Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

The results of an OECD Guideline 422 study with hydroxypropyl acrylate and 2-generation-studies (OECD Guideline 416) with the structural analogues acrylic acid (CAS No. 79-10-7) and methyl acrylate (CAS No. 96-33-3) in rats by the oral and inhalation route and the continuous breeding study with the metabolic cleavage product propylene glycol gave no indication of a fertility impairing effect.

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Chales River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Age at study initiation: 11 weeks (male animals); 10 weeks (female animlas)
- Housing: During the pretreatment period of the study, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Ho
henpeißenberg, Germany. During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water for a period of 7 days at room temperature were carried out.
Duration of treatment / exposure:
After the acclimatization period, the test substance was administered to the parental animals orally by gavage.
Frequency of treatment:
once daily at approximately the same time in the morning
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

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

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

Body weight data
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume;

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

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

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

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

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

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

Clinical Pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

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

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

Thyroid Hormones
Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
All generated serum samples were frozen at -80°C until measurement.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).
T4 ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Oestrous cyclicity (parental animals):
For all females in a pool of 50 animals, estrous cycle normality was evaluated before randomization (the estrous cycle data of these individuals are not reported and can be found in the raw data). For a minimum of 2 weeks prior to mating, estrous cycle length and normality was evaluated by daily analysis of vaginal smears for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, one additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Litter observations:
Litter observation:
Litter data
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Pup Necropsy observations“. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying
between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13.

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data
The pups were weighed on the day after birth (PND 1) as well as on PND 4 (before
standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females. „Runts“ were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

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

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

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

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All gross lesion, Adrenal glands, Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer`s patches, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid , glands, Trachea, Urinary bladder, Uterus, Vagina.
Postmortem examinations (offspring):
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing.
Statistics:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Reproductive indices:
The following indeces were determined: mating and fertility indes for both males and females, gestation index, live birth index and postimplantation loss for female.
Offspring viability indices:
The following indeces were determined: viability index, survival index, sex ration, anogenital index.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most animals of test group 3 as well as several animals of test group 2 (150 and 50 mg/kg bw/d) show ed salivation after treatment (grade: slight to severe) at some occasions during the study period. In female animals the incidence was generally higher during gestation and lactation periods, affecting most test group 3 and several test group 2 females. These observations were considered to be treatment-related.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups. Spontaneous findings were seen in one high-dose male (No. 33) which showed red discharge in the right eyeduring mating days 2 - 4, 8 - 11 and postmating days 0 – 1 as well as one sperm positive mid-dose female (No. 121) which did not deliver F1 pups.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups showed no significant difference to the concurrent control during the entire study.
The statistically significantly lower body weight change in the mid-dose females during PND 7 - 10 was considered as spontaneous in nature and not as treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the F0 males in all dose groups (15, 50 and 150 mg/kg bw/d) as well as low and mid-dose females was not influenced by the treatment throughout the study.
Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values during the entire premating period (up to 7%) while it remained unchanged during gestation and lactation periods.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test group 3 (150 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significant ly reduced, but the mean was within the historical control range (males, HQT 37.4 - 40.9 sec). Therefore, this alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test groups 1 and 2 (15 and 50 mg/kg bw/d) cholesterol values were significantly changed (test group 1 lower, test group 2 higher) compared to controls, but the alteration was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatmentrelated.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery (FOB)
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations: Two male animals of dose group 3 (Nos. 33 and 34 - 150 mg/kg bw/d) showed slight (area around the
mouth was moist) and severe (mouth very wet, wet paws) salivation, respectively. All other male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) did not show any abnormalities.
Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test
groups. The statistically significantly higher value of the landing foot splay test in females of dose group 2 was considered as spontaneous in nature and not treatment related.
Motor activity measurement (MA)
No treatment-related changes of motor activity data was observed in all male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) in comparison to the concurrent control group. Overall activity levels and habituation to the test environment corresponded to the age of these animals, if usual biological variation inherent in the strain of rats used for this experiment was considered.
The isolated statistically significantly decreased numbers of beam interrupts in the males of dose groups 1 and 2 during interval 12 were not related to the dose and did not influence the overall activity levels and habituation. Thus, they were not considered to be related to the test substance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in forestomach and duodenum of male and female animals of test groups 2 and 3.
Duodenum: In the duodenum, a thickening of the mucosa characterized by an increase in villus height, was observed, which correlated to the macroscopic finding “dilation”. One female animal of test group 3 showed a focal hyperplasia of the duodenal mucosa.
Forestomach: Diffuse squamous hyperplasia (correlating with the macroscopic findings “margo plicatus, thickened”) and the presence of erosion/ulcer (correlating often with the macroscopic finding “focus”) were noted in the forestomach of male and female animals.
No treatment-related findings were noted in the glandular stomach and no correlate was found for the macroscopically observed discoloration in two test group 3 male animals. All other findings occurred either individually or were biologically equally distributed over control
and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.The morphology of the testicular interstitium and the stages of spermatogenesis in the testes of males of the high dose (150 ppm) were comparable to those of the controls.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 4.0 days in test groups 0 - 3, respectively.
Reproductive performance:
no effects observed
Thyroid hormones:
In the F0 parental males of test groups 2 and 3 (50 and 150 mg/kg bw/d) T4 values were significantly higher compared to controls. However, the means were within the historical control range (males T4 44.87-88.29 nmol/L). Additionally, neither a change of thyroid weight nor any histopathological findings in the thyroids were noted. Therefore, the higher T4 values in parental males were regarded as incidental and not treatment-related.
In male and female pups at PND13 (test groups 11, 12 and 13; 15, 50 and 150 mg/kg bw/d), no treatment-related alterations of T4 levels were observed.

