from the F1 and F2 litters randomly selected at the time of weaning were submitted on PND 29 for a complete necropsy by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. Pups were anesthetized with CO2, weighed and euthanized by decapitation. Gross pathological examination was performed as described above for adults, except that the weanlings were not fasted overnight. Representative sample of grossly abnormal tissues and any known target organs were collected from all weanlings at the scheduled necropsy. In addition, one of the three pups/sex/litter was randomly selected from those examined grossly for the collection of brain, spleen, uterus, and thymus weights. Organ-to-body weight ratios were calculated. The brain, spleen, thymus, gross lesions and known target organs were preserved in neutral, phosphate-buffered 10% formalin. Dead or moribund pups were examined in a similar manner for possible defects and/or cause of death and were preserved in neutral, phosphate-buffered 10% formalin.
Statistics:
See below "Any other information on materials and methods incl. tables".
Reproductive indices:
Reproductive indices were calculated for all dose level groups as follows:
• Female mating index = (No. females with evidence of mating/No. paired) x 100
• Male mating index = (No. males with evidence of mating/No. paired) x 100
• Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
• Male conception index = (No. males siring a litter/No. mated) x 100
• Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
• Male fertility index = (No. males siring a litter/No. paired) x 100
• Gestation index = (No. females delivering a viable litter/No. females with evidence of pregnancy) x 100
• Gestation survival index = percentage of delivered pups alive at birth
• Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
• Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100
• Day 7, 14, 21 or 28 pup survival index = (No. viable pups on day 7, 14, 21 or 28/No. live after culling) x 100
Offspring viability indices:
• Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100
• Day 7, 14, 21 or 28 pup survival index = (No. viable pups on day 7, 14, 21 or 28/No. live after culling) x 100
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:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Screening Test- Treatment-related clinical signs in the 150 ppm P1 males and females were limited to a transient sneezing/huffing sound noted each day immediately following the end of exposure, occurring from day 36. This sound was observed until termination of the P1 males. In females, this sound was no longer present when exposure was stopped from GD 21 – LD 4, but was observed again upon resumption of exposure (LD 5-28),
albeit at a lesser severity and incidence. P1 males and females exposed to 150 ppm had treatment-related decreases in body weights, body weight gains and feed consumption that were observed during the pre-breeding, gestation and lactation phases. Similar, but less severe effects on body weight and feed consumption were seen in males and females exposed to 75 ppm. There was a dose-dependent decrease in the terminal body weights of rats exposed to methyl acrylate.

There were no treatment-related effects on any reproductive parameters, organ weights, or gross pathology. Dose-related histopathologic effects were present in the nasal tissues of males and females at all exposure concentrations. Degeneration with regeneration of the olfactory epithelium (very slight to severe) occurred in males and females at 150 ppm and in males at 75 ppm. Regenerative hyperplasia of the olfactory epithelium accompanied the degenerative change in multifocal sites. A lesser degree of olfactory epithelial degeneration (very slight), without accompanying regenerative hyperplasia, was noted in females at 75 ppm, and in males and females at 25 ppm. Very slight or slight degeneration of the olfactory nerve was present in males and females at 150 ppm only. Very slight or slight chronic active inflammation accompanied the olfactory epithelial degeneration in males and females at 150 ppm, and in females at 75 ppm. Very slight necrosis of individual olfactory epithelial cells, and multifocal, very slight or slight hyperlasia of the transitional epithelium that covers the nasal turbinates was present in rats exposed to 25, 75, or 150 ppm. There was a slight decrease in the PND 14 body weight of pups whose dams were exposed to 150 ppm methyl acrylate. No clinical signs were observed in the F1 males or females that were exposed from PND 28-35. F1 males and females exposed to 150 ppm had treatment-related decreases in body weights and feed consumption. Similar, but less severe effects on body weight and feed consumption were seen in F1 males and females exposed to 75 ppm.

Percent Difference in Terminal Body Weight Compared to Control
25 ppm 75 ppm 150 ppm
P1 Males -1% -7% -13%
P1 Females -1% -2% -12%
F1 Males +2% -4% -18%
F1 Females -1% -9% -17%

Chamber Concentration
Mean chamber concentration values during the study were 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm. Actual mean chamber concentration values deviated 0.5-6% from the targeted values of 0, 5, 25, and 75 ppm.

In-Life Observations
Examinations performed on all animals prior to the study start revealed that all animals were in good health for study purposes.

No treatment-related effects on behavior or demeanor were observed in any phase of the study at any dose level. A number of incidental observations bearing no relation to treatment were observed.

Feed Consumption
There was a treatment-related decrease in feed consumption of the P1 males in the 75 ppm exposure group when compared to controls, although these differences only reached statistical significance for three measurement intervals (TD 1-7, 7-14, and 56-63) throughout the first generation. During the 10-week premating period, there was a treatment-related decrease in feed consumption of the P1 females in the 75 ppm exposure group when compared to controls, and these differences reached statistical significance for most measurement intervals. Feed consumption of the 75 ppm females was also decreased throughout gestation (≤ 12%) when compared to controls. During lactation, feed consumption of the 75 ppm females was slightly decreased when compared to controls, although these differences only reached statistical significance for three measurement intervals (LD 14-17, 17-19, and 23-26). There were no treatment-related or statistical differences in feed consumption of P1 males and females exposed to 25 or 5 ppm methyl acrylate when compared to controls.

Selected P1 Male Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
TD 1-7 22.4 21.7 22.8 20.0*
TD 7-14 24.2 24.8 24.7 23.0*
TD 21-28 24.9 24.1 25.9 24.8
TD 42-49 26.8 26.1 26.1 25.7
TD 56-63 27.4 26.0 26.6 25.0*
TD 86-91 27.1 25.8 27.4 25.5
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P1 Female Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
Premating days 1-7 16.2 15.0 15.9 14.2$
Premating days 7-14 16.6 16.9 16.6 15.4*
Premating days 21-28 17.6 17.3 17.7 16.7
Premating days 42-49 18.8 18.0 17.8 17.3*
Premating days 56-63 18.0 17.2 17.2 13.3$
Premating days 63-70 18.1 17.4 17.3 18.8
GD 0-7 22.6 21.3 22.1 19.9*
GD 7-14 23.8 22.9 23.8 21.7*
GD 14-21 23.2 23.1 22.6 21.0*
LD 1-4 29.4 32.1 29.8 30.4
LD 7-11 40.6 41.8 41.2 38.1
LD 14-17 51.8 52.1 50.6 48.3$
LD 23-26 90.9 90.9 92.5 87.3$
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.
$ Statistically different from control mean by Wilcoxon’s test, alpha = 0.05.

There was a treatment-related decrease in feed consumption of the P2 males in the 75 ppm exposure group when compared to controls, and these differences reached statistical significance for the majority of measurement intervals throughout the second generation. During the 10-week premating period, there was a treatment-related decrease in feed consumption of the P2 females in the 75 ppm exposure group when compared to controls, and these differences also reached statistical significance for most measurement intervals. Feed consumption of the 75 ppm females was also decreased throughout gestation (≤ 10%) when compared to controls. During lactation, feed consumption of the 75 ppm females was slightly decreased when compared to controls, although these differences only reached statistical significance for two measurement intervals (LD 7-11 and 14-17). There were no treatment-related differences in feed consumption of P2 males and females exposed to 25 or 5 ppm methyl acrylate when compared to controls. The difference in feed consumption of 5 ppm males when compared to controls was statistically identified and decreased for two measurement intervals (TD 1-6 and 34-41), however, this was not considered to be a treatment-related effect due to the lack of both a dose-response relationship and temporal association.

Selected P2 Male Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
TD 1-6 24.3 22.6* 24.7 20.8*
TD 6-13 27.0 25.6 27.2 23.8*
TD 20-27 28.7 27.6 29.4 26.2*
TD 34-41 29.4 27.6* 29.5 26.9*
TD 41-48 28.7 27.6 29.9 27.0
TD 55-62 29.3 28.0 29.7 27.1*
TD 90-97 29.0 28.5 29.2 26.6*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P2 Female Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
Premating days 1-6 19.3 19.0 18.4 17.1$
Premating days 6-13 19.9 19.6 19.2 17.7*
Premating days 20-27 20.2 19.4 19.8 18.0*
Premating days 41-48 20.4 19.1 20.0 19.2
Premating days 55-62 18.6 19.1 18.9 18.8
Premating days 62-69 19.1 18.3 18.8 17.6*
GD 0-7 23.1 22.3 22.7 21.1*
GD 7-14 25.2 23.9 25.0 22.7*
GD 14-21 24.1 23.0 23.1 21.9*
LD 1-4 33.7 32.4 30.6 33.0
LD 7-11 46.0 44.7 43.7 41.9*
LD 14-17 55.3 53.7 53.0 51.1*
LD 23-26 95.0 93.2 92.5 91.9
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.
$ Statistically different from control mean by Wilcoxon’s test, alpha = 0.05.


