Isobutyl acrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No experimental data on isobutyl acrylate are available. Data on the structural analogue n-butyl acrylate and tert-butyl acrylate are included.

In the extended one generation reproductive toxicity study with n-butyl acrylate no adverse effects on fertility and reproductive organs after oral administration could be identified (RA TF, 2017). A combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test (OECD TG 413/ 422) with tert-butyl acrylate did not reveal any potential of the substance to impair fertility (BASF, 2004). Based on the data derived from the structural analogues n-butyl acrylate and tert-butyl acrylate, isobutyl acrylate is not expected to impair fertility.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

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:

- Name of test material: n-butyl acrylate
- 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 or test system 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 (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the range-finding study
- Fasting period before blood sampling for clinical biochemistry: yes, overnight prior to blood collection

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 Motile) 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 Motile) 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.

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.

Key result

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:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects (nonglandular stomach)

Key result

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.

Anogenital distance (AGD):

effects observed, non-treatment-related

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 differeces 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.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

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.

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):

- 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

Key result

Dose descriptor:

NOAEL

Generation:

F1

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

Generation:

F1

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)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Results of F0 Reproductive Performance Results of F0 Reproductive Performance

Parameter	Dosage Level (mg/kg bw/day)				CRL HC^aMean (Range)
	0	20	50	150
Male Mating Index (%)	96.7	93.3	96.7	93.3	95.8 (86.7-100.0)
Female Mating Index (%)	96.7	93.3	96.7	93.3	96.4 (86.7-100.0)
Male Fertility Index (%)	93.3	86.7	90.0	83.3	88.2 (70.0-100.0)
Female Fertility Index (%)	93.3	86.7	90.0	83.3	88.7 (70.0-100.0)
Male Copulation Index (%)	96.6	92.9	93.1	89.3	92.7 (70.0-100.0)
Female Conception Index (%)	96.6	92.9	93.1	89.3	92.7 (70.0-100.0)
Estrous Cycle Length (days)	4.3	4.1	4.4	4.4	4.6 (3.9-7.6)
Pre-Coital Interval (days)	2.7	2.7	2.6	2.1	3.0(1.8-4.7)

^aCharles River Ashland historical control data (version 2016.01).

 

Table 2: Incidence of Selected Histopathological Findings, F0 Generation

Dosage (mg/kg bw/day):	Males				Females
	0	20	50	150	0	20	50	150
Nonglandular Stomach^a	30	30	30	30	30	30	30	30
Hyperplasia. Epithelial Cell	0	4	22	30	0	1	8	30
Minimal	-	4	21	0	-	1	7	16
Mild	-	0	1	30	-	0	1	14
Hyperkeratosis	0	21	26	30	0	12	20	29
Minimal	-	21	22	0	-	12	20	3
Mild	-	0	4	20	-	0	0	19
Moderate	-	0	0	10	-	0	0	7

^aNumber of tissues examined from each group.

 

Table 3: Incidence of Selected Histopathological Findings, F1 Generation

Dosage (mg/kg bw/dav):	Males				Females
	0	20	50	150	0	20	50	150
Nonglandular Stomach^a	26	24	24	23	26	24	24	24
Hyperplasia. Epithelial Cell	0	0	7	23	0	0	3	24
Minimal	-	-	7	20	-	-	3	21
Mild	-	-	0	3	-	-	0	3
Hyperkeratosis	0	10	15	23	0	5	19	24
Minimal	-	10	15	7	-	5	19	15
Mild	-	0	0	16	-	0	0	9

^aNumber of tissues examined from each group.

Reference 2

Endpoint:

fertility, other

Remarks:

Repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 January 1977 to 25 April 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Group of rats (20 male and 20 female per dose and control group) were exposed to butyl acrylate vapors (6 h/ day) over a period of 13 weeks. Animals were observed for clinical signs, body weight gain, hematological and biochemical parameters. Necropsies were performed on all animals and weight of the testes were measured. Tissues, including seminal vesicles, prostate, epididymis/uterus, testes and ovary were examined histologically from all animals.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material: N-Butyl acrylate

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breed supplied, WIGA, Sulzfeld
- Age at study initiation: 42 days
- Weight at study initiation: males-159 g and females-130 g
- Housing: 2-3 rats per cage
- Diet (ad libitum): Altromin-R supplied by Altrogge, Lage/Lippe
- Water (ad libitum): tap water

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 pump, the liquid product was metered onto a heated vaporizer (temperature about 80 °C) at a constant rate and vaporized there. A stream of supply air measured by means of a rotameter took up the vapors. This vapor-air mixture, after passing through a mixing device, was introduced into an inhalation chamber with a volume of 200 liters.

TEST ATMOSPHERE
- Brief description of analytical method used: The n-butyl acrylate air mixture was measured continuously using a flame ionization detector (FID). Apparatus used was FID total hydrocarbons analyzer (CARLO ERBA, mod. 370).
- Samples taken from breathing zone: yes. Sampling was carried out by means of a diaphragm pump which continuously passes the n-butyl acrylate air mixture to the FID. A second diaphragm pump continuously sweeped the sample tubes that were not needed for measurement in the chamber up to the pneumatic valve. The duration of measurement was 10 minutes per chamber, and the sweeping time 7 minutes (measuring cycle).

Details on mating procedure:

not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical measurements of the theoritical concentrations of 20, 100, 200 and 550 ppm were 21, 108, 221 and 546 ppm, respectively. Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h/day, 5 d/week for 13 weeks (63 exposures)

Dose / conc.:

0.11 mg/L air (analytical)

Remarks:

21 ppm

Dose / conc.:

0.57 mg/L air (analytical)

Remarks:

108 ppm

Dose / conc.:

1.11 mg/L air (analytical)

Remarks:

221 ppm

Dose / conc.:

2.86 mg/L air (analytical)

Remarks:

546 ppm

No. of animals per sex per dose:

20

Control animals:

yes, sham-exposed

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 7 days before (sampling 0), and 4 (sampling 1), 8 (sampling 2) and 13 (sampling 3) weeks after the beginning of exposure.
- Parameters examined: hemoglobin content, erythrocytes, hematocrit, hemoglobin content per erythrocyte, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelets, leukocytes and differential blood count

CLINICAL CHEMISTRY: Yes
- Parameters examined: Total bilirubin, creatinine, urea, sodium, potassium, total protein, glucose, inorganic phosphate, calcium, chloride, triglycerides, cholesterol and enzymes (Glutamic pyruvic transaminase, Alkaline phosphatase, Glutamic oxalacetic transaminase)

URINALYSIS: Yes. Urine was sampled individually from all animals overnight after 4 1/2 weeks (urine collection 1), 8 1/2 weeks (urine collection 2) and 12 1/2 weeks (urinalysis 3) after the beginning of the study.
- Parameters examined: pH, protein, glucose, urobilinogen and sediment microscopy.