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

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until copulation was confirmed (GD 0) varied between 2.1 and 5.3 days without showing a statistically significant difference. The reason for the higher average in test group 3 were 3 mating pairs with copulation confirmed after 12 or 14 days of pairing, respectively. All 3 affected females had normal estrous cycles and normal pregnancies, and no findings were noted in the reproductive organs of the affected males and females. The very extensive period before copulation fits best to artificial pseudo-pregnancies provoked by the daily manipulations during vaginal lavage. None of this is considered to be treatment-related.
All female rats delivered pups or had implants in utero with the following exceptions: Mid-dose female No. 121 (mated with male No. 21) did not become pregnant.
The fertility index varied between 90% in test group 2 and 100% in the control and test groups 1 and 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non-pregnant female had no relevant gross lesions or microscopic findings.
The mean duration of gestation values varied between 21.9 (test group 1), 22.2 (control and test group 2) and 22.4 (test group 3), not indicating any influence by the test substance.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.8 / 13.0 / 13.2 and 14.3 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.1 / 12.6 / 12.6 and 13.9 pups/dam in test groups 0 - 3, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 100% and 99.3% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance Hydroxypropylacrylate did not adversely affect reproduction and delivery of the F0 generation parental animals.
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed.
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: irritation in forestomach and duodenum
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
PND 0: 1 male pup died in test group 0 and 3
PND 1-4: 1 male and 1 female pup died in test group 1; 2 male pups died in test group 2
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1 - 3 (15, 50 and 150 mg/kg bw/d).
One female runt was seen in the control, one male and two female runts were seen in test group 1, three male and two female runts were seen in test group 2 and one male and two female runts were seen in test group 3. These are spontaneous findings unrelated to the treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored testis (left, red) and dilated renal pelvis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Litter data
Pup number and status at delivery
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
Pup viability
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100%/ 98.6% /97.9% and 100% in test groups 0 - 3, respectively. The survival index indicating pup mortality until mid-lactation (PND 4 - 13) was 100% in all test groups.
Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Anogenital distance/anogenital index
No test substance-related effects were noted on anogenital distance or anogenital index in all treated F1 offspring (test groups 1 - 3 [15, 50 and 150 mg/kg bw/d]).
Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL for fertility and reproductive performance was 150 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/d.
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 19, 1992 to April 19, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar rats (Chbb = THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: (P): 35 ± 1 days
- Weight at study initiation: (P) Males: 140.0 (127 - 154) g; Females: 118.8 (106 - 130) g
- Housing: Single in type DK III stainless steel wire mesh cages, with the following exceptions: during mating periods, the males designated for mating were kept individually in Makrolon cages, type M III; for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages.
- Diet (ad libitum): Kliba maintenance diet rat/ mouse/hamster GLP 343 meal (KLINGENTALMUEHLE AG, Kaiseraugst, Switzerland)
- Water (ad libitum): Tap water
- Acclimation period: 8 days

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

Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 3 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of Acrylic acid in the aqueous solutions was determined by gas chromatography.
Duration of treatment / exposure:
Exposure period: F0: at least 70 days before the mating and afterwards during the gestation and lactation periods.
F1: at least 98 days before the mating and afterwards during the gestation and lactation periods.
Premating exposure period (males): at least 70 days
Premating exposure period (females): at least 70 days
Duration of test: approx. 12 months
Frequency of treatment:
continuously
Dose / conc.:
500 ppm
Remarks:
corresponding to approx. 53 mg/kg bw/day
Dose / conc.:
2 500 ppm
Remarks:
corresponding to approx. 240 mg/kg bw/day
Dose / conc.:
5 000 ppm
Remarks:
corresponding to approx. 460 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Positive control:
none
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. Particular attention was given to the nesting, littering and lactation behaviour of the dams, but only special findings were documented.

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

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


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


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

ITx = WCx * D / BWy

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


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

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

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

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

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

Postmortem examinations (parental animals):
SACRIFICE
Parental animals were killed by decapitation under CO2 anaesthesia and examined macroscopically.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys and stomach (non-glandular and glandular).
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by CO2 asphyxiation, examined externally, eviscerated and their organs assessed macroscopically.


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


HISTOPATHOLOGY
Vagina, cervix, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulation gland, oesophagus and duodenum.
Statistics:
The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: Dunnett test, Fisher`s exact test and Wilcoxon test.
Reproductive indices:
Mating and fertility indices were calculated according to following formulas:

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

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

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

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

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

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

Remark:
Day 4 after birth preceded standardization of the litters.
Day 21 after birth followed standardization of the litters.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
**** F0 GENERATION PARENTAL ANIMALS ****

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

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

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

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

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

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


GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity
Dose descriptor:
NOAEL
Effect level:
460 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: fertility
Remarks on result:
other: no adverse effects observed up to and including the highest tested dose
Critical effects observed:
no
**** F1 GENERATION PARENTAL ANIMALS *****

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

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

GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.
Dose descriptor:
NOAEL
Effect level:
460 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: fertility
Remarks on result:
other: no adverse effects observed up to and including the highest tested dose
Dose descriptor:
NOAEL
Effect level:
53 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
**** F1 GENERATION PUPS/LITTERS ****

VIABILITY
No substance-induced effects on pup mortality/viability were recorded during the lactation period. Both the viability index, as an indicator of the viability of the pups during the first 4 days after birth, and the lactation index, as an indicator how the pups were nursed during the rest of their rearing, do not show differences of biological relevance.
 
SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 post parturition (pp) did not show any substantial difference between controls and treated groups; all differences observed are regarded as spontaneous.
 
BODY WEIGHT
Mean body weights of F1 male and female pups were clearly reduced in 5000 ppm test group from day 14 pp onwards and impaired in the intermediate dose (2500 ppm) on day 21 pp when compared to the controls. On day 21 pp the pup weights (both sexes combined) in the high dose group were about 35% and those of the 2500 ppm pups about 11% lower than the corresponding control values. Body weight gains of the 2500 ppm and 5000 ppm pups were also statistically significantly decreased from days 7 (5000 ppm) or 14 pp (2500 ppm) up to weaning (day 21 pp). The reductions in pup body weights/body weight gains in the 5000 and 2500 ppm groups were attributed to the test substance administration. All other differences concerning pup body weights/body weight gains are without any biological relevance and lie within the biological range of variation.
 
CLINICAL OBSERVATIONS
- None of the F1 pups of any one group showed abnormal clinical findings during the lactation period.
- There were no biologically relevant differences between the control and the substance-treated F1 pups in the several morphological development stages monitored up to weaning.
- No remarkable differences between the groups were observed in the different behavioral tests which the pups underwent up to weaning.
- Only spontaneous findings were seen at necropsy (e.g. incisors sloped, hernia diaphragmatica, dilated renal pelvis) in very few of the pups examined. All findings were present in the concurrent control at a comparable frequency and/or did not show a clear relation to dosing.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
53 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
460 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: fertility
Remarks on result:
other: no adverse effects observed up to and including the highest tested dose
Critical effects observed:
no
**** F2 GENERATION PUPS/LITTERS ****

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

Mean Body Weight Changes (F0 parental animals), grams

Treatment week

 0 mg/kg bw/d

 53 mg/kg bw/d

 240 mg/kg bw/d

 460 mg/kg bw/d

male

female

male

female

male

female

male

female

0 - 1

52.6

23.8

52.2

24.8

52.3

23.6

46.7**

23.1

1 - 2

53.7

22.1

55.5

22.0

54.4

22.3

51.6

21.8

2 - 3

47.2

16.2

45.8

17.2

46.9

17.5

44.8

14.6

3 – 4

35.4

12.9

34.8

17.1*

35.1

14.9

32.4

17.8*

4 – 5

29.1

15.7

27.6

13.3

28.6

12.6

27.0

13.7

5 – 6

21.0

9.4

21.8

8.8

23.3

12.5

23.2

11.0

6 – 7

23.2

8.5

24.7

11.7

21.2

10.1

19.3*

10.7

7 – 8

20.4

7.7

17.7

10.3

19.8

7.8

18.5

8.6

8 – 9

19.3

9.8

19.1

7.1

18.0

7.2

16.6

8.1

9 – 10

13.1

4.7

13.0

4.5

14.6

7.9*

11.9

6.0

10 – 11

-7.0

 

-2.8

 

-2.8

 

-2.8

 

11 – 12

21.8

 