Body Weights/Body Weight Gains
There was a treatment-related decrease in the body weight of P1 males of the 75 ppm exposure group when compared to controls, although these differences only reached statistical significance on TD 7 and 14 throughout the entire first generation. Similarly, there was a treatment-related decrease in the body weight of P1 females of the 75 ppm exposure group, which reached statistical significance on three days (TD 21, 63, and 70) of the 10-week premating period. There was a treatment-related decrease in the body weight of P1 females of the 75 ppm exposure group across the entire
gestation and lactation period. However, gestation body weight gain of the 75 ppm females remained comparable to controls. During lactation, the 75 ppm females did not lose as much body weight as controls, which could have been related to their lower body weight at the start of lactation. There were no treatment-related differences in body weight of P1 males and body weight/body weight gain of P1 females exposed to 25 or 5 ppm methyl acrylate when compared to controls. The difference in body weight of 5 ppm males (TD 7) and gestation body weight gain of 5 ppm females (GD 0-7) was statistically identified and decreased when compared to their respective controls. However, this was not considered to be a treatmentrelated effect due to the lack of a dose-response relationship and the low incidence.

Selected P1 Male Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 193.9 190.4 193.6 191.9
TD 7 237.8 228.0* 235.9 226.3*
TD 14 291.1 286.8 284.9 276.7*
TD 28 369.3 358.5 366.7 358.6
TD 70 512.4 492.5 499.2 487.3
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P1 Female Pre-Breeding Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 139.4 136.4 138.9 139.8
TD 7 158.7 153.8 159.0 152.5
TD 21 203.8 203.0 204.6 191.5*
TD 63 273.1 266.1 272.4 248.1*
TD 70 281.7 272.0 276.6 258.5*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05

Selected P1 Gestation/Lactation Body Weights/Body Weight Gains
Gestation Mean Body Weight (g)
ppm 0 5 25 75
GD 0 287.4 280.1 284.3 261.8*
GD 7 319.3 302.7 315.8 289.0*
GD 14 350.1 337.2 347.5 318.5*
GD 21 433.8 429.4 435.9 404.6*

Gestation Mean Body Weight Gains (g)
GD 0-21 146.4 149.3 151.6 142.9
Lactation Mean Body Weight (g)
LD 1 330.9 316.4 321.6 297.7*
LD 4 343.8 335.6 337.2 312.6*
LD 7 328.0 323.0 324.4 297.9*
LD 14 336.9 334.8 335.6 308.8*
LD 28 313.4 306.6 309.3 294.6*
Lactation Mean Body Weight Gains (g)
LD 1-28 -17.5 -9.8 -12.3 -3.1*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

There was a treatment-related decrease in the body weight of P2 males of the 75 ppm exposure group when compared to controls, which was statistically identified throughout the entire second generation. Similarly, there was a treatment-related decrease in the body weight of P2 females of the 75 ppm exposure group, which reached statistical significance on all measurement days during the 10-week premating period. There was a treatment-related decrease in the body weight of P2 females of the 75 ppm exposure group across the entire gestation and lactation periods. The gestation body weight gains of the 75 ppm females were significantly lower than control values, however, this difference was equivocal when all dose groups were considered. As in the P1 females, the net body weight loss typical of lactating female rats was less in the 75 ppm females than it was in controls. There were no treatment-related differences in body weight of P2 males and body weight/body weight gain of P2 females exposed to 25 or 5 ppm methyl acrylate when compared to controls. Statistical differences in the body weight/body weight gain of 25 or 5 ppm females were not considered to be treatment-related due to the lack of a doseresponse relationship and/or the low incidence.

Selected P2 Male Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 169.7 158.0 170.9 148.4*
TD 6 212.6 200.7 213.6 181.2*
TD 13 274.6 258.9 277.9 239.6*
TD 27 373.7 365.2 379.0 330.4*
TD 69 538.4 512.4 540.3 472.3*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P2 Female Pre-Breeding Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 143.6 135.6 135.6 125.1*
TD 6 166.4 160.0 156.4 144.3*
TD 13 194.3 187.0 183.1 169.9*
TD 27 238.9 229.4 228.3 206.4*
TD 69 299.5 288.1 293.2 264.1*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05
Selected P2 Gestation/Lactation Body Weights/Body Weight Gains
Gestation Mean Body Weight (g)
ppm 0 5 25 75
GD 0 298.7 292.5 290.2 266.5*
GD 7 334.0 319.7 321.7 294.7*
GD 14 367.1 350.4 353.3 324.4*
GD 21 459.3 437.6 440.5 412.4*
Gestation Mean Body Weight Gains (g)
GD 0-21 160.6 145.1* 150.3 145.9*
Lactation Mean Body Weight (g)
LD 1 345.1 328.3 337.0 303.8*
LD 4 365.4 345.6* 350.4 323.0*
LD 7 352.6 333.7 336.4 311.2*
LD 14 358.3 338.3* 341.1 321.2*
LD 28 322.5 310.0 314.4 298.0*
Lactation Mean Body Weight Gains (g)
LD 1-28 -22.6 -18.2 -22.5 -6.1*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Organ Weights
P1 males and females exposed to 75 ppm had treatment-related lower final body weights (statistically identified in females at 75 ppm). The final body
weights of P1 males and females exposed to 75 ppm were 5.0% and 7.7% lower than controls, respectively. The relative weights of the testes and epididymides of males exposed to 75 ppm, and the relative weights of the liver and brain of females exposed to 75 ppm were statistically identified as higher than controls. The elevated relative organ weights of males and females exposed to 75 ppm were interpreted to be reflective of the lower body weights of these animals as the absolute weights of the organs were not different from the controls. The relative testes weight of males exposed to 5 ppm, and the relative liver weight of females exposed to 5 ppm were statistically identified as higher than controls. These organ weight alterations wereinterpreted to be unrelated to treatment due to the lack of a dose response. The absolute pituitary weights of males exposed to 5 or 25 ppm, and females exposed to 5, 25, or 75 ppm were statistically identified as lower than controls. In addition, the relative pituitary weight of males exposed to 25 ppm was statistically identified as lower than controls. The alterations in pituitary weights were interpreted to be unrelated to treatment because of the a lack of a clear dose response, the absence of any histopathologic correlates in males and females from the high-dose group, and because all of the pituitary weights were within historical controls ranges of studies recently conducted at this laboratory.

Organ Weight Data – P1 Adults
Concentration (ppm)
0 Historical1 5 25 75
Parameter MALES
Final Body Weight (g) 554.0 520.1-581.8 540.9 549.8 526.1a
Relative Testes (g/100g bw) 0.635 0.608-0.695 0.698* 0.673 0.709*
Relative Epididymides (g/100g bw) 0.248 0.238-0.277 0.264 0.261 0.277*
Absolute Pituitary (g) 0.0146 0.011-0.015 0.0133* 0.0130* 0.0138
Relative Pituitary (g/100g bw) 0.0026 0.002-0.002 0.0025 0.0024* 0.0026
Parameter FEMALES
Final Body Weight (g) 314.2 278.0-330.4 300.8 311.5 290.1*a
Relative Liver (g/100g bw) 2.878 2.994-3.491 3.103* 3.008 3.105*
Relative Brain (g/100g bw) 0.658 0.651-0.692 0.685 0.658 0.699$
Absolute Pituitary (g) 0.0200 0.012-0.018 0.0175* 0.0179* 0.0171*
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.
$Statistically different from control mean by Wilcoxon’s Test, alpha =0.05.
1Historical controls group mean range from seven dietary studies reported between 2002 and 2006.
a- Values interpreted to be treatment-related effects.