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Not examined.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
At the end of the study period, the surviving animals were sacrificed after a fasting period of 16 hours. For reasons of organization, only half of the male group received no feed. Only this small group was used for calculating the organ weights. The sacrifice was carried out under anesthesia with CO2 by exsanguination (opening of the bracchial vessels) and subsequent decapitation. Then the animals were necropsied and assessed by gross-pathology. Data on reproductive organ toxicity (testes weights as well as information on gross and microscopic pathology for testes, seminal vesicles, ovaries and uteri) were derived. The relative organ weights (organ weight/100 g body weight) were calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were observed in animals of dose groups 21 and 108 ppm. All animals in the 211-ppm dose group exhibited distinct discharge from the eyes and noses from the first inhalation onward, however, the animals recovered quickly each time. Animals in the 546-ppm dose group exhibited pronounced discharge from the eyes and noses, however animals no longer recovered from day 11 of the exposure, and also exhibited severe dyspnoea, bloody discharge from the eyes and noses, extremely aggressiveness and a stronger inclination to cannibalism.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality was observed in the 21, 108, and 211 ppm dose groups. Thirty one (16 male and 15 female) of 40 animals (77 %) in the 546 ppm dose group died between the 3rd and 13th week of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Throughout the study, there was no significant influence on the body weight change of the male and female animals in the 21 ppm and 108 ppm dose groups compared with the control group, except in one weighing after 7 days, only the female animals of 108 ppm dose group exhibited a significant decrease in body weight caused by a very slight body weight gain in 4 animals. In the 211 ppm dose group, the body weight of the female rats was decreased from day 7 to day 77 after the beginning of the study (at a significance of 95-99%). The animals showed no significant body weight changes at the weighing after 85 and 91 days. At the final weighing, the body weight was decreased (at a significance of 95%). In the male rats, the body weight was significantly decreased after 63 days until the end of the study (at a significance of 95-99%).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematocrit, hemoglobin content per erythrocytes, MCV (mean cell volume) and MCHC (mean corpuscular hemoglobin concentration) were unaffected. There was increase in hemoglobin content and erythrocytes in male and female animals of the 546 ppm dose group in blood sampling 1-3. The changes in the leukocyte and monocyte counts occurred only in one sex, hence cannot be considered test substance specific.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total bilirubin, Creatinine, Inorganic phosphate, Calcium, Chloride, Triglycerides and enzymes (Glutamic pyruvic transaminase and Glutamic oxalacetic transaminase) were unaffected. There was an increase in sodium level in male and female animals of the 546 ppm dose group in blood sampling 2 and 3. An increase in the level of Alkaline phosphatase was observed only in female animals of the 211 and 546 ppm dose groups, however a time- and dose-dependent trend was seen. There was a decrease in the levels of potassium, total protein, glucose and cholesterol in female animals only.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No adverse effects of the test substance could be seen from the urinary findings.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

546 ppm dose group: Histopathological changes were found mostly in the respiratory tract of the animals in the 546 dose ppm group that died intercurrently. In the male and female rats slight hyperemia of the nasal mucosa, edema and dysplasia of the epithelial mucosa was observed. In one female animal pronounced purulent rhinitis was noted. Hyperemia and edema were found also in the trachea in almost all males that died spontaneously and that were examined in this group; they were seen in only half of the females. Metaplasia of the mucosa (multi-rowed, cornified epithelium) was detected in most of the rats and predominantly in males. One single animal even had necrosis of the mucosa. Metaplasia was found as far as the bronchioles and was observed in both sexes. In many animals multi-focal infiltration with various degrees of extension emerged from metaplasia on the one hand and from proliferation in the alveolar zone on the other. It consisted of metaplastic epithelial cells still containing mucin and dark-nuclear cells (some of them mononuclear) and which had to be distinguished from the usual rat-specific infiltrates. Multifocal infiltration, which was found more in the females, changed into bronchopneumonia in nearly the same number of male and female animals. Gram-positive germs could be detected in the area of the pneumonic foci in animals which were characterized by extensive and advanced necrosis of the lungs. Male and female animals that died intercurrently were found to have hyperemia and necrosis in the liver (1 male and 1 female) together with fatty deposits predominantly in the peripheral zones of the lobules, these being more pronounced in the females. In one third of the male animals the glycogen content was slightly increased. The spleen of two male animals of the 546 ppm group showed hyperemia, of one female moderate fibrosis and the thymus of four males and one female hemorrhages. In the case of the bone marrow, erythropoesis prevailed in four male and three female rats and granulopoesis in four male and seven females, in some with a shift to the left. Hyperemia of the kidneys and meninges, and a cyst in the anterior lobe (twice) was found in the animals of the 546 ppm group that died spontaneously.

Other dose groups: Slight edema and erosions of the nasal mucosa were found in only 2 male animals of the 211 ppm dose group and in one female rat of the 108 ppm dose group. In female animals in the 211 ppm dose group, a slight increase in fatty deposits and the glycogen content in the liver were noted in comparison to the control group. In nearly half of all male animals of the 211, 108 and 0 ppm dose groups, the fat-free vacuoles (hematoxylin eosin stain), fatty deposits, mainly in Kupffer’s star cells, and increase of the glycogen content in the liver were attributed to the fact that these animals had not been fasted on the day before they were necropsied and therefore the findings are to be regarded normal. Using the Turnbull blue reaction, a slight decrease in the hematogenic iron pigment was seen above all in the females of the 211 and 108 ppm groups when compared with the control. No histopathological examinations were carried out in the 21 ppm group.

Data on reproductive organ toxicity were derived from a 3 month inhalation study. Respective results from animals exposed to the highest dose level of 546 ppm (2.86 mg/L) did not give evidence for any impairment of the investigated reproductive organs of both sexes.

Key result

Dose descriptor:

NOAEC

Effect level:

2.86 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: no effects on reproductive organs

Key result

Critical effects observed:

no

Reproductive effects observed:

not specified

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 Aug 2002 - 04 Dec 2003 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 413 (Sub-Chronic Inhalation Toxicity: 90-day Study)

Version / remarks:

May 12, 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 22, 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental)

Version / remarks:

712-C-00-368; July, 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

712-C-98-204; August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)

Version / remarks:

May 30, 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material: Tert.- Butylacrylat
- Physical state: colorless liquid
- Batch No. 2-011002-15/1 and B602

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: Males: 106.1-108.3 g; Females: 92.7-94.7 g (groupwise)
- Housing: individually in type DK II stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Underneath the DK III cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding (SSNIFF, Soest, FRG); from day 18 post coitum until sacrifice, the pregnant animals and their litters were housed in Markolon M III cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Pregnant females were provided with nesting material (cellulose wadding) towards the end of pregnancy.
- Diet: ad libitum, "GLP" (Provimi Kliba SA, Kaiseraugst, CH)
- Water: ad libitum, tap water
- Acclimation period: ca. 2 weeks

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

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: clean air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps (Sarstedt DESAGA) and glass vaporizers with thermostat (BASF AG)
- Method of holding animals in test chamber: The animals were kept singly in wire cages located in a glass-steel inhalation chamber, volume of 1.4 m3 (BASF AG).
- Method of conditioning air: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump. The vapor was generated with conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C) and passed into the inhalation system.
- Temperature, pressure in air chamber: 25 ± 3°C, -10 Pa
- Air flow rate: 27.5-28.5 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used:  The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control (Hewlett-Packard 5840 A). Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean  concentrations and standard deviations for the entire study were derived. The concentration constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer.
To ensure, that no liquid aerosols were formed at concentration levels as high as 180 ppm, a scattered light photometer was used to monitor the test atmosphere of the high dose group.
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: from study day 69 - study day 80
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Target concentrations were: 0.106, 0.319, and 0.956 mg/L (corresponding to 20, 60, and 180 ppm)
- Measured concentrations were: 0.107 ± 0.0061, 0.317 ± 0.0211, 0.958 ± 0.0481 mg/L

Duration of treatment / exposure:

- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)

Frequency of treatment:

6 hours/day, 5 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: 16 weeks
- After ten weeks of exposure, the parental animals were mated to produce a litter. Mating pairs were formed from the same concentration group. The parental animals were examined for their mating and reproductive performances.
- The pups were sexed and were weighed on the day after birth and on day 4 post partum. Their viability was recorded. All pups were necropsied on day 4 post partum and were examined macroscopically for external and visceral findings.