18.5

 

18.4

 

14.7**

 

12 – 13

14.8

 

13.3

 

13.7

 

11.1

 

13 – 14

8.0

 

10.7

 

9.7

 

6.1

 

14 – 15

9.2

 

7.0

 

7.5

 

8.1

 

15 – 16

10.5

 

10.4

 

8.0

 

9.9

 

16 – 17

5.7

 

7.9

 

7.3

 

4.6

 

17 – 18

2.7

 

4.6

 

1.7

 

1.4

 

18 – 19

1.1

 

0.2

 

1.3

 

1.6

 

19 - 20

4.1

 

6.7

 

5.8

 

5.0

 

*P<0.05

**P<0.01

Reproduction and litter data for F0 parents /F1 pups

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Parents

Females mated

25

25

25

25

Females pregnant

24

23

23

24

Females with delivery

24

23

23

24

Mean duration of gestation (d)

22.0

21.9

21.9

21.9

Litter means

Live births/litter

13.8

13.8

13.7

14.3

Survivors day 4 preculling

13.3

13.5

13.4

13.8

Survivors day 4 postculling

7.5

7.9

7.9

7.9

Survivors day 21

7.5

7.9

7.8

7.8

Weight at day 1 (g) M/F

6.4/6.1

6.6/6.2

6.5/6.2

6.5/6.2

Weight at weaning (g) M/F

52.3/50.01

52.1/49.4

46.6**/44.6**

34.2**/32.7**

Sex ratio of live newborns % M/F

51/49

49/51

55/45

53/47

Selected as parents for the next generation M/F

25/25

25/25

25/25

25/25

**P≤0.01

Reproduction and litter data for F1 parents /F2 pups

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Parents

Females mated

25

25

25

25

Females pregnant

23

23

23

24

Females with delivery

23

23

23

24

Mean duration of gestation (d)

22.0

21.9

21.9

21.9

Litter means

Live births/litter

12.5

11.6

12.0

13.8

Survivors day 4 preculling

11.8

10.6

10.8

12.5

Survivors day 4 postculling

7.7

7.0

7.5

7.8

Survivors day 21

7.7

6.9

7.5

7.4

Weight at day 1 (g) M/F

6.3/5.9

6.5/6.2

6.1/5.8

6.4/6.1

Weight at weaning (g) M/F

50.4/48.4

52.1/49.4

44.6**/42.4**

34.5**/33.2**

Sex ratio of live newborns % M/F

47/53

55/45

53/47

49/51

**P≤0.01

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

Parameter

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Historical control range

Pinna unfolding

93.6 (16.65)

93.5 (20.87)

80.7 (33.37)

86.9 (26.87)

74-100

Auditory canal opening

98.8 (3.87)

95.1 (20.90)

91.4*(18.09)

94.2 (12.26)

81-100

Eye opening

93.2 (18.08)

92.4 (21.89)

92.4 (21.89)

86.5*(21.15)

85-100

*P≤0.05

Figures in parentheses indicate standard deviations.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Anogenital distance, a triggered end point as per test guidelines, was not measured in the F2 pups because there were no significant effects observed on F1 sex ratio or age at vaginal opening or preputial separation.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
See above
Qualifier:
according to
Guideline:
other: JMAFF, Guideline 2-1-17, Reproduction Study (2000)
Deviations:
yes
Remarks:
See above
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River Laboratories Inc. (Portage, Michigan)
Age: Approximately six weeks at initiation of treatment.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Results Continued in Remarks Section (below)
Dose descriptor:
NOEC
Remarks:
reproductive toxicity
Effect level:
ca. 0.269 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 75 ppm; the highest concentration tested.
Remarks on result:
other: no adverse effects observed up to and including the highest dose tested
Dose descriptor:
NOEC
Remarks:
parental local toxicity
Effect level:
ca. 0.019 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 5 ppm; based on histologic changes in nasal tissues.
Dose descriptor:
NOEC
Remarks:
repoductive toxicity
Effect level:
ca. 0.269 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 75 ppm; the higest concentration tested
Remarks on result:
other: no adverse effects observed up to and including the highest dose tested
Dose descriptor:
NOEC
Remarks:
parental local toxicity
Effect level:
ca. 0.019 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 5 ppm; based on histologic changes in nasal tissues.
In-Life Observations

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Gross Pathology
There were no treatment-related gross pathologic observations in F1 weanlings at any exposure level. In F2 weanlings, 2/81 males and 3/78 females from the 75 ppm exposure group had necrosis of the tail. This observation may have been related to treatment, but the significance of the tail necrosis is not known. All other gross pathologic observations from F1 and F2 weanlings were considered to be spontaneous alterations, unassociated with exposure to methyl acrylate.
Dose descriptor:
NOEC
Remarks:
developmental toxicity
Generation:
F1
Effect level:
ca. 0.092 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 25 ppm; based on decreases in pup weight at 75 ppm which were secondary to prenatal toxicity
Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Generation:
F2
Effect level:
ca. 0.092 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 25 ppm; based on decreases in pup weight at 75 ppm which were secondary to parental toxicity.
Reproductive effects observed:
no

Results- Continued

Histopathologic Nasal Tissue Effects

P1 Females

Dose (ppm) 0 5 25 75

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

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

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

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

                                                                -slight 0 0 0 19a

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

                                                                                                     -moderate 0 0 0 19a

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

                                                                         -slight 0 0 2a 3a

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

                                                                                                                -slight 0 0 13a 1a

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

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

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

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

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

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

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

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

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

 

Histopathologic Nasal Tissue Effects

P2 Males

Dose (ppm) 0 5 25 75

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

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

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

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

                                                                -slight 0 0 0 13a

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

                                                                                                    -moderate 0 0 0 14a

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

                                                                         -slight 0 0 2a 19a

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

                                                                                                                -slight 0 0 0 3a

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

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

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

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

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

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

Histopathologic Nasal Tissue Effects

P2 Females

Dose (ppm) 0 5 25 75

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

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

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

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

                                                                -slight 0 0 0 12a

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

                                                                                                     -slight 0 0 0 14a

                                                                                                     -moderate 0 0 0 12a

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

                                                                        -slight 1 0 0 1a

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

                                                                                                                 -slight 3 1 5 2

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

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

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

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

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

---

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

 

Sperm Parameters

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

 

Estrous Cyclicity

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

 

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

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

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

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

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

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

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

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

Endpoint:
two-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, published in peer-reviewed literature, minor restrictions in design and reporting, but otherwise adequate for assessment.
Qualifier:
according to
Guideline:
other: NTP Reproductive Assessment by Continuous Breeding (RACB)
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY.
- Age at study initiation: 6 weeks
- Housing: group housed by sex in solid-bottom polypropylene or polycarbonate cages with stainless-steel wire lids, during the quarantine and the 1-week premating periods. subsequently, the animals were housed as breeding pairs or individually
- Diet (e.g. ad libitum): ground rodent chow, ad libitum
- Water (e.g. ad libitum): deionized/filtered water

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

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each concentration was mixed separately at periodic intervals.