P2 males and females exposed to 75 ppm had statistically identified treatment-related lower final body weights. The final body weights of P2 males and females exposed to 75 ppm were 13.1% and 9.6% lower than controls, respectively. The relative weights of the brain, testes, seminal vesicles (with coagulating glands) and epididymides of males exposed to 75 ppm, and the relative weights of the adrenals and brain of females exposed to 75 ppm were statistically identified as higher than controls. The absolute weights of the adrenals, kidneys, spleen, pituitary gland, and thyroid gland of males exposed to 75 ppm, and the absolute weights of the kidneys, spleen and thyroid gland of females exposed to 75 ppm were statistically identified as lower than controls. The organ weight alterations of males and females exposed to 75 ppm were interpreted to be reflective of the lower body weights of these animals. Males exposed to 25 ppm had statistically identified lower absolute and relative pituitary weights, and females exposed to 25 ppm had a statistically identified lower absolute thyroid weight. Males exposed to 5 ppm had a statistically identified higher relative testes weight, and females exposed to 5 ppm had statistically identified higher absolute and relative adrenal weights. The organ weight alterations from the 5 and 25 ppm dose groups were interpreted to be unrelated to treatment due to the lack of a clear dose response and/or the values were within historical controls
ranges of studies recently conducted at this laboratory.

Organ Weight Data – P2 Adults
Concentration (ppm)
0 Historical1 5 25 75
Parameter MALES
Final Body Weight (g) 624.4 606.9-674.8 599.9 627.6 542.9*a
Absolute Adrenal Glands (g) 0.055 0.062-0.073 0.054 0.056 0.048*
Absolute Kidneys (g) 4.058 3.924-4.350 4.010 4.002 3.671*
Relative Brain (g/100g bw 0.346 0.333-0.386 0.363 0.344 0.386*
Absolute Spleen (g) 0.895 0.845-0.963 0.905 0.867 0.783*
Relative Testes (g/100g bw) 0.582 0.571-0.641 0.636* 0.593 0.662*
Relative Seminal Vesicle (g/100g bw) 0.319 0.266-0.319 0.322 0.312 0.367*
Relative Epididymides (g/100g bw) 0.223 0.224-0.248 0.238 0.229 0.255*
Absolute Pituitary (g) 0.0149 0.009-0.017 0.0141 0.0137* 0.0134*
Relative Pituitary (g/100g bw) 0.0024 0.002-0.003 0.0024 0.0022* 0.0025
Absolute Thyroid Gland (g) 0.0253 0.0224-0.0284 0.0255 0.0269 0.0224*
Parameter FEMALES
Final Body Weight (g) 326.3 296.3-347.2 313.7 318.4 295.0*a
Absolute Adrenal Glands (g) 0.062 0.070-0.111 0.068* 0.064 0.067
Relative Adrenal Glands (g/100g bw) 0.019 0.023-0.035 0.022* 0.020 0.023*
Absolute Kidneys (g) 2.274 2.187-2.424 2.259 2.149 2.108*
Relative Brain (g/100g bw) 0.620 0.588-0.703 0.644 0.628 0.669*
Absolute Spleen (g) 0.585 0.593-0.620 0.543 0.562 0.513*
Absolute Thyroid Gland (g) 0.0203 0.0166-0.0202 0.0182 0.0180* 0.0180*
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.
1Historical controls group mean range from seven dietary studies reported between 2002 and 2006.
a-Values interpreted to be treatment-related effects.
Gross Pathology
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be incidental findings, unassociated with exposure to methyl acrylate.

Histopathology
Treatment-related histopathologic effects were present in the nasal tissues of P1 and P2 males and females given 25 or 75 ppm. The incidence and severity of the nasal effects were dose-related. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. The degeneration consisted of thinning and disarray of the olfactory epithelial cells, which was most prevalent in the anterior and dorsal aspects of the nasal passages. Regenerative hyperplasia of the olfactory epithelium accompanied the degenerative change in multifocal sites. A lesser degree of multifocal olfactory epithelial degeneration (very slight), without accompanying regenerative hyperplasia, was noted in 7/27 P1 females exposed to 25 ppm, and in 6/27 P2 males and 8/27 P2 females exposed to 25 ppm. One P1 female and one P2 female exposed to 5 ppm also had very slight multifocal olfactory epithelial degeneration. However, the degeneration was located in only two sites of the nose for both of these animals, and therefore was interpreted to be comparable with spontaneous focal olfactory epithelial degeneration that was noted in 2/27 control group P1 females and 3/27 control group P2 females, and not an effect of treatment.

There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. This effect was characterized by thinning of the axons and reduction in the diameter of the olfactory nerve fascicles in areas of olfactory epithelial degeneration. Very slight or slight multifocal chronic-active inflammation accompanied the olfactory epithelial degeneration in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or two males and females from both generations exposed to 25 ppm. The inflammation
consisted of neutrophils in the olfactory epithelium, with or without the presence of a mucopurulent exudate. Very slight multifocal necrosis of individual olfactory epithelial cells, with or without exfoliation of necrotic cells into the lumen of the nasal passages, was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. One P1 female exposed to 5 ppm also had very slight multifocal necrosis of individual olfactory epithelial cells. However, the necrosis was located in only two sites of the nose in this animal, and therefore was interpreted to be comparable with spontaneous focal olfactory epithelial cell necrosis that was noted in one control group P1 male, one control group P1 female, and one control group P2 female, and not an effect of treatment.

A treatment-related increase in the incidence of very slight or slight multifocal hyperplasia of the transitional epithelium that covers the nasal turbinates was present in P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of transitional epithelial hyperplasia in P1 and P2 males and females exposed to 5 ppm was comparable to controls.

A treatment-related increase in the incidence of very slight or slight diffuse hyperplasia and hypertrophy of the respiratory epithelium that covers the nasal septum and dorsal portion of the anterior nasal cavity was present in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm. The incidence and severity of respiratory epithelial hyperplasia and hypertrophy in P1 and P2 males and females exposed to 5 ppm was comparable to controls.

Treatment-related very slight focal or multifocal mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. The mineralization was present in areas olfactory epithelial degeneration. One P1 female exposed to 75 ppm also had slight multifocal mineralization of the nasal respiratory epithelium that was interpreted to be treatment related. Other treatmentrelated nasal effects consisted of very slight multifocal squamous metaplasia of the transitional epithelium in 5/27 P1 males exposed to 75 ppm, and ulceration of the olfactory epithelium in four P1 males, one P1 female, and one P2 female exposed to
75 ppm.

All other histopathologic observations were considered to be spontaneous alterations, or caused by accidental trauma, unassociated with inhalation exposure of methyl acrylate. There were no histopathologic systemic effects in P1 or P2 rats at any exposure level. The NOEC for histopathologic nasal effects was 5 ppm.

Histopathologic Nasal Tissue Effects – P1 Males
Dose (ppm) 0 5 25 75
NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)
Degeneration, olfactory nerve, multifocal -very slight 0 0 1a 11a
-slight 0 0 0 14a
Degeneration with Regeneration, olfactory epithelium, multifocal
-slight 0 0 1a 12a
-moderate 0 0 0 15a
Hyperplasia, transitional epithelium; multifocal -very slight 3 4 17a 12a
-slight 0 0 0 15a
Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse
-very slight 1 0 4a 1
-slight 0 0 1a 22a
Inflammation, chronic active, olfactory epithelium, multifocal
-very slight 1 0 2 15a
-slight 0 0 0 1a
Metaplasia, squamous, transitional epithelium, multifocal -very slight 0 0 0 5a
Mineralization, olfactory epithelium, focal -very slight 0 0 0 1a
Mineralization, olfactory epithelium, multifocal -very slight 0 0 1a 5a
Necrosis, individual cell, olfactory epithelium, focal -very slight 1 0 1 0
Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 0 1a 26a
Ulcer, olfactory epithelium, focal -very slight 0 0 0 4a
a- Indicates the effects judged to be treatment-related.

Results Continued in Remarks Section (below)
Dose descriptor:
NOEC
Remarks:
developmental toxicity
Effect level:
ca. 0.092 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 25 ppm; based on decreases in pup weight at 75 ppm which were secondary to parental toxicity.
Remarks on result:
other: Generation: F1, F2 (migrated information)
Dose descriptor:
NOEC
Remarks:
reproductive toxicity
Effect level:
ca. 0.269 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 75 ppm; the highest concentration tested.
Remarks on result:
other: Generation: P1 and P2 (migrated information)
Dose descriptor:
NOEC
Remarks:
parental local toxicity
Effect level:
ca. 0.019 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 5 ppm; based on histological changes in nasal tissues.
Remarks on result:
other: Generation: P1 and P2 (migrated information)
In-Life Observations

No treatment-related effects on behavior or demeanor were observed in any phase of the study at any dose level. A number of incidental observations bearing no relation to treatment were observed.