Dose / conc.:

0.106 mg/L air (nominal)

Remarks:

equals 20 ppm

Dose / conc.:

0.319 mg/L air (nominal)

Remarks:

equals 60 ppm

Dose / conc.:

0.956 mg/L air (nominal)

Remarks:

equals 180 ppm

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
0.106 mg/L (ca. 20 ppm) : as the expected no observed adverse effect level
0.319 mg/L (ca. 60 ppm) : as the intermediate dose level
0.956 mg/L (ca. 180 ppm): as the dose level where toxic effects were expected

Preflow period of 4 days

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on each workday in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT:
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well as prior to gross necropsy.

FOOD CONSUMPTION:
- Time schedule: day -7, on day -4 (start preflow period), on day 0 (start of exposure period) and then in weekly intervals.
- It was calculated as mean food consumption in grams per animal and day.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular, haemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, bile acids

URINALYSIS:
- Time schedule for collection of urine: not reported
- Analysis only performed in males
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION:
- Detailed clinical observation (DCO) were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable. The following parameters were examined: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, faeces (appearance/consistency), urine, pupil size
- A functional observational battery (FOB) was carried out on the assigned animals (5 males and 5 females/ test group) on study days 56 and 57 for males and females, respectively. On the days of neurofunctional tests there was no exposure of the concerning animals as well as the other 5 animals of the same test group. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. During the home cage observations attention was paid to posture, tremor, convulsions, abnormal movements, impairments of gait and other findings. During the open field observations the following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within 2 minutes and other findings. After the open field test animals were subjected to the following sensorimotor or reflex tests: approach response, touch response, vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behaviour during “handling”, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test and other finding. All findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity (MA) was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order . The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages ; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring: number and sex of pups, weight gain (The pups were weighed on the day after birth (day 1 p.p.) and on day 4 after birth), stillbirths, live births, postnatal mortality, presence of gross anomalies.

GROSS EXAMINATION OF DEAD PUPS:
yes, for abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals 3 weeks after mating
- Maternal animals: All surviving animals after d4 of gestation

GROSS PATHOLOGY:
The animals were sacrificed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. To prevent post mortem autolysis, the animals that died intercurrently were necropsied as soon as possible after death.

ORGAN WEIGHTS:
The following weights parameters from all animals sacrificed were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, thymus, spleen, brain, heart, lungs

HISTOPATHOLOGY:
-The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumber cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (mandibular and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), skull (with nasal cavities, larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lacrimal glands. Testes, epididymides and ovaries of animals that were killed as scheduled were fixed in Bouin's solution and embedded in paraplast, thereafter . Testes, epididymides and ovaries of animals that died intercurrently were fixed in 4% formaldehyde solution.
- After the organs were fixed, histotechinical processing and examination was were performed as follows: Nasal cavities (level I- IV), Larynx (level I- III), Trachea (longitudinal, with carina), Lungs (5 lobes) and thyroid glands/parathyroid glands in all animals; all gross lesions in all affected animals; evaluations of all other organs and tissues fixed were only performed in animals of the control and high dose group

Postmortem examinations (offspring):

GROSS NECROPSY
All surviving pups (after sacrifice on day 4 p .p . by means of C02), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

Two-sided Dunnett test for food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups
delivered per litter.
Pairwise comparison by the Fisher´s exact test for male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy and urine analysis except vlume, color, turbidity and specific gravity.
Pairwise comparison by the Wilcoxon test for the proportions of affected pups per litter with necropsy observations.
Non-parametric Kruskal-Wallis test (two-sided)/Wilcoxon test for feces, rearing, grip strength forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity for the different time intervals, clinical pathology parameters except differential blood count and organ weights.

Reproductive indices:

- Male mating index (%) = number of males with confirmed mating / number of males places with females x 100
- Male fertility index (%) = number of males proving their fertility / number of males place with females x 100
- Female mating index (%) = number of females mated / number of females placed with males x 100
- Female fertility index (%) = number of females pregnant / number of females mated x 100
- Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100

Offspring viability indices:

- Viability index (%) = number of live pups on day 4 after birth / number of liveborn pups on the day of birth x 100
- Sex ratio (%) – number of live male or female pups on day (0/4) / number of live male and female pups on day (0/4) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the study period one male animal of the control group showed injuries laterally at right flank and on the right head. This injury was of mechanic matter and was therefore not related to the study. One female animal of the low concentration group showed alopecia on dorsal body region and on both forelimbs. This was most likely to be incidental, because alopecia was not observed in other animals of the low and mid concentration group. At the high concentration (0.956 mg/L) male and female animals showed various clinical abnormalities comprising slight to moderate visually increased respiration, eyelid closure, salivation, eye discharge (red) indicating that the test substance was irritating to eyes and upper respiratory tract at this high concentration. Other findings like aggressiveness, apathy (1 female), as well as alopecia and piloerection were more of general nature indicating the bad general state of the animals.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Unspecific clinical symptoms indicative for some irritation and systemic toxicity (comprising visually increased respiration, salivation, piloerection, eyelid closure, eye discharge, alopecia, aggressiveness, hyperactivity and apathy)

Mortality:

mortality observed, treatment-related

Description (incidence):

Two female animals exposed to the high concentration died on study day 88 and 91 (day 18 and 20 of gestation), respectively. Both animals were found pregnant at death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development of the high concentration animals was substantially impaired by the exposure to the test substance. Although the mean body weights on some examination days were not of statistical significance, the significantly reduced mean body weight gains proved the existence of this effect. The retardation of the body weight development is not only of secondary matter due to the reduced food consumption.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Retarded body weight development of the males
- mean body weight: - 8.1 % to - 15.5 % of the control from study day 9 onward (statistically significant to a level of 99 %)
- mean body weight gain: -23.2 % to - 36.3 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Retarded body weight development of the females
- mean body weight: - 3.0 % to -8.0 % of the control from study day 9 onward (statistically significant on study day 51 to a level of 95 %)
- mean body weight gain: -19.9 % to - 41.8 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Significantly (to a level of 99 %) decreased mean terminal body weight in males and in females (- 17.2 % and -9.4 %, resp.)
Reduced body weight development in the dams during pregnancy and lactation
- Average weight gain 61 % less than the control between days 0-20 of pregnancy
- Mean body weight on day 20 of pregnancy 24% below control
- Body weights persisted being 19-23% below control during lactation days 0-4, though weight gain was about 60% above control in these females after cessation of exposure on gestation day 18.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of the high concentration animals was either decreased only marginally and of transient matter, or increased slightly compared to the control.

Food efficiency:

no effects observed

Description (incidence and severity):

The food efficiency of the high concentration animals was, when compared with the control, only reduced transiently at the beginning of the exposure (day 9).

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There are no treatment-related changes in the haematological parameters measured. Clotting analysis revealed prolonged prothrombin times in the blood of the males of the high concentration group at the end of the study.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Regarding clinical pathology findings, in the high concentration group females, indications of a reduced general state was seen, characterized by significantly decreased serum creatinine, total protein, albumin and globulin levels. No treatment-related effects were observed in the clinical pathology parameters of the animals of the low and mid concentration groups.

Summary of the treatment-related findings, test group 3 (0.956 mg/l = 180 ppm):
-Increased prothrombin times, urea*, magnesium*, urinary specific gravity and urinary casts* in the males
-Decreased triglycerides** and urinary volume in the males
-Decreased chloride, creatinine*, total bilirubin, total protein**, albumin* and globulins** in the females
(* statistically significant to a level of 95 %
** statistically significant to a level of 99 %)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Regarding clinical pathology findings, treatment-related effects were observed only in the high concentration groups. The investigations revealed mild impairment of renal function in the males, substantiated by slightly increased urea concentrations in the serum, excretion of decreased urinary volume with increased specific gravity and the presence of urinary casts.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment related findings were observed in the functional observation battery and motor activity examinations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology detected treatment-related hyperplasia of the respiratory epithelium in the anterior part of the nasal cavity (cut level I) of males (higher incidence) and of females (higher grades of severity). One male of the high dose group showed hyperplasia of the respiratory epithelium also in cut level III of the nasal cavity.
Hyperplasia of the respiratory epithelium in the nasal cavity was interpreted as an adaptive, reversible reaction to the inhaled test article.