VEHICLE
Water
- Concentration in vehicle: 1.00, 2.50 and 5.00%
Details on mating procedure:
In the continuous breeding study mice were exposed to the test substance for 7-day premating period, and were then randomly grouped as mating pairs and cohabited and treated continuously for 98 days. Data were collected on all newborns during this period within 12 hours of birth, after which each litter was discarded. After the 98-day cohabitation, the pairs were separated but continued on treatments. During the next 21 days, any final litters were delivered and kept for at least 21 days (weaning). The mother was dosed through weaning and F1 mice were dosed until mated at 74 ± 10 days of age. For this, male offspring were mated to female off-spring from the same treatment group (n = 20/group/sex) and the F2 litters were examined for litter size, sex and pup weight.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
14 days in the dose range-finding study; 7 days pre-mating period, 98 days (14 weeks) cohabitation, 21 days post-cohabitation. Any litters delivered during these 21 days (second generation animals) were kept for at least 21 days (weaning) and continued receiving the chemical treatment initially through lactation (mother was dosed throughout) and then through drinking water. Second generation animals were mated at 74 ± 10 days of age.
Frequency of treatment:
Daily
Details on study schedule:
Other: A 14-day dose-setting study utilized one control group and 5 groups of dosed animals (8/sex/dose). The endpoints for this study were clinical signs, mortality, body weight gain and consumption of food and water.
In the main study, the litters were removed and examined as soon as possible after delivery was completed. The offsprings were sexed, weighed and killed so that the female may be impregnated immediately. This approach maximized the number of litters which could be produced in the 14-week breeding phase.
Remarks:
Doses / Concentrations:
0, 1.82, 4.80 and 10.10 g/kg bw/day (main study)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 0.5, 1.0, 2.5, 5.0 and 10% in water (dose-range finding study)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
5% in water (second generation animals)
Basis:
nominal in water
No. of animals per sex per dose:
Dose range-finding study: 8/sex/dose
Main study: 20/sex/dose in each treatment group; 40/sex/dose in the control group
Second generation animals: 20/sex/dose (only the highest dose (5% in water) was tested).
Control animals:
yes
Details on study design:
- Dose selection rationale: based on a 14-day dose-selecting study
- Rationale for animal assignment: stratified randomization procedure based on body weights.

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

Positive control:
Diethylstilbestrol and ethylene glycol monoethyl ether
Parental animals: Observations and examinations:
Clinical signs, mortality, body weight gain and consumption of food and water were assessed during the 14-day dose-setting study.
Oestrous cyclicity (parental animals):
Not performed.
Sperm parameters (parental animals):
As no adverse effects on fertility were observed in the continuous breeding study, the subsequent substudy (crossover mating with subsequent examination of male reproductive function in F0 animals) was not performed.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
mean No. litters per pair, mean No. live pups per pair, mean No. live male pups per litter, mean No. live female pups per litter, proportion of pups born alive, sex of pups born alive, mean live pup weight per litter, mean live male pup weight per litter, mean live female pup weight per litter; adjusted mean live pup weight per litter; adjusted mean live male pup weight per litter; adjusted mean live female pup weight per litter, body weight.



Postmortem examinations (parental animals):
As no adverse effects on fertility were observed during the continious breeding study, subsequent crossover mating with subsequent necropsies of F0 animals was not performed.
Postmortem examinations (offspring):
Not performed.
Statistics:
Dose-related trend in fertility: the Cochran-Armitage test (Armitage, 1971)
Pairwise comparisons involving mating and fertility indices: Fisher's exact test
Overall differences in number of litters, number of live pups, proportion of live pups and the sex ratio for dose group means: Kruskal-Wallis test (Conover 1980)
Ordered differences in number of litters, number of live pups, proportion of live pups and the sex ratio for dose group means: Jonckheere's test (Jonckheere, 1954)
Pairwise comparisons of treatment group means: Wilcoxon-Mann-Whitney U test
Average pup weight: Kruskal-Wallis test
Treatment differences in average pup weight: analysis of covariance (Neter and Wasserman, 1974)
Pairwise comparisons: two-sided t test
Organ weights: analysis of covariance F test (overall equality) and t test (pairwise equality)
Reproductive indices:
Fertility index was determined as (No. fertile/No.cohabited) x 100
Mating index was determined as No. females with plugs/No. cohabited
Offspring viability indices:
No data.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No chemical-related deaths were observed in either a dose-range finding study or during the 14 weeks cohabitation period in the main study. No significant chemical related clinical signs of toxicity were noted during the main study cohabitation phase.

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

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

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

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

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

Dose descriptor:
NOAEL
Remarks:
toxicity
Effect level:
10 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects reported at the highest dose tested.
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Propylene glycol had no apparent effect on pup survival.

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

ORGAN WEIGHTS (OFFSPRING)
For the second generation animals, the absolute as well as adjusted organ weights for the liver, kidney, seminal vesicles, right cauda, prostate, right testis and right epididymis were not affected by propylene glycol treatment.
Dose descriptor:
NOAEL
Remarks:
fertility
Generation:
F1
Effect level:
10 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects on fertility of F1 generation were observed at the highest dose.
Dose descriptor:
NOAEL
Remarks:
developmental effects
Generation:
F2
Effect level:
10 100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No effects on litter size, sex and pup weight in F2 pups were observed at the highest dose.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD Guideline 422:

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

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

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

OECD Guideline 416:

No reproductive toxicity studies including more generations are available for hydroxypropyl acrylate. From the structurally analogous substances acrylic acid (CAS No. 79-10-7) and methyl acrylate (CAS No. 96-33-3) 2-generation-studies are available.

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

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

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

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

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

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

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

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

Data from the structural analogue methyl acrylate (CAS No. 96-33-3):

In a two-generation study according to OECD TG 416 groups of 27 male and female Crl:CD(SD) rats were whole-body exposed to methyl acrylate vapours at target concentrations of 0, 5, 25, and 75 ppm for six hours/day, seven days/week, resulting in actual average concentrations of 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm, respectively (corresponding to approx. 0, 0.019, 0.092, and 0.269 mg/L).Rats were exposed daily for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. Maternal rats were not exposed after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Exposure of maternal rats continued from LD 5 – LD 28. In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Treatment-related effects in parental rats exposed to 75 ppm included decreased body weight and feed consumption in males and females throughout most of the two generation study. There were no effects on body weight or feed consumption at 25 or 5 ppm. Treatment-related, adverse histopathologic effects were present in the nasal tissues of P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of the nasal effects were concentration dependent. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. Very slight olfactory epithelial degeneration, without accompanying regenerative hyperplasia, was noted in some of the P1 and P2 females and P2 males exposed to 25 ppm. There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. Very slight or slight chronic-active inflammation was present in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or two males and females from both generations exposed to 25 ppm. Very slight necrosis of individual olfactory epithelial cells was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. Very slight mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, and in 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. Other nasal effects consisted of an increase in the incidence of very slight or slight hyperplasia of the transitional epithelium in P1 and P2 males and females exposed to 25 or 75 ppm, and an increase in the incidence of very slight or slight hyperplasia and hypertrophy of the respiratory epithelium in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm. There were no treatment-related histopathologic effects in P1 or P2 animals exposed to 5 ppm.