Observations made on F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings, which included a small number of observations in the control, low-, middle-, and high-dose groups, were seen with no evidence of a dose-response relationship. Included among these incidental findings was a single high dose pup which exhibited a head tilt and circling behavior associated with overgrown incisors. This pup was euthanized on lactation day 25.

Reproductive Indices, Pup Survival and Sex Ratio
There were no effects of treatment at any exposure level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating, gestation length, pup survival or pup sex ratio in either generation.

Litter Size
There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size at any time interval in any exposure group for either generation.

Pup Body Weight
F1 pup body weights from all exposure groups were comparable to control until PND 14. Male and female pups from the 75 ppm exposure group had decreased body weights that were statistically identified when compared to control on PNDs 14, 21, and 28. There were no treatment-related findings for F1 pup body weights from the 25 or 5 ppm exposure groups when compared to control. On PND 7, there was a statistically identified decrease in pup body weights of F1 females from the 5 ppm exposure group. This was considered spurious and unrelated to treatment due to the lack of a dose response relationship and because it was not repeated in the next generation.

Treatment-related effects on the body weights of F2 pups were similar to what was seen in the previous generation. F2 pup body weights from all exposure groups were comparable to control until PND 14. Male and female pups from the 75 ppm exposure group had decreased body weights that were statistically identified when compared to control on PNDs 14, 21, and 28. There were no treatment-related or statistical findings for F1 pup body weights from the 25 or 5 ppm exposure groups when compared to control.

These findings are likely secondary to maternal toxicity in the form of decreased maternal body weights of ~10% and severe nasal irritation. This conclusion is supported by a feed restriction study where a 10-20% decrease in maternal body weight can lead to decreased pup weights by as much as 21% (Carney, et al., 2004).

Selected F1 Pup Body Weights (g)
0 ppm 5 ppm 25 ppm 75 ppm
PND 14 Males 27.0 26.7 26.3 24.3*
Percent from Control NA -1% -3% -10%
PND 14 Females 26.5 25.8 25.6 23.7*
Percent from Control NA -3% -3% -11%
PND 21 Males 44.2 42.3 42.6 39.5*
Percent from Control NA -4% -4% -11%
PND 21 Females 43.8 41.1 41.4 39.3*
Percent from Control NA -6% -6% -10%
PND 28 Males 83.1 82.2 82.0 77.3*
Percent from Control NA -1% -1% -7%
PND 28 Females 79.4 76.6 76.6 73.1*
Percent from Control NA -4% -4% -8%
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.

Selected F2 Pup Body Weights (g)
0 ppm 5 ppm 25 ppm 75 ppm
PND 14 Males 30.3 29.8 29.6 27.4*
Percent from Control NA -2% -2% -10%
PND 14 Females 29.7 28.7 29.0 26.7*
Percent from Control NA -3% -2% -10%
PND 21 Males 49.0 48.7 47.5 44.1*
Percent from Control NA -1% -3% -10%
PND 21 Females 48.3 46.5 46.8 42.9*
Percent from Control NA -4% -3% -11%
PND 28 Males 89.7 89.7 87.5 83.4*
Percent from Control NA 0% -2% -7%
PND 28 Females 84.2 82.3 82.0 77.8*
Percent from Control NA -2% -3% -8%
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.

Puberty Onset
Age at vaginal opening and age at preputial separation were similar in all exposure groups, indicating no effect of treatment on these end points despite the lower body weight of the 75 ppm animals.

Organ Weights
The final body weights of F1 weanling males and females from the 75 ppm group were approximately 6% lower than controls, and although not statistically identified, were considered treatment-related due to the immediately preceding decrease in pup body weights from PND 14-28. There were no
treatment-related alterations in organ weights of F1 weanlings at any dose level.

Final Body Weight Data – F1 Weanlings
Concentration (ppm)
0 5 25 75
Parameter MALES
Final Body Weight (g) 88.1 86.7 87.2 82.6a
Parameter FEMALES
Final Body Weight (g) 81.9 77.8 80.6 76.7a
a- Values interpreted to be treatment-related effects.

F2 weanling males and females from the 75 ppm group had treatment-related lower final body weights (statistically identified in females at 75 ppm). The final body weights of F2 weanling males and females from the 75 ppm group were 5.8% and 7.7% lower than controls, respectively. As in the 75 ppm group F1 pups, these decreases in final body weights (PND 29) were reflective of the statistically significant decreases in pup body weights during the preceding two weeks. There were no treatment-related alterations in organ weights of F2 weanlings at any dose level. The only statistically identified organ weight alteration was a higher absolute brain weight in F2 male weanlings from the 5 ppm exposure group, which was unrelated to treatment due to the lack of a dose response.

Final Body Weight Data – F2 Weanlings
Concentration (ppm)
0 5 25 75
Parameter MALES
Final Body Weight (g) 94.6 93.0 94.6 89.1a
Parameter FEMALES
Final Body Weight (g) 87.2 84.7 84.5 80.4*a
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.
a- Values interpreted to be treatment-related effects.

Gross Pathology
There were no treatment-related gross pathologic observations in F1 weanlings at any exposure level. In F2 weanlings, 2/81 males and 3/78 females from the 75 ppm exposure group had necrosis of the tail. This observation may have been related to treatment, but the significance of the tail necrosis is not known. All other gross pathologic observations from F1 and F2 weanlings were considered to be spontaneous alterations, unassociated with exposure to methyl acrylate.
Reproductive effects observed:
not specified

Results- Continued

Histopathologic Nasal Tissue Effects

P1 Females

Dose (ppm) 0 5 25 75

NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)

Degeneration, olfactory epithelium, focal -very slight 2 1 4 0

Degeneration, olfactory epithelium, multifocal -very slight 0 1 7a 0

Degeneration, olfactory nerve, multifocal -very slight 0 0 0 8a

                                                                -slight 0 0 0 19a

Degeneration with Regeneration, olfactory epithelium, multifocal-slight 0 0 0 8a

                                                                                                     -moderate 0 0 0 19a

Hyperplasia, transitional epithelium; multifocal -very slight 2 1 23a 24a

                                                                         -slight 0 0 2a 3a

Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse-very slight 0 1 8a 10a

                                                                                                                -slight 0 0 13a 1a

Inflammation, chronic active, olfactory epithelium, multifocal-very slight 0 0 1 20a

Inflammation, chronic active, respiratory epithelium, multifocal-very slight 0 0 1 5a

Mineralization, olfactory epithelium, focal -very slight 0 0 0 2a

Mineralization, olfactory epithelium, multifocal -very slight 0 0 0 2a

Mineralization, respiratory epithelium, focal -slight 0 0 0 1a

Necrosis, individual cell, olfactory epithelium, focal -very slight 1 0 3 0

Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 1 2a 26a

Ulcer, olfactory epithelium, focal -very slight 0 0 0 1a

a-Indicates the effects judged to be treatment-related.

 

Histopathologic Nasal Tissue Effects

P2 Males

Dose (ppm) 0 5 25 75

NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)

Degeneration, olfactory epithelium, focal -very slight 0 0 1 0

Degeneration, olfactory epithelium, multifocal, -very slight 0 0 6a 0

Degeneration, olfactory nerve, multifocal -very slight 0 0 0 14a

                                                                -slight 0 0 0 13a

Degeneration with Regeneration, olfactory epithelium, multifocal-slight 0 0 0 13a

                                                                                                    -moderate 0 0 0 14a

Hyperplasia, transitional epithelium; multifocal -very slight 4 4 18a 8a

                                                                         -slight 0 0 2a 19a

Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse-very slight 0 0 0 6a

                                                                                                                -slight 0 0 0 3a

Inflammation, chronic active, olfactory epithelium, multifocal-very slight 0 0 0 14a

Mineralization, olfactory epithelium, focal -very slight 0 0 1a 1a

Mineralization, olfactory epithelium, multifocal -very slight 0 0 1a 15a

Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 0 1a 24a

Ulcer, olfactory epithelium, focal -very slight 1 0 0 1a

a-Indicates the effects judged to be treatment-related.