All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at low or at comparable incidence and graded severity in control and high dose males and/or females. Hence, they were all regarded to have developed fortuitously and unrelated to treatment.

Summary of the treatment-related findings, test group 3 (0.956 mg/l = 180 ppm):
Hyperplasia of the respiratory epithelium in the nasal cavity at level I in male (incidence) and in female rats (graded severity, only)
Hyperplasia of the respiratory epithelium in the nasal cavity at level III in one male rat.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Male cohabitation data
For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index reached 100% in all groups, including the controls.
Fertility could be proven for nearly all F0 parental males within the scheduled mating interval for F1 litter. One mid dose male and one high dose male did not generate F1 pups. Thus, the male fertility index ranged between 89% and 100%. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. For none of the affected male rats conclusive histopathological findings were gathered which could account for the observed impaired fertility.

- Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% for all groups.
The mean cohabitation time (duration until sperm was detected (i.e. day 0 p.c.)) amounted to 3.3 days/2.3 days/2.5 days/2.0 days (0, 0.106, 0.319 and 0.956 mg/l). These values reflect the normal range of biological variation inherent in the strain used in this study. Consequently, the differences between the groups are assessed as spontaneous in nature and without any biological relevance.
All sperm positive rats delivered pups with the following exceptions: one mid dose F0 parental female and one high dose female did not become pregnant. Therefore, the fertility indices ranged between 89% and 100%.

The mean duration of gestation was very similar in the test groups 0-3 (between 21.8 and 21.9 days).

The gestation index was 100% in test groups exposed to 0, 0.106 and 0.319 mg/l, but was markedly reduced to 67% in the high dose group (0.956 mg/L). Only 4 out of 6 surviving presumed pregnant females had liveborn pups in the high dose group.

The number of liveborn and stillborn pups was comparable between the control and test groups 1 and 2, while there were a statistically significantly decreased number of liveborn and an increased number of stillborn pups in test group 3. Thus, the live birth index amounted to 100% in test groups exposed to 0, 0.106 and 0.319 mg/l and 80% in test group 3 (0.956 mg/L).

Dose group (mg/L): 0 - 0.106 - 0.319 - 0.956
cohabitation time (days): 3.3 - 2.3 - 2.5 - 2.0
male mating index (%) : 100 - 100 - 100 - 100
male fertility index (%): 100 - 100 - 90 - 89
female mating index (%): 100 - 100 - 100 - 100
female fertility index (%) 100 100 90 89
duration of gestation (days) 21.8 21.9 21.9 21.8
gestation index (%) 100 100 100 67
pups delivered 11.0 9.7 10.4 8.3
live birth index (%) 100 100 100 80**
Stillborn (%) 0 0 0 20**

(** significant to a level of 99 %)

Dose descriptor:

NOAEC

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

urinalysis

histopathology: non-neoplastic

Remarks on result:

other: marked maternal toxicity, including mortality

Key result

Dose descriptor:

NOAEC

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Test group 3 (0.956 mg/L ): markedly lower viability index (20% vs 97% in the control)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (0.956 mg/L ): lower pup body weights on day 1 p .p . (28% below control ). Average pup body weights and body weight gain significantly below control (42% on
day 4 p.p. and 67% for days 1-4 p.p., respectively)

Sexual maturation:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

0.956 mg/L air (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

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

319 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Study according to OECD TG 413/422, GLP

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No experimental data on isobutyl acrylate are available. Data on the structural analogue, tert-butyl and n-butyl acrylate are included.

 

An extended one generation reproductive toxicity study according to OECD 443 and GLP was performed with the structural analogue n-butyl acrylate. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route.

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 addition another structural analogue,tert-butyl acrylate, was tested in a combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test by inhalation in rats (based on OECD-guidelines 413 and 422). Groups of ten male and ten female Wistar rats were exposed to vapours of tert-butyl acrylate for 6 hours/day and 5 days/week to concentrations of 20, 60 and 180 ppm (corresponding to 0.106, 0.319 and 0.956 mg/L). A concurrent control group was exposed to clean air. The males were treated for approx. 13 weeks (10 weeks premating, 3 weeks mating and post mating). In females treatment lasted from 10 weeks premating, during mating and gestation through day 4 after delivery (approx. 15 weeks). No effects were seen in the low (20 ppm) and mid (60 ppm) dose group. Due to the significant decrease of the mean terminal body weight in animals (males -17.4 %; females - 9.4 %) of the high dose group (180 ppm), some mean relative organ weights were increased, e.g. the relative testes and epididymis weight of males. No morphologic alterations were noted in those organs. For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index reached 100% in all groups, including the controls. One mid dose male and one high dose male did not generate F1 pups. Thus, the male fertility index ranged between 89% and 100%. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The female mating index calculated after the mating period for F1 litter was 100% for all groups. The gestation index was markedly reduced to 67% in the high dose group, only 4 out of 6 surviving presumed pregnant females had liveborn pups. The number of liveborn and stillborn pups, the live birth index amounted to 100% in test groups exposed to 0, 20 and 60 ppm and 80% in the high dose group (180 ppm). Thus, the NOAEC for fertility of the parent animals was determined to be 0.319 mg/L (60 ppm) (BASF, 2004).

Furthermore, a repeated dose toxicity study with n-butyl acrylate according to OECD TG 413 is available. Sprague Dawley rats (20 animals per sex and dose) were exposed by inhalation to measured concentrations of 0, 21, 108, 211 and 546 ppm (corresponding to approx. 0, 0.11, 0.57, 1.11, 2.86 mg/L) for 6 hours per day, 5 days/week for 13 weeks. Clinical, clinico-chemical, haematological, gross-pathological, and histopathological examinations revealed no substance-related effects in the 21 and 108 ppm dose groups. At 211 ppm, the test substance caused eye irritation and irritation of the nasal mucosa. Significant reductions in body weight changes (13.3 %) were observed. In clinico-chemical examinations of females, decreased potassium values and an increase in alkaline phosphatase activity were observed. In the 546 ppm dose group, 31 of 40 animals (77 %) died. Hemorrhagic discharge from eyes and noses and severe dyspnoea were observed, which became constantly more severe. Many clinico-chemical and haematological parameters were affected in animals of this dose group. The animals died during exposure due to strong irritation of the respiratory tract. Metaplasia of the respiratory epithelium as far as the terminal bronchioles and proliferation of the bronchoalveolar epithelium could be detected in histopathological examinations. The NOAEC for this study is 108 ppm (0.57 mg/L/day) and the LOAEC is 211 ppm (1.11 mg/L/day) based on body weight decrease, clinico-chemical changes, and changed organ weights. The NOAEC for local effects (histological changes in the nasal mucosa and olfactory epithelium) is 21 ppm (0.11 mg/L/day) and the LOAEC is 108 ppm (0.57 mg/L/day).No substance-related effect could be determined by organ weight determinations, gross - and histopathology in the reproductive organs of the male and female animals at any concentration. The NOAEC for effects on reproductive organs was 546 ppm (2.86 mg/L), the highest concentration investigated (BASF, 1978).