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

In summary, the no-observed-effect concentration (NOAEC) for parental systemic toxicity was determined to be 5 ppm (= ca. 0.018 mg/L) and was based on histologic changes in the nasal tissues seen at higher concentrations. The NOAEC for developmental toxicity was 25 ppm (= ca. 0.089 mg/L), based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOAEC for reproductive toxicity was 75 ppm (= ca. 0.268 mg/L), the highest concentration tested.

Continuous breeding study

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

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

Repeated dose toxicity: Oral route

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

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

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

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

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

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

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

Conclusion:

The results of the OECD Guideline 422 study with hydroxypropylacrylate and 2-generation-studies (OECD Guideline 416) with the structural analogues acrylic acid (CAS No. 79-10-7) and methyl acrylate (CAS No. 96-33-3) in rats by the oral and inhalation route gave no indication of a fertility impairing effect caused by hydroxypropyl acrylate. In addition, no indications of a possible reproductive toxicity were determined in a continuous breeding study with the metabolic cleavage product propylene glycol.

Effects on developmental toxicity

Description of key information

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

No indications of a developmental toxic or teratogenic effect were seen in a rat study with hydroxypropyl acrylate, which is supported by developmental toxicity studies in rabbits as a second species, conducted with the structural analogues acrylic acid (CAS No. 79-10-7) and methyl acrylate (CAS No. 96-33-3) and the data in rats, mice, rabbits and hamsters with propylene glycol CAS No 57-55-6).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication, comparable to current guidelines
Principles of method if other than guideline:
Groups of 20-29 bred female rats (17-25 pregnant) were exposed to the compound 6h/day on days 6 through 20 of gestation by inhalation. Control animals were exposed concurrently to filtered room air. The test concentrations were 1, 5, and 10 ppm (corresponding to approx. 5.4, 27.0, and 54.0 µg/L)*.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l' Arbresle, France)
- Age at study initiation: Young, nulliparous females
- Weight at study initiation: 200-220 g
- Housing: Single in clear polycarbonate cages with stainless-steel wire lids and hardwood shaving as bedding.
- Diet: Food pellets (UAR Alimentation Villemoisson, France), ad libitum
- Water: Filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
EXPOSURE
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6-20 m3/h). The chamber temperature was set at 23 ± 2°C, and the relative humidity at 50 ± 5 %. The exposure system delivered, with an infusion pump, a constant rate of liquid chemical from the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a countercurrent fashion to the liquid flow. The vapourized compound was introduced into the main air inlet pipe of the exposure chambers.
Concentrations of acrylate ester were monitored continuously with a gas-chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with dichloromethane. The resulting samples were analyzed by gas chromatography using appropriate internal standards. Since 2-HPA has a rather low vapour pressure (0.07 mm Hg at 20 °C), the presence of liquid particles was evaluated at the highest concentration generated (i.e. 25 ppm). Airborn particles were measured with an Aerodynamic Particle Sizer.
No differences in particle counts were observed between the clean filtered air (control) and the vapour-laden air in the exposure chambers.


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


TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentrations (mean ± SD):
1.0 ± 0.1 ppm (nominal: 1 ppm)
5.1 ± 0.3 ppm (nominal: 5 ppm)
10.3 ± 0.8 ppm (nominal: 10 ppm)
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2-3
- Length of cohabitation: Overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 to 20 of gestation
Frequency of treatment:
daily, 6h/d
Duration of test:
until gestation day 21
Remarks:
Doses / Concentrations:
1, 5, and 10 ppm (corresponding to approx. 5.4, 27.0, and 54.0 µg/L)
Basis:
nominal conc.
No. of animals per sex per dose:
20
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
Exposure concentrations were based on preliminary studies in which severe maternal toxicity (i.e. weight loss during GD 6-13 and pronounced reduction in weight gain during GD 13-21) was observed at 25 ppm hydroxypropyl acrylate.
Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day (GD) 0, 6, 13 and 21.


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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10 %) to detect very early resorptions.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin's solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70 %), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.

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

Details on maternal toxic effects:
No maternal deaths were observed. Maternal weight gain was significantly less than control during the first half of exposure at 10 ppm and absolute weight gain at 5 and 10 ppm. Food consumption was slightly reduced during treatment at 10 ppm.
Dose descriptor:
NOAEC
Effect level:
ca. 0.005 mg/L air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of implantation sites and live fetuses, and the incidence of non-live implants and resorptions were comparable among groups. There was no effect on fetal weights at any exposure level. Visceral malformations were observed in single fetuses in the control and the 5 ppm groups. Several visceral and skeletal variations were observed, with no significant differences between treated and control groups.
Dose descriptor:
NOAEC
Effect level:
ca. 0.054 mg/L air (nominal)
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: no adverse effects observed up to and including the highest tested dose
Abnormalities:
no effects observed
Developmental effects observed:
no

Maternal body weights:

 

Concentration [ppm/6h/d]

Maternal body weight GD 6 [g]

Absolute weight gain [g]

0

266 ± 16

27 ± 13

1

272 ± 15

27 ± 9

5

271 ± 13

19 ± 11*

10

272 ± 15

16 ± 12**

 

 

Reproductive parameters:

 

Conc. [ppm/6h/d]

No. of litters

No. of implantation sites/litter

% of non-live implants/litter

% of resorption sites/litter

No. of live fetuses/litter

Average fetal body weight/litter [g]

0

21

14.48 ± 3.06

6.10 ± 8.37

6.10 ± 8.37

13.62 ± 3.29

5.48 ± 0.21

1

20

14.40 ± 2.23

9.76 ± 15.58

9.76 ± 15.58

13.00 ± 2.90

5.61 ± 0.29

5

22

14.27 ± 4.09

10.83 ± 21.79

10.83 ± 21.79

14.00 ± 3.22

5.58 ± 0.33

10

21

14.52 ± 3.74

6.70 ± 5.90

6.70 ± 5.90

13.57 ± 3.70

5.48 ± 0.27

 

 

Concentration [ppm/6h/d]

0

1

5

10

Mean % of fetuses with:

 

 

 

 

- any malformations/litter

0.43 ± 1.98

0

0.26 ± 1.21

0

- external variations/litter

0

0

0

0

- visceral variations/litter

5.05 ± 12.39

7.45 ± 13.11

7.66 ± 13.08

4.65 ± 6.82

- skeletal variations/litter

10.40 ± 11.37

11.70 ± 19.45

23.76 ± 28.69

7.37 ± 9.35

- any variations/litter

7.68 ± 8.51

9.56 ± 10.87

15.67 ± 18.03

5.92 ± 5.70

 

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

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products, Inc. (Denver, PA)
- Age at study initiation: 5.5-6 months old
- Weight at study initiation: 2.8-4.0 kg
- Housing: Single in in stainless steel, wire-mesh cages (61 x 61 x 41 cm)
- Diet (ad libitum): AGWAY® PROLAB® Animal Diet (Agway Inc.) Except during exposures
- Water (ad libitum): Tap water (except during exposures)