Histopathologic Nasal Tissue Effects

P2 Females

Dose (ppm) 0 5 25 75

NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)

Degeneration, olfactory epithelium, focal -very slight 3 2 4 0

Degeneration, olfactory epithelium, multifocal -very slight 0 1 8a 0

Degeneration, olfactory nerve, multifocal -very slight 0 0 0 14a

                                                                -slight 0 0 0 12a

Degeneration with Regeneration, olfactory epithelium, multifocal-very slight 0 0 0 1a

                                                                                                     -slight 0 0 0 14a

                                                                                                     -moderate 0 0 0 12a

Hyperplasia, transitional epithelium; multifocal -very slight 9 6 23a 25a

                                                                        -slight 1 0 0 1a

Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse-very slight 7 8 11 16a

                                                                                                                 -slight 3 1 5 2

Inflammation, chronic active, olfactory epithelium, multifocal-very slight 0 0 1a 8a

Mineralization, olfactory epithelium, multifocal -very slight 0 0 1a 14a

Necrosis, individual cell, olfactory epithelium, focal -very slight 1 0 1 0

Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 0 4a 27a

a-Indicates the effects judged to be treatment-related.

---

One P1 male exposed to 75 ppm (animal number 08A1350) died on test day 70. The cause of death was not determined. One P1 male exposed to 75 ppm (animal umber 08A1351) was euthanized moribund on test day 106. The cause of moribundity was urolithiasis, with associated inflammation and transitional cell hyperplasia of the urinary bladder and kidneys. One P1 male from the control group (animal number 08A1269) was euthanized moribund on test day 98. The cause of moribundity was lymphoid cell leukemia. Another P1 male from the control (animal number 08A1250) was euthanized on test day 113 due to accidental fracture of the upper jaw. One P2 female exposed to 25 ppm (animal number 08A5190) was euthanized on test day 57 due to severe inflammation of the hind feet. One P2 male exposed to 5 ppm (animal number 08A5040) was euthanized moribund on test day 87. The cause of moribundity was severe inflammation of the periodontal tissue associated with fracture of the upper incisors. One P2 female from the control group (animal number 08A5134) was euthanized on test day 70 due to accidental fracture of the nose. Histologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects of treatment. There were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females exposed to 75 ppm as compared to females from the control group.

 

Sperm Parameters

There were no treatment-related effects of methyl acrylate on any sperm analysis parameter at any exposure level in either generation. There was a statistically identified increase in epididymal and testicular sperm counts of P1 males of the 75 ppm exposure group when compared to controls, which was due to two males in the control group (1258 and 1263) with very low sperm counts.

 

Estrous Cyclicity

There was no evidence of an effect on estrous cyclicity at any dose level of methyl acrylate in either generation.

 

Conclusions:
The no-observed-effect concentration (NOEC) for parental systemic toxicity was determined to be 5 ppm and was based on histologic changes in the nasal tissues seen at higher concentrations. The NOEC for developmental toxicity was 25 ppm, based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOEC for reproductive toxicity was 75 ppm, the highest concentration tested.
Executive summary:

The purpose of this two-generation inhalation reproduction toxicity study was to evaluate the potential effects of methyl acrylate on male and female reproductive function, as well as the survival, growth and development of the offspring. Groups of 27 male and 27 female Crl:CD(SD) rats were whole-body exposed to target concentrations of 0, 5, 25, or 75 ppm vaporized methyl acrylate for six hours/day, seven days/week, resulting in actual average concentrations of 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm, respectively. Rats were exposed daily for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. Maternal rats were not exposed after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Exposure of maternal rats continued from LD 5 – LD 28. In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Treatment-related effects in parental rats exposed to 75 ppm included decreased body weight and feed consumption in males and females throughout most of the two generation study. There were no effects on body weight or feed consumption at 25 or 5 ppm.

Treatment-related, adverse histopathologic effects were present in the nasal tissues of P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of the nasal effects were concentration dependent. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. Very slight olfactory epithelial degeneration, without accompanying regenerative hyperplasia, was noted in some of the P1 and P2 females and P2 males exposed to 25 ppm. There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. Very slight or slight chronic-active inflammation was present in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or

two males and females from both generations exposed to 25 ppm. Very slight necrosis of individual olfactory epithelial cells was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. Very slight mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, and in 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. Other nasal effects consisted of an increase in the incidence of very slight or slight hyperplasia of the transitional epithelium in P1 and P2 males and females exposed to 25 or 75 ppm, an increase in the incidence of very slight or slight hyperplasia and hypertrophy of the respiratory epithelium in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm, very slight squamous metaplasia of the transitional epithelium in 5/27 P1 males exposed to 75 ppm, and

ulceration of the olfactory epithelium in four P1 males, one P1 female, and one P2 female exposed to 75 ppm. There were no treatment-related histopathologic effects in P1 or P2 animals exposed to 5 ppm.

No treatment-related effects were seen in reproductive function or pup survival. However, pup body weights of the 75 ppm exposure group were decreased on PND 14-28 in both generations. There were no effects on pup body weight in rats exposed to 25 or 5 ppm. The effects on pup body weight, as well as the changes in parental body weight and feed consumption, likely were secondary changes all stemming from nasal irritation and resultant stress.

In summary, the no-observed-effect concentration (NOEC) for parental systemic toxicity was determined to be 5 ppm and was based on histologic changes in the nasal tissues seen at higher concentrations. The NOEC for developmental toxicity was 25 ppm, based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOEC for reproductive toxicity was 75 ppm, the highest concentration tested.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Study according to OECD TG 443, GLP
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
286 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guideline study performed under GLP conditions. No effects on reproduction parameter.
This is supported by the two generation study with the structural analog methyl acrylate, which led to no effects on reproductive performance including fertility after whole body inhalative exposure. The NOAEC for reproductive function was 75 ppm (= ca. 269 mg/m³), the highest concentration investigated.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

For n-butyl acrylate an extended one generation study according to OECD 443 and GLP was performed. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (Acrylate Reach TF, 2017).

There were no test substance-related effects on survival for F0 and F1 animals at any dosage level. No test substance-related clinical observations were noted for F0 and F1 animals at any dosage level. Mean body weights, body weight gains, food consumption, and food efficiency in the 20, 50, and 150 mg/kg/day F0 and F1 males and females were unaffected by test substance administration. No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cyclicity or spermatogenic parameters (testicular and epididymal sperm concentrations, sperm production rate, sperm motility, and sperm morphology) at any dosage level.

There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance,

offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation).

No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) were noted in F0 and F1 males or females or F1 pups (on PND 4 and 21). Test substance-related histologic changes were observed in all dosage groups in the F0 generation and F1 males and females in Cohort 1A. Epithelial hyperplasia and/or hyperkeratosis was observed in the nonglandular stomach in all test substance-treated groups examined. Mild to moderate changes in the 150 mg/kg/day group males and females of the F0 and F1 generations were considered adverse in this study. Microscopic changes in the stomach were associated with the gross observation of thickened nonglandular stomach, but were not associated with any clinical pathology, organ, or body weight changes. In the F0 generation, a nonadverse increased incidence of biliary hyperplasia (males and females) and random hepatocellular necrosis (males) were observed in the liver in the 150 mg/kg/day group. Additionally, nonadverse test substance-related microscopic findings (increased severity of mineralization at the

corticomedullary junction) were observed in the kidneys of the 150 mg/kg/day group F0 females. Thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females at the scheduled necropsies for Cohort 1B; this finding was considered test substance-related and adverse in the 150 mg/kg/day group males and females. No other test substance-related internal findings were observed at any dosage level for F1 Cohort 1B animals. No test substance-related effects on the mean number of F0 implantation sites or number of unaccounted-for sites were noted at any dosage level. No test substance-related macroscopic findings were observed in F1 pups that were found dead, culled on PND 4, or examined at the scheduled necropsy on PND 21; F1 pup organ weights on PND 21 were unaffected by test substance administration. No test substance-related effects on ovarian primordial follicle counts were noted in the F0 females suspected of reduced fertility or F1 Cohort 1A females. There were no test substance-related effects on organ weights noted for F0 and F1 males and females at any dosage level.