 

Conclusion:

Based on the data derived from the extended one generation reproductive toxicity study and the sub-chronic toxicity study with n-butyl acrylate adverse effects could neither be observed on the reproductive performance of the animals nor on any reproductive organ. This is supported by a combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test with tert-butyl acrylate. No impairment of reproduction was determined. By cross-reading from the data on tert-butyl acrylate and n-nutyl acrylate it is expected that isobutyl acrylate itself does not exhibit fertility impairing effects, too.

For isobutyl acrylate the systemic DNEL for long-term exposure is based on the OEL of 2 ppm for n-butyl acrylate derived from a 2-year inhalation toxicity study by the German MAK Commission and the EU Scientific Committee on Occupational Exposure Limits (SCOEL). In this study, local irritation of the olfactory epithelium of the nasal mucous membranes at 15 ppm was identified as the most sensitive endpoint. Based on the above mentioned studies, not indicating any suspicion for a reproductive toxic effect at much higher concentrations, a further animal study with isobutyl acrylate will not provide more safety to workers.

Effects on developmental toxicity

Description of key information

No experimental data on isobutyl acrylate are available. Data on the structural analogue n-butyl acrylate and tert-butyl acrylate are included.

Tert-butyl acrylate was tested in a prenatal developmental toxicity study according to OECD TG 414 in rats by oral gavage. Intrauterine growth and survival and fetal morphology (external, visceral, and skeletal) were unaffected by test substance administration at all dosage levels. The NOAEL for embryo/fetal developmental toxicity was considered to be 120 mg/kg/day (BASF 2017).

N-butyl acrylate was tested in a prenatal developmental toxicity study according to OECD TG 414 in rabbit by oral gavage. No test substance-related external, visceral, and skeletal malformations or developmental variations were noted in the offspring at any dosage level. Therefore, a NOAEL of 400 mg/kg bw is derived for both maternal and developmental toxicity (RA TF, 2017).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key 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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 Jan 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of the test material: n-butyl acrylate
- 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 or test system 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:

- Dose selection rationale:
Dosage levels were selected based on the range-finding study. In that study, rabbits were dosed from gestation days 7-28 at dosage levels of 50, 125, 250, and 400 mg/kg/day. There were no test substance-related deaths at any dosage level. Lower mean body weight gains (approximately 18%) and food consumption (approximately 19%) combined with decreased defecation were noted in the 400 mg/kg/day group compared to the control group throughout the treatment period (gestation days 7-29). There were no significant clinical observations noted and no treatment-related findings noted at the macroscopic examinations at any dosage level. Based on these results, the following doses were selected for the current study: 400 mg/kg/day as the high-dosage level because it was expected to produce some maternal toxicity (i.e., decreased body weight gain), 150 mg/kg/day as the mid-dosage level, and 50 mg/kg/day as the low-dosage level because it was not expected to cause any toxic effects.

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 gestation 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. No histopathological examinations were conducted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

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.

Key result

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

Key result

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.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on fetuses

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Summary of maternal survival and pregnancy status

 

Dose Group [mg/kg bw/d]	0		50		150		400
	No.	%	No.	%	No.	%	No.	%
Females On Study	25		25		25		25
Females that aborted or delivered	0	0.0	0	0.0	0	0.0	0	0.0
Females that died	0	0.0	0	0.0	0	0.0	0	0.0
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Females that were euthanized	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Females examined at scheduled necropsy	25	100.0	25	100.0	25	100.0	25	100.0
Nongravid	0	0.0	1	4.0	0	0.0	1	4.0
Gravid	25	100.0	24	96.0	25	100.0	24	96.0
with resorptions only	0	0.0	0	0.0	0	0.0	0	0.0.
with viable fetuses	25	100.0	24	100.0	25	100.0	24	100.0
Total females gravid	25	100.0	24	96.0	25	100.0	24	96.0

 

Table 2: Summary of fetal data at scheduled necropsy

 

Group [mg/kg bw/d]		Sex		Viable fetuses	Dead fetuses	Resorptions		Post implantation loss	Implantation sites	Corpora Lutea	Pre implantation loss	Fetal weight [g]	No. gravid females
		M	F			early	late
0	Total	97	122	219	0	4	1	5	224	249	25	NA	25
	Mean	3.9	4.9	8.8	0.0	0.2	0.0	0.2	9.0	10.0	1.0	42.2
	S.D.	1.88	1.79	2.05	0.00	0.37	0.20	0.41	2.09	1.34	1.68	5.61
	S.E.	0.38	0.41	0.00	0.00	0.07	0.04	0.08	0.42	0.27	0.34	1.12
50	Total	104	110	214	0	7	1	8	222	248	26	NA	24
	Mean	4.3	4.6	8.9	0.0	0.3	0.0	0.3	9.3	10.3	1.1	41.0
	S.D.	1.93	2.19	1.77	0.00	0.62	0.20	0.64	1.75	1.66	1.06	3.78
	S.E.	0.39	0.45	0.36	0.00	0.13	0.04	0.13	0.36	0.34	0.22	0.77
150	Total	105	94	199	0	8	1	9	208	240	32	NA	25
	Mean	4.2	3.8	8.0	0.0	0.3	0.0	0.4	8.3	9.6	1.3	43.1
	S.D.	1.94	1.92	2.44	0.00	0.48	0.20	0.49	2.58	2.10	2.09	4.19
	S.E.	0.39	0.38	0.49	0.00	0.10	0.04	0.10	0.52	0.42	0.42	0.84
400	Total	102	112	214	0	12	1	13	227	250	23	NA	24
	Mean	4.3	4.7	8.9	0.0	0.5	0.0	0.5	9.5	10.4	1.0	41.0
	S.D.	1.65	1.61	1.47	0.00	0.51	0.20	0.59	1.56	1.56	1.30	3.66
	S.E.	0.34	0.33	0.30	0.00	0.10	0.04	0.12	0.32	0.32	0.27	0.75

NA = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, fetal weights compared using Dunnett's test.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2017 to Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 Jan 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Name of the test material: t-Butyl Acrylate
Lot No. 70780336W0
Exp. date: 05 Jan 2018

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC; time-mated rats were received on Gestation Day 1, 2, 3, or 4.
- Age at study initiation: 10–12 weeks
- Housing: individually
- Diet (ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): Reverse osmosis purified (on site) drinking water
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70
- 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 vehicle and test substance formulations were administered orally by gavage, via appropriately sized flexible, disposable, plastic feeding tubes (Instech Laboratories, Inc., Plymouth Meeting, PA), once daily during Gestation Days 6–20. The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Details on analytical verification of doses or concentrations:

Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first 6, 12, and 24 mg/mL dosing formulations and from the middle stratum of the first control group formulation. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated gas chromatography method with flame ionization detection. The analyzed concentrations was 74.5 % to 102 % of the target concentration.

Details on mating procedure:

The time-mated rats were received on Gestation Day 1, 2, 3, or 4, a breeding record was provided by the supplier.

Duration of treatment / exposure:

once daily during Gestation Days 6–20

Frequency of treatment:

once daily during Gestation Days 6–20

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the range-finding study. In this study, rats were dosed from Gestation Day 6 through 20, inclusively, at dosage levels of 60, 120, 240, and 360 mg/kg/day. Mortality and clinical findings of ataxia, clonic convulsions, impaired equilibrium, cool extremities, and/or pale body were noted at 240 and 360 mg/kg/day. Lower body weight gains or body weight losses with corresponding lower food consumption were also noted at 240 and 360 mg/kg/day throughout the dosing period. Microscopic findings were observed in the nonglandular stomach in all test substance-treated groups. Remarkable findings were limited to squamous cell hyperplasia and hyperkeratosis at 60 and 120 mg/kg/day. In the 240 and 360 mg/kg/day groups, increased severity of squamous cell hyperplasia and hyperkeratosis was accompanied by chronic active inflammation and intraepithelial pustules. Focal ulceration of the nonglandular stomach was also observed at 360 mg/kg/day. As a result, dosage levels of 30, 60, and 120 mg/kg/day were selected for the current study.
The selected high dose for the present study represented half of this lethal dose. This approved procedure of decreasing a lethal dose by a factor of two to become the high dose in a subsequent regulatory study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in compliance with EU Directive 86/609/EEC on animal protection.