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


Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Females assigned to the study were exposed to acrylic acid vapour or filtered air for 6 hours/day during the period of major organogenesis (gestation day -gd- 6 through 18). Filtered air was bubbled through a glass reservoir containing liquid acrylic acid. For all vapour concentrations, a Dwyer Flowmeter was used to measure airflow prior to passing the air through the acrylic acid. The vapour, was introduced into the exposure chambers through 1 inch glass tubing containing stainless steel wool.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of acrylic acid vapour in each exposure chamber was monitored throughout the 15 days of exposure by sampling with XAD-8 sorbent tubes and subsequent analysis using high performance liquid chromatography (HPLC) analysis. The HPLC system was composed of a Model 981 Lambda Max LC Spectrophotometer, a Programmable Systems 680 Gradient Controller, a 712 WISP, and a Model 501 Solvent Delivery System. A Spectra Physics SP4270 computing Integrator provided a record of the chromatograms, chromatographic analyses, and peak height measurement. The concentration in each exposure chamber atmosphere was determined approximately 3 times during each 6-hour exposure. The control chamber was sampled once daily. The nominal concentration was calculated by dividing the total quantity of acrylic acid delivered to the chamber by the chamber airflow rate.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
Duration of treatment / exposure:
From day 6 to day 18 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
29 days
Dose / conc.:
25 ppm
Remarks:
corresponding to approx. 0.075 mg/L). Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]
Dose / conc.:
75 ppm
Remarks:
corresponding to approx. 0.224 mg/L). Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]
Dose / conc.:
225 ppm
Remarks:
corresponding to approx. 0.673 mg/L). Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]
No. of animals per sex per dose:
16
Control animals:
yes, sham-exposed
Maternal examinations:
CAGE SIDE OBSERVATIONS: not specified

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

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

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

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


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

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


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

Details on maternal toxic effects:
No animal died in any group. In the 225 ppm group, individual clinical signs observed included: perinasal wetness beginning as early as the first day of exposure and ending by the second day after the last exposure; perinasal encrustation (from days 14 through 22); and nasal congestion observed during and subsequent to exposures (through Day 22). Nasal congestion was observed in a single dose from the 75 ppm group on day 12 only. There were no clinical signs observed during or subsequent to exposures to 25 ppm acrylic acid vapour.
Dose descriptor:
NOAEC
Effect level:
0.075 mg/L air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no evidence of developmental toxicity including teratogenicity at any exposure concentration.
Dose descriptor:
NOAEC
Effect level:
0.673 mg/L air (nominal)
Basis for effect level:
other: fetotoxicity
Remarks on result:
other: no adverse effects observed up to and including the highest tested dose
Dose descriptor:
NOAEC
Effect level:
0.673 mg/L air (nominal)
Basis for effect level:
other: teratogenicity
Remarks on result:
other: no adverse effects observed up to and including the highest tested dose
Abnormalities:
no effects observed
Developmental effects observed:
no

Maternal food consumption:

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

Gestation day

Group mean food consumption (g)

0 ppm

25 ppm

75 ppm

225 ppm

6 – 7

191.42

166.96

147.31**

101.55**

7 – 8

193.42

169.23

149.51**

110.35**

8 – 9

195.13

163.75*

150.69**

121.66**

9 – 10

185.38

166.73

153.59*

132.49**

10 – 11

184.98

200.79

154.79*

134.49**

12 – 13

180.85

123.21*

203.10

136.18**

16 – 17

187.39

186.42

161.23

152.77*

18 – 19

208.56

178.61

167.23*

147.33**

23 - 24

158.19

176.43

204.25**

199.75*

28 - 29

134.35

167.32*

160.41

188.19**

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

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

Maternal body weights and weight changes:

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

Gestation day

Gestational mean body weight changes (g)

0 ppm

25 ppm

75 ppm

225 ppm

0 - 3

-34.08 (150.76)

-7.47 (152.69)

-6.83 (150.42)

-43.38 (214.46)

3 - 6

203.77 (96.19)

240.45 (76.84)

206.55 (96.92)

163.45 (251.41)

6 - 12

68.43 (65.47)

-18.87 (93.75)

-37.67 (93.47)

-41.06 (201.25)

12 - 18

146.67 (69.62)

160.54 (72.07)

123.51 (63.56)

148.45 (65.59)

18 - 24

112.36 (73.58)

150.13 (109.11)

178.41 (73.84)

176.15 (86.22)

24 - 29

26.65 (89.84)

51.55 (125.51)

64.32 (87.17)

143.49**(96.91)

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

Numbers in parentheses indicate standard deviation

Maternal Necropsy:

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

Gestational parametres:

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

Concentration (ppm)

0

25

75

225

Corpora lutea (mean)

8.4

9.1

9.5

8.8

Implantations (mean)

8.6

8.8

8.2

8.5

Viable implants (mean)

8.4

8.6

7.5

7.9

Percent live fetuses (mean)

97.8

98.4

91.3

95.0

Dead implantations (mean)

0.1

0.1

0.0

0.1

Mean weight of live fetuses (g)

43.99

42.44

42.44

42.93

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals; Proposal for updating Guideline 414: Prenatal Developmental Toxicity Study (22 Jan 2001)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 216, pp. 227-235 (29 Apr 2004)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 16-24 weeks
- Weight at study initiation (pregnant animals): 2202-2829 g
- Housing: Singly in type 12.2395.C stainless steel wire mesh cages. During exposure, the animals were kept singly in special mesh cages (40x13x16cm) from BASF SE.
- Diet: Pelleted “Kliba maintenance diet for rabbit & guinea pig, GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum. During exposure, no food was supplied.
- Water: Tap water ad libitum. During exposure, no drinking water was supplied.
- Acclimation period: approx. 7-14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: conditioned supply air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass steel inhalation chamber, volume of 1.4 m³ (BASF SE)
- Method of holding animals in test chamber: animals were kept singly in wire cages located in the inhalation chamber
- System of generating inhalation atmospheres: Generator systems: two-component atomizers (BASF SE); continuous infusion pumps PERFUSOR (B. Braun); Glass mixing stages (BASF SE); glass vaporizers with thermostat (BASF SE). Generation procedure: The test substance was used unchanged. For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the piston metering pump. The vapor was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.