Due to the absence of systemic toxicity noted for F0 and F1 males and females throughout the study, a dosage level of 150 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when n-butyl acrylate was administered orally by gavage to Crl:CD(SD) rats. Epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the 150 mg/kg/day group F0 and F1 males and females were considered adverse; based on these results, 50 mg/kg/day was considered to be the NOAEL and 150 mg/kg/day was considered to be the lowest-observed-adverse-effect level (LOAEL) for local effects in the F0 and F1 generations. Based on the lack of effects noted for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity. There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day.

A subchronic inhalation study in rats demonstrated no indication of a fertility impairing potential of n-butyl acrylate.

A two-generation reproduction toxicity study with the structural analogue methyl acrylate, which was considered as reliable surrogate for n-butyl acrylate, led to no effects on reproductive performance including fertility after whole body inhalative exposure. The NOAEC for reproductive function was 75 ppm (= ca. 0.269 mg/L), the highest concentration investigated.

Conclusion:

Based on the data derived from the extended one genereation study and the sub-chronic and chronic toxicity studies, n-butyl acrylate is not expected to impair reproduction. Also a two-generation study with the structural analogue methyl acrylate did not give any indications for a fertility impairing effect.

Effects on developmental toxicity

Description of key information

Inhalation exposure in two prenatal developmental toxicity studies to butyl acrylate caused unspecific embryo-/fetotoxic effects only at maternal toxic concentration, while morphological alterations even at concentrations of clear maternal toxicity did not occur consistently in both studies, especially no indication of teratogenicity was noted.

An oral developmental toxicity study in rabbits according to OECD414 and GLP was performed (Acrylate REACH TF, 2017). 25 inseminated New Zealand White rabbits were exposed orally (gavage) to 50, 150 and 400 mg/kg bw/day during gestation days 7 through 28. A NOAEL of 400 mg/kg bw is derived for both maternal and developmental toxicity based on the absence of adverse effects.

n-Butyl acrylate was also tested in an extended one generation study according to OECD 443 and GLP. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (Acrylate Reach TF, 2017).There was no indication of a developmental toxic effect.
Oral administration of butyl acrylate to mice by gavage at very high doses (2500 – 3000 mg/kg bw), clearly exceeding the current limit dose of 1000 mg/kg bw up to 3fold, caused malformations only at maternally lethal dose levels and other forms of clear maternal toxicity. At lower dose levels, still clearly maternally toxic including deaths, no morphological alterations were noted but developmental toxicity in form of body weight impairment. At concentrations without maternal toxicity, butyl acrylate did not cause any developmental toxicity in the exposed mice.
A prenatal developmental toxicity study in rabbits with inhalation exposure, conducted with the structural analogue methyl acrylate, revealed no indication for a developmental toxic or teratogenic potential, even at maternally toxic concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 Feb 1979 - 13 Mar 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: Guidelines for reproduction studies for safety evaluation of drugs for human use, FDA, Jan. 1966 and Guidance on reproduction studies from the Association of the British Pharmaceutical Industry, 1975.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Iffa Credo, Lyon, France
- Body weight at study initiation:
The mean body weight ± SD in dose groups 0, 25, 135 and 250 ppm were 209±12, 214±14, 214±9 and 219±16, respectively.
- Age at study initiation: 9-11 week
- Diet (e.g. ad libitum): Herilan Mrh-Zucht, H. Eggermann KG, Rinteln.
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±5
- 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:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
By means of a continuous infusion apparatus (UNITA I, B. Braun, Melsungen, Federal Republic Germany) constant amounts of the liquid product were supplied to a heated (about 80°C) evaporator. The n-butyl acrylate vapors were diluted with dust-free, conditioned fresh air and passed through 200 L inhalation chambers (all-glass construction with steel frame) under dynamic airflow conditions at a flow rate of 20 changes of air per hour (4000 L/h; 200 L chamber). A mean temperature of 24.5°C and a mean relative humidity of 53% were measured during exposure.


TEST ATMOSPHERE
- Brief description of analytical method used: The n-butyl acrylate test atmosphere concentrations were monitored analytically by means of a total
hydrocarbon analyzer (R 5 of RATFISCH, Munich). The total hydrocarbon analyzer was calibrated using an infrared analyzer Miran I (WILKS) calibrated with standards of known concentrations of n-butyl acrylate.
- Samples taken from breathing zone: yes


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical concentrations (Mean ± SD) of the dose groups 25, 135 and 250 ppm were 25 ± 1, 137 ± 4 and 251 ± 3 ppm, respectively.
Details on mating procedure:
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6-15 of gestation
Frequency of treatment:
6 hours per day
Duration of test:
21 days
Remarks:
Doses / Concentrations:
0, 25, 135 and 250 ppm (corresponding to 0, 0.13, 0.71 and 1.31 mg/L) Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57.
Basis:
nominal conc.
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: day on which sperm had been detected (day 0) and on the 6th, 16th and 20th days post coitum.



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on the 20th day post coitum.



Ovaries and uterine content:
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
The weights and the length of fetuses were determined. After fixation in Bouin's solution, 1/3 of the fetuses were examined for organ changes according to the method of Wilson and Warkany (1965), and after staining of the skeleton (Dawson, 1926) 2/3 of the fetuses were investigated for skeletal changes.
Wilson, J.G. and Warkany, J. (1965). Teratology: Principles and Techniques. The University of Chicago Press, Chicago and London.
Dawson, A.B. (1926). Stain Technol. 1:123.
Statistics:
A trend analysis based on the generalization of the t-test according to Williams (1971, 1972) was carried out for the variables of maternal body weight and body weight gain, fetal weight and length, and placental weight in each case. The U-test (Krauth, 1971; Stucky and Vollmar, 1976) was carried out for the parameters of implantations per pregnant animal, live and dead embryos as percent per pregnant animal, and anomalies, variations and retardations as percent of live fetuses per litter.
Williams, D.A. (1971). Biometrics 27:103-117.
Williams, D.A. (1972). Biometrics 28:519-531.
Krauth, J. (1971). Ann. Math. Statist. 42:1949-1956.
Stucky, W. and Vollmar, J. (1976). J. Statist. Comput. Simul. 5:73-81.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
25 ppm were tolerated without any impairment of body weight. The body weight gain was significantly reduced after inhalation of 135 and 250 ppm during the period of treatment. In the period after the end of treatment (gd 16 - 20) the steepness of the body weight curve obtained after 135 and 250 ppm was similar to that of the control group. During the exposure 135 ppm led to distinct discharge from the eyes and noses and to ruffled fur. After inhalation of 250 ppm these symptoms were even more pronounced. No mortality occurred.
It is noteworthy to mention that no histopathological examination on the respiratory tract of the dams was performed, as it was not required by the guideline. However, with regards to the observed respiratory irritation in the subchronic inhalation study at comparable concentrations (see 7.5.2 BASFAGXXVI/352), the occurrence of clear effects on the respiratory tract as evidence for maternal toxicity can be considered as very likely, especially as irritation was already noted clinically.
Dose descriptor:
NOAEC
Effect level:
ca. 0.13 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The necropsy of the animals did not reveal any gross-pathological changes of the internal organs which could be attributed to the test substance. The number of corpora lutea and the number of implantations did not show any differences between the individual groups. After inhalation of 135 and 250 ppm the percentage of resorptions (i.e. post-implantation loss) per pregnant animal was dose-dependent reduced. No adverse effect on the weight and length of the fetuses was observed. No treatment related morphological alterations, especially no malformations were observed in the fetuses at any concentration.
Dose descriptor:
NOAEC
Effect level:
ca. 0.13 mg/L air (nominal)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEC
Effect level:
ca. 1.31 mg/L air (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Maternal body weight development (mean values ± standard deviation):

 

Concentration [ppm]

Maternal body weight GD 0 [g]

Maternal body weight GD 20 [g]

Maternal body weight gain GD 0-20 [g]

0

209.38 ± 11.83

354.01 ± 36.66

144.64 ± 33.08

25

213.96 ± 13.94

359.62 ± 36.81

145.66 ± 29.87

135

213.68 ± 9.30

335.54 ± 43.00

121.87 ± 37.62*

250

218.64 ± 16.19

318.11 ± 42.79**

99.47± 33.68**

*  p < 0.05

** p < 0.01

Reproductive parameters:

 

Conc.  (ppm)   

no. pregnant/  total animals 

live fetuses/ animal 

resorptions (%)

weight of fetuses (g)