Maternal examinations:

Clinical Observations and Survival
All rats 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 21 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

Body Weights and Gravid Uterine Weights
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) and 6–21 (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 6–9, 9–12, 12–15, 15–21, and 6–21.
Gravid uterine weight was collected and net body weight (the Gestation Day 21 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–21 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Food Consumption
Individual food consumption was recorded on Gestation Days 6–21 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals

Gestation Day 21 Laparohysterectomy
The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rats were euthanized on Gestation Day 21 by carbon dioxide inhalation. 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 correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. 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. The individual uterine distribution of implantation sites was documented using the following procedure. 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.
The stomach (nonglandular and glandular portions) was preserved in 10% neutral-buffered formalin for microscopic examination as described in Section 4.1.5. In addition, gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

Histology and Microscopic Examinations
After fixation, the stomach (glandular and nonglandular portions) was trimmed according to Charles River SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin.
Microscopic examination was performed on all stomachs (glandular and nonglandular portions) from all animals in the control and 120 mg/kg/day groups at the scheduled necropsy. The nonglandular stomach was also examined in the 30 and 60 mg/kg/day groups

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
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 correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. 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. The individual uterine distribution of implantation sites was documented using the following procedure. 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.

Fetal examinations:

Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the Charles River study number, the female number, and the fetus number. 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 and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
The sex of each fetus was confirmed by internal examination. Approximately one-half of the viable fetuses in each litter were subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. Fetal kidneys were examined and graded for renal papillae development. Heads were placed in Harrison’s fixative for subsequent soft tissue examination by the Wilson sectioning technique
The remaining one-half of fetuses in each litter were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

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, and skeletal), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal Wallis nonparametric ANOVA test to determine intergroup differences. If the 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 observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Observations noted in the treated groups, including brown material around the urogenital area, hair loss on the forelimb(s), and red material around the nose or mouth, 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, 30, 60, and 120 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 30, 60, and 120 mg/kg/day groups were unaffected by test substance administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 30, 60, and 120 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) higher mean food consumption was noted in the 30 mg/kg/day group during Gestation Days 6–9, 15–21 (g/kg/day value only), and when the entire treatment period (Gestation Days 6–21) was evaluated compared to the control group. These differences were sporadic in nature, had no effect on mean body weights or body weight changes, and did not occur in a dose-related manner, and therefore were not considered test substance-related

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:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the nonglandular stomach of all test substance-administered groups. Test substance-related microscopic changes consisted of squamous epithelium hyperplasia and hyperkeratosis with a clear dose-related response. Microscopic findings were most prominent in the 120 mg/kg/day group and consisted of mild squamous epithelium hyperplasia and hyperkeratosis; changes in the 60 mg/kg/day group consisted of minimal to mild squamous epithelium hyperplasia and hyperkeratosis; and microscopic changes in the 30 mg/kg/day group were limited to minimal squamous epithelium hyperplasia and hyperkeratosis. These findings were considered nonadverse. Finally, an individual female in the 60 mg/kg/day group contained minimal mononuclear cell infiltrates in the submucosa; this finding is most likely to be incidental as infiltrates were not identified in the stomach sections of any other animal.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

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):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 30, 60, and 120 mg/kg/day. Parameters evaluated included postimplantation loss, number and percentage of viable fetuses, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: systemic toxicity

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Key result

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:

no effects observed

Description (incidence and severity):

No test substance-related external malformations were noted for fetuses at any dosage level. An omphalocele (several loops of the intestine protruded through an opening in the umbilicus with remnants of a membranous sac) was noted for one fetus in the 60 mg/kg/day group. This finding was not considered test substance-related because it occurred in a single fetus, was not noted in a dose-related manner, and/or the difference in mean litter proportion was not statistically significant compared to the concurrent control group. There were no other external malformations noted in this study.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test substance-related skeletal malformations were noted for fetuses at any dosage level. Fused sternebrae (the left halves of sternebra Nos. 1 and 2 were fused, no costal cartilage associated with the sternum at the normal No. 2 position, left costal cartilage No. 2 associated with sternum in normal No. 3 position, and all subsequent left costal cartilages associated with sternum 1 number position higher than normal) was observed for one feuts in the 120 mg/kg/day group. This finding was not considered test substance-related because it occurred in a single fetus and the difference in the mean litter proportion was not statistically significant compared to the concurrent control group. No other skeletal malformations were noted in this study.
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, and/or the differences in the mean litter proportions were not statistically significant compared to the concurrent control group.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test substance-related external malformations were noted for fetuses at any dosage level. An omphalocele (several loops of the intestine protruded through an opening in the umbilicus with remnants of a membranous sac) was noted for one fetus in the 60 mg/kg/day group. This finding was not considered test substance-related because it occurred in a single fetus, was not noted in a dose-related manner, and/or the difference in mean litter proportion was not statistically significant compared to the concurrent control group. There were no other external malformations noted in this study.

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 303(25), 282(24), 308(25), and 289(24) in the control, 30, 60, and 120 mg/kg/day groups, respectively. Malformations were observed in 0(0), 0(0), 1(1), and 1(1) fetuses (litters) in the same respective groups, and were considered to be spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance treated groups, occurred infrequently or at a frequency similar to that in the control group, and/or did not occur in a dose-related manner. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on fetuses

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Summary of maternal survival and pregnancy status

 

Dose Group [mg/kg bw/d]	0		30		60		120
	No.	%	No.	%	No.	%	No.	%
Females On Study	25		25		25		25
Females that aborted or delivered	0	0.0	0	0.0	0	0.0	0	0.0
Females that died	0	0.0	0	0.0	0	0.0	0	0.0
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Females that were euthanized	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Females examined at scheduled necropsy	25	100.0	1	4.0	25	100 .0	25	100.0
Nongravid	0	0.0	24	96.0	0	0.0	1	4.0
Gravid	25	100.0	0	0.0	25	100 .0	24	96.0
with resorptions only	0	0.0	24	100.0	0	0.0	0	0.0.
with viable fetuses	25	100.0	24	96.0	25	100 .0	24	100.0
Total females gravid	25	100.0	1	4.0	25	100 .0	24	96.0

Table 2: Summary of fetal data at scheduled necropsy

 

Group [mg/kg bw/d]		Sex		Viable fetuses	Dead fetuses	Resorptions		Post implantation loss	Implantation sites	Corpora Lutea	Pre implantation loss	Fetal weight [g]	No. gravid females
		M	F			early	late
0	Total	160	143	303	0	17	1	18	321	355	34	NA	25
	Mean	6.4	5.7	12.1	0.0	0.7	0.0	0.7	12.8	14.2	1.4	5.9
	S.D.	2.45	2.28	2.45	0.00	0.90	0.20	0.89	2.39	2.43	2.84	0.29
	S.E.	0.49	0.46	0.49	0.00	0.18	0.04	0.18	0.48	0.49	0.57	0.06
30	Total	148	134	282	0	19	0	19	301	328	27	NA	24
	Mean	6.2	5.6	11.8	0.0	0.8	0.0	0.8	12.5	13.7	1.1	5.9
	S.D.	2.41	2.28	2.07	0.00	1.22	0.00	1.22	1.77	1.76	2.01	0.33
	S.E.	0.49	0.47	0.42	0.00	0.25	0.00	0.25	0.36	0.36	0.41	0.07
60	Total	150	158	308	0	12	0	12	320	350	30	NA	25
	Mean	6.0	6.3	12.3	0.0	0.5	0.0	0.5	12.8	14.0	1.2	6.0
	S.D.	2.48	2.44	2.78	0.00	1.23	0.00	1.23	2.52	2.31	1.55	0.40
	S.E.	0.50	0.49	0.56	0.00	0.25	0.00	0.25	0.50	0.46	0.31	0.08
120	Total	150	139	289	0	20	0	20	309	327	18	NA	24
	Mean	6.3	5.8	12.0	0.0	0.8	0.0	0.8	12.9	13.6	0.8	5.9
	S.D.	2.29	2.04	1.88	0.00	1.01	0.00	1.01	1.73	1.91	0.90	0.30
	S.E.	0.47	0.42	0.38	0.00	0.21	0.00	0.21	0.35	0.39	0.18	0.06