TEST ATMOSPHERE
- Brief description of analytical method used: GC analysis of absorption samples
- Samples taken from breathing zone: yes, immediately adjacent to the animals' noses
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Calculation of nominal concentrations: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.
Online monitoring of the atmospheric concentration: The constancy of concentrations in the inhalation atmospheres were monitored continuously by calibrated online total hydrocarbon analyzer (TESTA FID 2010T in test group 0 and TESTA FID 123 in test groups 1-3) in all test groups including control. Using line recorders the measurements were recorded (see examples in Figure 003 in the APPENDIX) and the signals were transferred to the automated measuring system. The long-term drift of the online devices were checked by a weekly measurement of certificated test gas. To control the correctness of the calibrated devices and the identity of the test substance in the atmosphere, weekly two absorption samples per concentration were drawn from the atmospheres and were analyzed by gas chromatography (GC). The measured values of test group 0 served as blind control. The blind values were mainly the animals' gas emission and, in minor amount, directly from the atmosphere. Therefore, the concurrent blind values were substracted from the measured values. From the corrected daily mean values of each concentration, mean concentrations and standard deviations for the entire study were calculated.
Details on mating procedure:
- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
6 hours on workdays over a time period of 23 consecutive days (gestation days (GD) 6–28)
Frequency of treatment:
daily on workdays
Duration of test:
Until GD 29
Dose / conc.:
5 ppm
Remarks:
corresponding to approx. 0.0174 mg/L
Dose / conc.:
15 ppm
Remarks:
corresponding to approx. 0.0553 mg/L
Dose / conc.:
45 ppm
Remarks:
corresponding to approx. 0.1556 mg/L
No. of animals per sex per dose:
25 inseminated female Himalayan rabbits
Control animals:
yes, sham-exposed
Maternal examinations:
- Mortality
Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0 29).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0 29). During pre-flow period and on the day of necropsy the animals were examined for clinical symptoms at least once a day. During the exposure period, a clinical inspection of each animal was performed at least three times a day (before, during and after exposure). During the exposure procedure a groupwise examination was conducted.


BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).


FOOD CONSUMPTION: Yes
- The food consumption was determined daily on GD 1–29. Because of the malfunction of a balance, the food consumption was not properly recorded on 24 June 2008and the values from this day were not used for the calculation of means and were specified as “not measured / no value determinable”.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: weight assessment: lungs; histopathology: nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as: 1) live fetuses; 2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non pregnant animals and the empty uterus horn in the case of single horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
- Calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, half per litter
Statistics:
DUNNETT-test (two-sided), FISHER'S EXACT test (one-sided), WILCOXON-test (one-sided), KRUSKAL-WALLIS test (two-sided)
Details on maternal toxic effects:
Details on maternal toxic effects:
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data. On female each of test group 0 (0 ppm), test group 2 (15 ppm), and test group 3 (45 ppm)were excluded from the above-mentioned calculations since they were not pregnant. Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

- Mortality: There were no test substance-related or spontaneous mortalities in any group.
- Clinical symptoms: No test substance-related clinical signs or any disturbances of the general behavior were observed in any rabbit during the entire study period.
- Food consumption: The average food consumption was comparable to the control group in all test groups (5, 15 and 45 ppm) and did not show a test substance-related impairment. Differences between control rabbits and test substance-treated rabbits did not show a relation to dosing and were considered to be without biological relevance. This overall statement is true in spite of the slightly but significantly lower high-dose value on GD 23-24.
- Body weight data: The average body weights and body weight gain were comparable among control and treated groups (5, 15 and 45 ppm) during the entire study. All observable differences in the treated groups in comparison to the controls are without any biological relevance. This statement includes the statistically significatly decreased body weight gain in test group 3 on GD 9-11.
- Corrected (net) body weight gain: The results of the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) revealed no difference of biological relevance between the test substance-treated groups and the control group. Mean carcass weights remained also unaffected by the treatment.
- Uterus weight: The mean gravid uterus weights of the animals of all test groups (5, 15 and 45 ppm) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. Considering the fluctuations in the mean number of live fetuses/doe, they reflect the normal degree of variation for rabbits of the strain used in this study.
- Reproduction data of does: The conception rate reached 96% in test groups 0, 2 and 3 (0, 15 or 45 ppm) and 100% in test group 1 (5 ppm). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus. There were no test substance related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Generally, gestational parameters in the various test groups were within the normal range for animals of this strain and age, see also Part III (Supplement) for historical control data.
Dose descriptor:
NOAEC
Effect level:
ca. 0.055 mg/L air (analytical)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Sex distribution of fetuses: The sex distribution of the fetuses in test groups 1-3 (5, 15 and 45 ppm) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of placentae: The mean placental weights in test groups 1, 2 and 3 (5, 15 and 45 ppm) were comparable to the controls.
- Weight of fetuses: The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male fetal weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.
- Fetal external malformations: Three external malformations, which were associated with corresponding soft tissue and skeletal malformations, were recorded for single fetuses of test groups 1 and 2 (5 or 15 ppm). Because a dose-response relationship was missing these findings were considered to be spontaneous in nature.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of all treated groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: One unclassified external observation, i.e. blood coagulum around placenta, was recorded for one fetus of the control group and was spontaneous in nature.
- Fetal soft tissue malformations: The examination of the soft tissues revealed three malformations in single fetuses of all treated groups (5, 15 and 45 ppm). Although no findings were observed in the control and the rate of affected fetuses per litter was significantly higher in the low- and mid-dose groups, no dose related increase of the incidence of soft tissue malformations was noted. Apart from the single incidental case of small cerebrum the findings occurred at incidences comparable to the historical control data. Thus an association of the soft tissue findings to the treatment is not assumed.
- Fetal soft tissue variations: Three soft tissue variations, such as absent lung lobe (lobus inferior medialis), malpositioned carotid branch and dilated cerebral ventricle, were detected in each test group including the controls without relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as discolored kidney, blood coagulum around urinary bladder and hemorrhagic ovary, were recorded for some fetuses of all test groups (0, 5, 15 and 45 ppm). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance-induced effect is not assumed.
- Fetal skeletal malformations: Malformations of the fetal skeletons were observed in fetuses of all test groups including the controls (0, 5, 15 and 45 ppm). Neither statistically significant differences between treated groups and control nor a dose-response relationship were noted. When calculated on a fetus per litter basis, the overall incidence of skeletal malformations was comparable to the historical control data. This is also true for the severely fused sternebrae (bony plate), where the incidence of affected fetuses per litter in the high-dose group was also slightly but statistically significantly higher than the concurrent control.
- Fetal skeletal variations: For all test groups, variations were detected in different skeletal structures with or without effects on corresponding cartilaginous structures. The observed skeletal variations were related to various parts of the fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data. Two isolated skeletal variations were statistically significantly higher than the concurrent and/or outside the historical control (on a fetus per litter basis). These findings are delays of ossification which is reversible and does not affect the morphology of the cervical vertebra as it becomes obvious by the unchanged underlying cartilage. Such slight retardations of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain. Also, the increased incidences are not at all related to the dose. Thus, these findings are regarded to be of no biological relevance.
- Fetal skeletal unclassified cartilage observations: Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were considered to be spontaneous in nature.
- Summary of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. Furthermore, the overall incidences were comparable to the historical control data. Thus, non of the malformations was considered to be related to the treatment. One external (paw hyperflexion), three soft tissue (absent lobus inferior medialis, malpositioned carotid branch and dilated cerebral ventricle) and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter did not show a relation to dosing. In addition, they can be found at a comparable frequency in the historical control data. Therefore, they were not considered to be related to the treatment.
Dose descriptor:
NOAEC
Effect level:
ca. 0.156 mg/L air (analytical)
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: no adverse effects up to and including the highest dose tested
Abnormalities:
no effects observed
Developmental effects observed:
no