0

22/30

11.5 ± 5.34     

11.6

3.85 ± 0.41

25

23/30

10.6 ± 4.94     

13.8

4.08 ± 0.39

135

18/30

8.8 ± 5.14        

23.6*

4.09 ± 0.23

250

19/30

8.4 ± 5.68        

31.6*

4.08 ± 0.47

*: p<0.05

Conc.  (ppm)

% fetuses per litter with anomalies

% litters with fetuses showing anomalies

% fetuses per litter with variations/ retardations

% litters with fetuses showing variations/ retardations

0

2.7

23.8

19.7

81.0

25

0.9

9.1

11.2

59.1

135

1.9

18.8

10.2

43.8

250

0

0

8.2

43.8

 

0 ppm

25 ppm

135 ppm

250 ppm

Skeletal findings

Anomalies:

- Cleft vertebral centra

7/170

2/162

4/105

0/107

Variations/retardations:

- incomplete ossification of skull bone

0/170

0/162

1/105

0/107

- aplasia of sternebrae

16/170

11/162

3/105

3/107

-incomplete ossification of sternebrae

30/170

18/162

14/105

9/107

- asymmetric sternebrae

4/170

1/162

0/105

0/107

-accessory rib, bilateral

2/170

3/162

0/105

0/107

-accessory rib, bilateral and rudimentary

1/170

0/162

0/105

0/107

-accessory rib, unilateral and rudimentary

1/170

0/162

0/105

0/107

-general incomplete ossification of bones

1/170

2/162

1/105

1/107

Organ findings

Variations/retardations:

-dilatation of pelvis, unilateral

2/82

0/81

1/54

0/53

Units given as number found/number examined

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20-5-2016 to 25-7-2016 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
GLP compliance:
yes
Specific details on test material used for the study:
- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hra:(NZW)SPF
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA
- Age at study initiation: The animals were approximately 7 months old upon receipt.
- Weight at study initiation: 2922 - 3949 g
- Housing: All rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Nesting material was not required because the females were euthanized prior to the date of expected parturition. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly. Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study.
- Diet: The basal diet used in this study, PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322, was a certified feed with appropriate analyses performed by the manufacturer. The basal diet was offered in 25-g increments 3 times per day on the day of arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the dose administration period; basal diet was offered ad libitum thereafter.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum during the study.
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 80 mg/mL (corresponding to dosage levels of 0, 50, 150 and 400 mg/kg/day)
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 10, 30, and 80 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on on the first, approximate middle, and last days of preparation on which all groups were dosed. Analysis was performed using a validated gas chromatography method using flame ionization detection. The analyzed concentrations was 85% to 115% of the target concentration.
Details on mating procedure:
The time-mated rabbits were received on gestation day 2, 3, or 4; a breeding record was provided by the supplier.
Duration of treatment / exposure:
gestation days 7 through 28
Frequency of treatment:
once daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were selected based on the range-finding stud (see any other information on materials and methods)
Maternal examinations:
CAGE SIDE OBSERVATIONS
All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from the day of receipt through gestation day 29 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHT
Individual maternal body weights were recorded on gestation days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 5, and 7-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-10, 10-13, 13-21, 21-29, and 7-29.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 5-29. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

POST-MORTEM EXAMINATIONS
The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rabbits were euthanized on estation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were co related with the antemortem observations, and any abnormalities were recorded. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Gravid uterine weight was collected and net body weight (the gestation day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Number of corpora lutea: The number of corpora lutea on each ovary was recorded.
- Number of implantations: The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in
10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
- Fetal examinations were performed blind to treatment group.
- External examinations: Each viable fetus was examined externally, individually weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.
- Soft tissue examinations: Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development
- Skeletal examinations: Following fixation in alcohol, each fetus was stained with Alizarin Red S8 and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- Head examinations: Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group.
- Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group.
- Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
Indices:
Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group / No. Gravid Females/Group

Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group

Where: Postimplantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter/ No. Implantation Sites/Litter x 100

Summation per Group (%) = Sum of Viable Fetuses Affected/Litter (%)/ No. Litters/Group

Where: Viable Fetuses Affected/Litter (%) = No. Viable Fetuses Affected/Litter/ No. Viable Fetuses/Litter x 100
Historical control data:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Findings noted in the test substance-treated groups, including decreased defecation and brown material and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy on gestation day 29.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day group, an absence of a mean body weight gain (0 g) was noted on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 (57 g, 54 g, and 64 g in the control, 50, and 150 mg/kg/day groups, respectively, compared to 12 g in the 400 mg/kg/day group). Mean body weight gains in the 400 mg/kg/day group were similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean body weight gain at the beginning of the treatment period, a lower mean body weight gain was noted at 400 mg/kg/day compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal body weight gains in the 50 and 150 mg/kg/day groups and mean body weights, net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day group, lower mean food consumption was noted during gestation days 7 -10 compared to the control group; mean food consumption in this group was similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean food consumption at the beginning of the treatment period, lower mean food consumption was noted in the 400 mg/kg/day group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences at 400 mg/kg/day were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50 and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on gestation day 29, no test substance-related internal findings were observed at dosage levels of 50, 150, and 400 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal systemic toxicity & maternal developmental toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related external malformations were noted for fetuses in this study. In the 150 mg/kg/day group, one fetus was noted with omphalocele (a portion of the liver protruded through an opening in the umbilicus, remnants of a membranous sac). The aforementioned malformation noted at 150 mg/kg/day occurred in a single fetus, did not occur in a dose-related manner, and the mean litter proportion was not statistically significantly different from the concurrent control group and was within the test lab historical control data range (version 2016.01); therefore, it was not considered test substance-related. No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related skeletal malformations were noted for fetuses at any dosage level. Vertebral anomaly with or without an associated rib anomaly (extra or fused ribs; extra and/or malpositioned arches; extra, malpositioned, absent, small, fused, and/or misshapen centra) was noted for 2 and 1 fetuses in the 50 and 150 mg/kg/day groups, respectively. The aforementioned malformation at 50 and 150 mg/kg/day occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range; therefore, it was not considered test substance-related.
- No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related visceral malformations were observed for fetuses at any dosage level. Lobular agenesis of the lungs (right accessory lobe absent) was noted for and 3 fetuses in the 50 and 150 mg/kg/day groups, respectively. Because this finding occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range, this finding was not considered test substance-related. In the control group, one fetus was noted with an absent left kidney and ureter and one fetus was noted with an absent right thyroid gland.
- No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
- A distended stomach was noted for one fetus in the 50 mg/kg/day group. This finding was not classified as either a malformation or developmental variation, was not considered to be test substance-related because it occurred infrequently and in a manner that was not dose-related.
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 219(25), 214(24), 199(25), and 214(24) in the control, 50, 150, and 400 mg/kg/day groups, respectively. Malformations were observed in 2(1), 4(4), 5(4), and 0(0) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Study according to OECD TG 414, GLP
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
133 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral application:

An oral developmental toxicity study in rabbits according to OECD414 and GLP was performed with n-butyl acrylate (Acrylate REACH TF, 2017).

25 inseminated New Zealand White rabbits were exposed orally (gavage) to 50, 150 and 400 mg/kg bw/day during gestation days 7 through 28. All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

A test substance-related absence of a mean body weight gain (0 g) was noted in the 400 mg/kg/day group on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 and a lower mean body weight gain when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. In addition, lower mean food consumption was noted in the 400 mg/kg/day group during gestation days 7-10 and resulted in lower mean food consumption in this group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated; however, these differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. Mean maternal body weights, body weight gains, and food consumption in the 50 and 150 mg/kg/day groups and mean body weights,net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. There were no substance-related macroscopic findings noted at the scheduled necropsy on gestation day 29 in the 50, 150, and 400 mg/kg/day groups. Intrauterine growth and survival in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. In addition, no test substance-related external, visceral, and skeletal malformations or developmental variations were noted at any dosage level.

Nonadverse lower mean body weight gains and corresponding lower mean food consumption were noted in the 400 mg/kg/day group. No evidence of developmental toxicity was noted at 50, 150, and 400 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day, the highest dosage level tested, was considered to be the no‑observed‑adverse‑effect level (NOAEL).

For n-butyl acrylate an extended one generation study according to OECD 443 and GLP was performed. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (Acrylate Reach TF, 2017).