NA = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, fetal weights compared using Dunnett's test.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 Feb 1979 - 13 Mar 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Guidelines for reproduction studies for safety evaluation of drugs for human use, FDA

Version / remarks:

1966

Qualifier:

according to guideline

Guideline:

other: Guidance on reproduction studies from the Association of the British Pharmaceutical Industry

Version / remarks:

1975

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material: n-Butyl acrylate

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system 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 (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:

air

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

Dose / conc.:

0.13 mg/L air (nominal)

Remarks:

25 ppm

Dose / conc.:

0.71 mg/L air (nominal)

Remarks:

135 ppm

Dose / conc.:

1.31 mg/L air (nominal)

Remarks:

250 ppm

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.

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of corpora lutea and the number of implantations did not show any differences between the individual groups.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

After inhalation of 135 and 250 ppm the percentage of live implantations per pregnant animal was dose-dependent reduced. In the 135 and 250 ppm groups, the percentage of dead resorptions was significantly increased.

Key result

Dose descriptor:

NOAEC

Effect level:

0.13 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEC

Effect level:

0.13 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

early or late resorptions

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on the weight and length of the fetuses was observed.

External malformations:

no effects observed

Description (incidence and severity):

The necropsy of the animals did not reveal any gross-pathological changes of the internal organs which could be attributed to the test substance.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No treatment related malformations and no signs of organ changes or skeletal abnormalities were observed in the fetuses at any concentration.

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment related malformations and no signs of organ changes or skeletal abnormalities were observed in the fetuses at any concentration.

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 live implanations per pregnant animal was dose-dependent reduced. In the 135 and 250 ppm groups, the percentage of dead resorptions were significantly increased. No adverse effect on the weight and length of the fetuses was observed. No treatment related malformations and no signs of organ changes or skeletal abnormalities were observed in the fetuses at any concentration.

Key result

Dose descriptor:

NOAEC

Effect level:

0.13 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Dose descriptor:

NOAEC

Effect level:

1.31 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

 

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 20-29 bred female rats (17-25 pregnant) were exposed to the compound 6h/day on days 6 through 20 of gestation. Control animals were exposed concurrently to filtered room air. The test concentrations of n-butyl acrylate were 100, 200, and 300 ppm (corresponding to approx. 0.52, 1.05, and 1.57 mg/L).

GLP compliance:

not specified

Specific details on test material used for the study:

- Test substance: n-butyl acrylate

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

EXPOSURE
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6-20 m3/h). The chamber temperature was set at 23 ± 2°C, and the relative humidity at 50 ± 5 %. The air-flow rate passed through the fritted disk of a heated bubbler containing the test chemical. Concentrations of acrylate ester were monitored continuously with a gas-chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with carbon disulfide. The resulting samples were analyzed by gas chromatography using appropriate internal standards.

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical concentrations (mean ± SD):
103.3 ± 6.7 ppm (nominal: 100 ppm)
202.8 ± 9.7 ppm (nominal: 200 ppm)
302.5 ± 10.1 ppm (nominal: 300 ppm)

Details on mating procedure:

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

Duration of treatment / exposure:

days 6 to 20 of gestation

Frequency of treatment:

6 hours/day

Duration of test:

until gestation day 21

Dose / conc.:

0.52 mg/L air (nominal)

Remarks:

100 ppm

Dose / conc.:

1.05 mg/L air (nominal)

Remarks:

200 ppm

Dose / conc.:

1.57 mg/L air (nominal)

Remarks:

300 ppm

No. of animals per sex per dose:

20

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
Exposure concentrations were based on preliminary studies in which marked decreases in maternal weight gain were observed at 200 and 300 ppm of butyl acrylate. Results from previous prenatal inhalation toxicity studies on butyl acrylate were also considered (Merkle and Klimisch, 1983). The high concentrations of butyl acrylate for the definitive study (200 and 300 ppm, respectively) were chosen to maximize the opportunity of identifying embryolethal or teratogenic potential.

Maternal examinations:

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

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

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

Ovaries and uterine content:

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

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

Fetal examinations:

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

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

Statistics:

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

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All female rats survived the test.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average maternal body weight gain (gd 6 - 21) was 141, 128, 84 and 18 g for the 0; 100, 200 and 300 ppm dose groups, respectively. These maternal weight gains were markedly lower in the 200 and 300 ppm dose groups as compared to the controls (p < 0.01). Absolute weight gain of dams, expressed as the day 21 body weight minus the gravid uterus weight and minus the day 6 body weight, was significantly reduced in the 100*, 200** and 300** ppm groups, respectively (* for p<0.05; ** for p<0.01), with the absolute weight gains reported to bei 32, 18, -16 and -60 g for the 0, 100, 200 and 300 ppm groups, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Early or late resorptions:

no effects observed

Description (incidence and severity):

No treatment-related effects were reported in terms of numbers of implanation sites or non-live implants.

Dead fetuses:

no effects observed

Details on maternal toxic effects:

No treatment-related effects were reported in terms of numbers of implanation sites, live fetuses, non-live implants or resorptions.

Key result

Dose descriptor:

LOAEC

Effect level:

0.52 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal body weight was significantly reduced at 200 ppm (for both sexes combined and males) and at 300 ppm (both sexes combined, males and females). These decreases amounted to 7-8 % (p<0.05) and 26-28 % (p<0.01) of control values for the 200 and 300 ppm groups, respectively.

Reduction in number of live offspring:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A few sporadic malformations were seen in the 300 ppm and the control group. There was no evidence of treatment-related effects on the incidence of external variations.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of vertebral centra) was similiar in the control and treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A few sporadic malformations were seen in the 300 ppm and the control group. There was no evidence of treatment-related effects on the incidence of external variations.

Key result

Dose descriptor:

NOAEC

Effect level:

0.52 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Maternal body weights:

Concentration [ppm/6h/d]	Maternal body weight GD 6 [g]	Absolute weight gain [g]
0	294 ± 23	32 ± 15
100	289 ± 23	18 ± 14*
200	299 ± 24	-16 ± 20**
300	292 ± 23	-60 ± 26**

Absolute weight gain: (Day 21 body weight) - (gravid uterus weight) - (Day 6 body weight)

Reproductive parameters:

Conc. [ppm/6h/d]	No. of litters	No. of implantation sites/litter	% of non-live implants/litter	% of resorption sites/litter	No. of live fetuses/litter	Average fetal body weight/litter [g]
0	25	15.68 ± 3.17	10.9 ± 15.49	10.64±15.62	14.12 ± 4.01	5.74 ± 0.43
100	24	15.58 ± 3.05	6.82 ± 10.19	6.82 ± 10.19	14.71 ± 3.57	5.71 ± 0.35
200	24	15.08 ± 4.23	4.72 ± 5.96	4.72 ± 5.96	14.46 ± 4.20	5.33 ± 0.40*
300	25	15.40 ± 5.24	6.48 ± 15.94	6.48 ± 15.94	14.68 ± 5.38	4.25 ± 0.94**

Concentration [ppm/6h/d]	0	100	200	300
Mean % of fetuses with:
- any malformations/litter	2.00 ± 7.33	0	0	0.62 ± 2.65
- external variations/litter	1.33 ± 6.67	0	0	0.22 ± 1.11
- visceral variations/litter	8.81 ± 14.64	0	0	4.17 ± 20.41
- skeletal variations/litter	13.70 ± 15.48	17.01 ± 14.53	18.71 ± 24.21	24.65 ± 20.69
- any variations/litter	12.60 ± 10.80	13.27 ± 15.07	10.10 ± 10.32	15.90 ± 19.98

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study according to OECD TG 414, GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

130 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Study reliable with restrictions, acceptable for assessment

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No experimental data on isobutyl acrylate are available. Data on the structural analogue, tert-butyl and n-butyl acrylate are included.