- Study means and standard deviations of test substance concentrations:

Test group

Target concentration

Measured concentration (FID)

Nominal concentration (mg/m³)

Effectiveness of vapor generation
(%)

(ppm)

(mg/m³)

ppm

mg/m³

Mean

SD

Mean

SD

0

0

0

-

-

-

-

-

-

1

5

17.6

4.9

1.1

17.4

3.9

21.9

79.5

2

15

52.8

15.7

1.8

55.3

6.2

57.2

96.8

3

45

158.4

44.2

1.2

155.6

4.3

176.5

88.2

- = not measured

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

Finding

Test group 0
0 ppm

Test group 1
5 ppm

Test group 2
15 ppm

Test group 3
45 ppm

HCD

Mean %

(range)

Incomplete ossification of cervical centrum; unchanged cartilage

1.9

8.9**

4.9

6.1

2.2

(0.0–5.0)

Incomplete ossification of sacral arch; cartilage present

0.0

0.7

1.9*

1.2

0.3

(0.0–2.2)

ppm = parts per million; HCD = Historical control data

* = p≤0.05, ** = p≤0.01 (Wilcoxon-Test [one-sided])

- Total fetal malformations: 

 

 

Test group 0
0 ppm

Test group 1
5 ppm

Test group 2
15 ppm

Test group 3
45 ppm

Litter
Fetuses

N
N

24
144

25
171

24
150

24
136

Fetal incidence

 

N (%)

 

3 (2.1%)

 

8 (4.7%)

 

7 (4.7%)

 

6 (4.4%)

Litter incidence

 

N (%)

 

3 (13%)

 

6 (24%)

 

6 (25%)

 

6 (25%)

Affected fetuses/ litter

 

Mean%

 

1.9

 

4.0

 

4.6

 

3.7

ppm = parts per million; N = number; % = per cent

- Total fetal variations:

 

 

Test group 0
0 ppm

Test group 1
5 ppm

Test group 2
15 ppm

Test group 3
45 ppm

Litter
Fetuse
s

N
N

24
144

25
171

24
150

24
136

Fetal incidence

 

N (%)

 

96 (67%)

 

123 (72%)

 

119 (79%)

 

83 (61%)

Litter incidence

 

N (%)

 

24 (100%)

 

25 (100%)

 

24 (100%)

 

23 (96%)

Affected fetuses/litter

 

Mean%

 

68.8

 

71.9

 

77.9

 

60.5

ppm = parts per million; N = number; % = per cent

Conclusions:
The administration of Methyl Acrylate (MA) to pregnant Himalayan rabbits via inhalation had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). The NOAEL for maternal toxicity is 15 ppm. The NOAEL for prenatal developmental Toxicity is 45 ppm. No adverse fetal findings of toxicological relevance were evident at any dose.
Executive summary:

The study was performed according to the proposal for updating OECD Guideline 414: Prenatal Developmental Toxicity Study (22 Jan 2001) in compliance with GLP.

Methyl Acrylate (MA) was administered to pregnant Himalayan rabbits via inhalation from implantation to one day prior to the expected day of parturition (GD 6-28).

There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis this is considered to be a significant adverse effect on maternal organism.

Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Methyl Acrylate (MA) had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm).

Conclusion: The NOAEL for maternal toxicity is 15 ppm. The NOAEL for prenatal developmental Toxicity is 45 ppm. No adverse fetal findings of toxicological relevance were evident at any dose.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity:

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

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

There was no significant difference in the numbers of implantation sites and live fetuses, in the incidence of non-live implants and resorptions, or in the fetal sex ratio, or fetal body weight between control and treated animals. No treatment related increase in embryo/foetal lethality or fetal malformations was observed at any dose level. The incidence of external, visceral, and skeletal variations was similar to controls.

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

In addition, developmental studies in rabbits are available for the structural analogues acrylic acid (CAS No. 79-10-7) and methyl acrylate (CAS No. 96-33-3):

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

Groups of 16 pregnantrabbits were exposed (6 h/d, whole-body) to atmospheres containing acrylic acid at 0, 25, 75, and 225 ppm (corresponding to approx. 0.075, 0.224, 0.673 mg/L) during days 6-18 of gestation (BAMM, 1993; Neeper-Bradley et al., 1997). All dose groups were observed daily for morbidity and mortality. During the exposure period, animals were observed for clinical signs preceding and subsequent to daily exposures and from outside during actual exposures. Maternal body weights were measured on gestation day 0, 3, 6, 12, 24, and 29. Food consumption was measured daily throughout the study beginning on gestation day 3. After sacrifice on gestation day 29, maternal liver and kidney weights were determined. All foetuses were weighed and examined for external malformations and variations, for thoracic and abdominal visceral abnormalities including internal sex organs, for craniofacial abnormalities and for skeletal malformations and variations.

Dose-related clinical signs (as perinasal/perioral wetness and nasal congestion, as well as reduced body weight gain and food consumption) were observed in the 75 and 225 ppm groups. The overall pregnancy rate was equivalent for all groups (94-100 %). No dose-related effects were observed in the reproduction function of the dams. There were no effects on the number of ovarian corpora lutea, the number of total viable or non-viable (early and late resorptions and dead foetuses) implantations/litter. Percentage live foetuses and sex ratio were equivalent across groups. Foetal body weights were unaffected by test substance exposure. There were no exposure-related increases in the incidences of external, visceral or skeletal malformations or variations.

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

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

Data from the structural analogue methyl acrylate (CAS No. 96-33-3):

A prenatal developmental toxicity study in rabbits as second species was conducted according to OECD TG 414 for the Acrylate Task Force (BASF SE 2009). 25 inseminated female Himalayan rabbits per group were whole-body exposed for 6 hrs/day, 5 days/week over a time period of 23 consecutive days (gestation days (GD) 6–28) to methyl acrylate vapours at target concentrations of 0, 5, 15, and 45 ppm. Analytical concentrations of 4.9, 15.7, 44.2 ppm (corresponding to approx. 0.0174, 0.0553, 0.1556 mg/L) were measured. On gestation day 29 the does were sacrificed and submitted to gross and histopathological examination (nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions). Examinations of ovaries and uterine content of the does included: determination of the weight of the unopened uterus, of the number of corpora lutea, of the number and distribution of implantation sites, and calculations of conception rate and pre- and post-implantation losses. Fetal examinations were performed on all fetuses per litter (external, soft tissue, skeletal) except head examinations that were done on half of the fetuses per litter.

There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects, such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis, this is considered to be a significant adverse effect on the maternal organism. The NOAEC for maternal toxicity was 15 ppm (0.0553 mg/L).

Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Methyl Acrylate (MA) had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). Thus, the NOAEC for developmental effects (fetotoxicity) and the NOAEC for developmental effects (teratogenicity) was the highest concentration tested of 45 ppm (0.1556 mg/L).

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

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

 

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

Justification for classification or non-classification

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