There were no test substance-related effects on survival for F0 and F1 animals at any dosage level. No test substance-related clinical observations were noted for F0 and F1 animals at any dosage level. Mean body weights, body weight gains, food consumption, and food efficiency in the 20, 50, and 150 mg/kg/day F0 and F1 males and females were unaffected by test substance administration. No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cyclicity or spermatogenic parameters (testicular and epididymal sperm concentrations, sperm production rate, sperm motility, and sperm morphology) at any dosage level. There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance, offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation). No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) were noted in F0 and F1 males or females or F1 pups (on PND 4 and 21). Test substance-related histologic changes were observed in all dosage groups in the F0 generation and F1 males and females in Cohort 1A. Epithelial hyperplasia and/or hyperkeratosis was observed in the nonglandular stomach in all test substance-treated groups examined. Mild to moderate changes in the 150 mg/kg/day group males and females of the F0 and F1 generations were considered adverse in this study. Microscopic changes in the stomach were associated with the gross observation of thickened nonglandular stomach, but were not associated with any clinical pathology, organ, or body weight changes. In the F0 generation, a nonadverse increased incidence of biliary hyperplasia (males and females) and random hepatocellular necrosis (males)

were observed in the liver in the 150 mg/kg/day group. Additionally, nonadverse test substance-related microscopic findings (increased severity of mineralization at the corticomedullary junction) were observed in the kidneys of the 150 mg/kg/day group F0 females. Thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females at the scheduled necropsies for Cohort 1B; this finding was considered test substance-related and adverse in the 150 mg/kg/day group males and females. No other test substance-related internal findings were observed at any dosage level for F1 Cohort 1B animals. No test substance-related effects on the mean number of F0 implantation sites or number of unaccounted-for sites were noted at any dosage level. No test substance-related macroscopic findings were observed in F1 pups that were found dead, culled on PND 4, or examined at the scheduled necropsy on PND 21; F1 pup organ weights on PND 21 were unaffected by test substance administration. No test substance-related effects on ovarian primordial follicle counts were noted in the F0 females suspected of reduced fertility or F1 Cohort 1A females. There were no test substance-related effects on organ weights noted for F0 and F1 males and females at any

dosage level.

Due to the absence of systemic toxicity noted for F0 and F1 males and females throughout the study, a dosage level of 150 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when n-butyl acrylate was administered orally by gavage to Crl:CD(SD) rats. Epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the 150 mg/kg/day group F0 and F1 males and females were considered adverse; based on these results, 50 mg/kg/day was considered to be the NOAEL and 150 mg/kg/day was considered to be the lowest-observed-adverse-effect level (LOAEL) for local effects in the F0 and F1 generations. Based on the lack of effects noted for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity. There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day.

In a prenatal developmental toxicity study with low reliability due to excessive dose levels by far exceeding the maximum dose recommended by the respective OECD guidelines and doses causing maternal lethality, pregnant CD-1 mice received butyl acrylate dissolved in cottonseed oil at dose levels of 0, 100, 1000, 1500, 2000, 2500, 3000 and 4000 mg/kg bw orally by gavage from gestation day 6 to day 15. No animal survived at the high dose. At 3000 and 2500 mg/kg bw 2/30 animals died; at 2000 mg/kg bw 1/29 died; at 1500 mg/kg bw 1/27 died; and at 1000 mg/kg bw 1/30 died. At 1500 mg/kg bw and above, mean maternal body weight gain and concurrently, mean fatal body weights were significantly reduced. Most likely due to the maternal stress, the percentage of resorptions was significantly increased at 2500 and 3000 mg/kg bw. In the control group as well as in at 100, 1000, 1500 and 2000 mg/kg bw, variations and malformations occurred sporadically at different sides (i.e. single cases of cleft palate, fused ribs, fused sternebrae, fused arches, extra arches, branched ribs) in a non-dose-dependent manner. At maternally lethal dose levels of 2500 mg/kg bw and 3000 mg/kg bw the number of fetuses with external and skeletal malformations and variations (cleft palate, exencephaly, open eyes, fused arches, fused ribs) was significantly increased (Rohm and Haas Co. 1982). The NOAEL for maternal toxicity was 100 mg/kg bw. The NOAEL for developmental toxicity was 1000 mg/kg bw and the NOAEL for teratogenicity was 2000 mg/kg bw. It is noteworthy to mention that currently the internationally accepted limit dose level is 1000 mg/kg bw. Thus, the NOAEL for developmental toxicity was at this limit dose level, while the NOAEL for teratogenicity was 2fold above.

Inhalation:

Sprague Dawley rats were exposed to butyl acrylate vapour concentrations of 25, 135 and 250 ppm (corresponding to approx. 0.13; 0.71; 1.31 mg/L) for 6 hours per day on days 6 to 15 of gestations (BASF AG, 1979). Inhalation of 135 and 250 ppm caused a significant reduction in maternal body weight gain, as well as irritation to the nose and eyes due to the known irritative nature of this test substance to mucous membranes. Following the exposure until termination the animals recovered and the weight gain was comparable to the controls. The two highest concentrations caused a dose-dependent increase in post-implantation loss (i.e. increase in resorptions). 25 ppm did not lead to any signs of maternal toxicity and had no effect on the implants. No substance-related morphogical alterations, especially no malformations were observed in the fetuses at any concentration. The NOAEC for maternal and developmental toxicity was 25 ppm (0.13 mg/L), and the NOAEC for teratogenicity was 250 ppm (1.31 mg/L), the highest concentration examined. It is noteworthy to mention that no histopathological examination of the respiratory tract of the dams was performed, as it was not required by the guideline. However, with regards to the observed respiratory irritation in the subchronic inhalation study at comparable concentrations (see 7.5.2 BASFAGXXVI/352), the occurrence of clear effects on the respiratory tract as evidence for maternal toxicity can be considered as very likely, especially as irritation was already noted clinically.

Subsequently, a comparative prenatal developmental toxicity study equivalent to OECD 414 on seven acrylates was performed in the same strain of Sprague-Dawley rats with a prolonged exposure period (Saillenfait, 1999). Groups of 25 pregnant rats were exposed to 0, 100, 200 and 300 ppm butyl acrylate (corresponding to approx.0.52, 1.05, and 1.57 mg/L) for 6 hrs/day from day 6 through day 20 of gestation. The body weight gain was concentration-dependent reduced in all dose groups. In contrast to the above study, no treatment-related effects occurred for the numbers of implantation sites, live fetuses, non-live implants or resorptions but fetal body weight was significantly reduced at 200 and 300 ppm. There were no treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations was similar in the control and treated groups. A NOAEC for maternal toxicity could not be derived with regards to the impaired body weight gain, the LOAEC for was 100 ppm (0.52 mg/L). The NOAEC for developmental effects was 100 ppm (0.52 mg/L), and the NOAEC for teratogenicity was the highest tested concentration of 300 ppm (1.57 mg/L).

A prenatal developmental toxicity study in rabbits as second species was conducted with the structural analogue methyl acrylate according to OECD 414 for the Acrylate Task Force (BAMM 2009). 25 inseminated female Himalayan rabbits per group were whole-body exposed for 6 hrs/day, 5 days/week over a time period of 23 consecutive days (gestation days (GD) 6–28) to methyl acrylate vapours at target concentrations of 0, 5, 15, and 45 ppm. Analytical concentrations of 4.9, 15.7, 44.2 ppm (corresponding to approx. 0.0174, 0.0553, 0.1556 mg/L) were measured. On gestation day 29 the does were sacrificed and submitted to gross and histopathological examination (nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions). Examinations of ovaries and uterine content of the does included: determination of the weight of the unopened uterus, of the number of corpora lutea, of the number and distribution of implantation sites, and calculations of conception rate and pre- and post-implantation losses. Fetal examinations were performed on all fetuses per litter (external, soft tissue, skeletal) except head examinations that were done on half of the fetuses per litter. There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects, such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis, this is considered to be a significant adverse effect on the maternal organism. The NOAEC for maternal toxicity was 15 ppm (0.0553 mg/L). Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Methyl Acrylate (MA) had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). Thus, the NOAEC for developmental effects (fetotoxicity) and the NOAEC for developmental effects (teratogenicity) was the highest concentration tested of 45 ppm (0.1556 mg/L).


Justification for classification or non-classification

CLP classification (Regulation (EC) No 1272/2008):

no classification required