 

Tert. butyl acrylate was tested in a prenatal developmental toxicity study according to OECD TG 414 in rats by oral gavage. The dose selection of 30, 60, and 120 mg/kg/day was done based on a maternal range-finding study (higher doses caused mortality). All females in the control, 30, 60, and 120 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. Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 30, 60, and 120 mg/kg/day groups were unaffected by test substance administration. At the scheduled necropsy on gestation Day 21, no gross observations were considered to be associated with administration of the test substance. Test substance-related squamous epithelium hyperplasia and hyperkeratosis of the nonglandular stomach were observed in the 30, 60, and 120 mg/kg/day groups. These microscopic findings ranged from minimal to mild and increased in severity with increasing dosage levels, but were not considered adverse. No other test substance-related microscopic findings were noted. Intrauterine growth and survival were unaffected by test substance administration. In addition, no test substance-related external, visceral, and skeletal malformations and developmental variations were noted at any dosage level. There were no adverse effects on maternal survival, clinical observations, body weights, food consumption, and gross necropsy or microscopic findings at any dosage level. Based on these results, a dosage level of 120 mg/kg/day was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity when tert‑butyl acrylate was administered orally by gavage to time-mated Crl:CD(SD) rats. Mild epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the mid and high dose level, therefore the NOEL for local effects was set at 30 mg/kg/day. Intrauterine growth and survival and fetal morphology (external, visceral, and skeletal) were unaffected by test substance administration at all dosage levels. Based on these results, a dosage level of 120 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity. (BASF, 2017)

 

Additionally, data regarding developmental toxicity are available for the structural analogue n-Butyl acrylate:

 

For n-butyl acrylate a developmental toxicity study according to OECD TG 414 and GLP was performed. 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 temporary 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 slightly lower mean body weight gain in this group compared to the control group during gestation days 7-10. Mean body weight gains in the 400 mg/kg/day group were similar to the control group for the remainder of the treatment period and therefore were considered non-adverse. 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. In addition, mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all 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 in the offspring at any dosage level. Therefore, a NOAEL of 400 mg/kg bw is derived for both maternal and developmental toxicity based on the absence of adverse effects (BASF, 2017).

 

In addition, two inhalation developmental toxicity studies on rats and one oral developmental toxicity study on mice with n-butyl acrylate are available.

 

Sprague Dawley rats were exposed to n-butyl acrylate vapour concentrations of 25, 135 and 250 ppm (corresponding to approx. 0.13; 0.71; 1.31 mg/L; whole body) for 6 hours per day on days 6 to 15 of gestations. Inhalation of 135 and 250 ppm of the test substance caused a significant reduction in maternal body weight gain, as well as irritation to the nose and eyes. At the end of the exposure period, the weight gain was comparable to the controls. The two highest exposure concentrations caused embryo lethality, as evidenced by a dose-dependent increase in post-implantation loss. The 25-ppm dose did not lead to any signs of maternal toxicity or embryo lethality. No signs of organ changes or skeletal abnormalities 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) (BASF, 1979).

 

In a study where the developmental toxicity of seven acrylates was investigated, groups of 25 pregnant rats were exposed to 0, 100, 200 and 300 ppm n-butyl acrylate (corresponding to approx.0.52, 1.05, and 1.57 mg/L, whole body) for 6 hrs/day from days 6 through 20 of gestation. A NOAEC for maternal toxicity could not be determined since the absolute weight gain was significantly reduced in all dose groups in a concentration-related manner. No treatment-related effects were reported in terms of numbers of implantation sites, live fetuses, non-live implants or resorptions. Fetal body weight was significantly reduced at 200 and at 300 ppm. A few sporadic malformations were seen in the 300 ppm and the control group. There was no evidence of treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of vertebral centra) was similar in the control and treated groups. No NOAEC could be derived for maternal toxicity. The LOAEC for maternal toxicity was 100 ppm (0.52 mg/L), the NOAEC for developmental effects (fetotoxicity) was 100 ppm (0.52 mg/L), and the NOAEC for developmental effects (teratogenicity) was the highest concentration tested of 300 ppm (1.57 mg/L) (Saillenfait, 1999).

 

In a gavage study, pregnant CD-1 mice were administered n-butyl acrylate dissolved in cottonseed oil. Concentrations of 0, 100, 1000, 1500, 2000, 2500, 3000 and 4000 mg n-butyl acrylate/kg bw were administered from gestation day 6 to 15. No animals survived in the high dose group. At 3000 and 2500 mg/kg bw/day 2 of 30 animals died; at 2000 mg/kg bw/day 1 of 29 died; at 1500 mg/kg bw/day 1 of 27 died; and at 1000 mg/kg bw/day 1 of 30 died. At the 1500 mg/kg bw/day dose and higher, average maternal body weight gain was significantly reduced. Fetal body weights were significantly reduced at doses of 1500 mg/kg bw/day and above. At 2500 and 3000 mg/kg bw/day, the percentage of resorptions was significantly increased. At 100, 1000, 1500 and 2000 mg/kg bw/day, and in the control group, variations and malformations occurred sporadically on 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, with a slight dose-dependent increase when taking the sum of all events per dose group together. In the 2500 mg/kg bw/day and 3000 mg/kg bw/day groups, the number of fetuses with external and skeletal malformations and variations (cleft palate, exencephaly, open eyes, fused arches, fused ribs) was significantly increased. Taking maternal mortality and reduced weight gain into account the NOAEL for maternal toxicity was 100 mg/kg bw/day. The NOAEL for developmental toxicity was 1000 mg/kg bw/day and the NOAEL for teratogenicity was 2000 mg/kg bw/day. The observed effects on the offspring are most likely the result of excessive maternal toxicity and therefore a secondary effect (Rohm and Haas, 1979).

 

Conclusion

The overall evidence based on the data on the read-across substances tert-butyl and n-butyl acrylate provides no indications that these substances exhibit a teratogenic potential. Only in overt maternal toxic concentrations /doses fetotoxic effects were described. Therefore, isobutyl acrylate is also considered not to impair embryonic and fetal development at dose levels not inducing maternal toxicity.

 

For isobutyl acrylate the systemic DNEL for long-term exposure is based on the OEL of 2 ppm for n-butyl acrylate derived from a 2-year inhalation toxicity study by the German MAK Commission and the EU Scientific Committee on Occupational Exposure Limits (SCOEL). In this study, local irritation of the olfactory epithelium of the nasal mucous membranes at 15 ppm was identified as the most sensitive endpoint. Based on the above mentioned studies, not indicating any suspicion for a teratogenic effect in much higher concentrations, a further animal study with isobutyl acrylate will not provide more safety to workers.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data on reproductive and developmental toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the substance is considered to be classified neither for reproductive nor for developmental toxicity under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.