methyl acrylate; methyl propenoate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD guideline 416.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

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.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

yes

Remarks:

See above

Test guideline 3

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Methyl acrylate

EC Number:

202-500-6

EC Name:

Methyl acrylate

Cas Number:

96-33-3

Molecular formula:

C4H6O2

IUPAC Name:

methyl prop-2-enoate

Details on test material:

Test material name: Methyl acrylate
Chemical name: 2-Propenoic acid methyl ester
Synonyms: Acrylic acid methyl ester, Methyl-2-propenoate
Source: The Dow Chemical Company, Bayport, Texas
Reference: Lot: VF1301RFM1
Purity: 99.9%
Characterization: The purity of the test material was determined to be 99.9 ± 0.001% by area percent gas chromatography flame ionization detection, 300 ± 17 ppm water by Karl Fischer titration with identification by gas chromatography mass spectrometry and nuclear magnetic resonance (Binkley et al., 2007).
Appearance: Colorless liquid
Vapor Pressure: 68 mm Hg at 20°C
Odor: Acrid odor
Molecular Formula: C4H6O2
Molecular Weight: 86.1

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system 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.

Administration / exposure

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).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 5, 25, and 75 ppm
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 5.3 ± 0.2, 24.9 ± 0.4, and 73.4 ± 1.8 ppm
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm (corresponding to approx. 0, 0.019, 0.092, and 0.269 mg/L)
Basis:
analytical conc.
Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (86.09 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG, 2005]

No. of animals per sex per dose:

27/sex/dose

Control animals:

yes, sham-exposed

Details on study design:

In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Positive control:

None

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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)

Effect levels (P0)

1

Key result

Dose descriptor:

NOEC

Remarks:

developmental toxicity

Effect level:

ca. 0.092 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: corresponding to 25 ppm; based on decreases in pup weight at 75 ppm which were secondary to parental toxicity.

Remarks on result:

other: Generation: F1, F2 (migrated information)

2

Key result

Dose descriptor:

NOEC

Remarks:

reproductive toxicity

Effect level:

ca. 0.269 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: corresponding to 75 ppm; the highest concentration tested.

Remarks on result:

other: Generation: P1 and P2 (migrated information)

3

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.

Remarks on result:

other: Generation: P1 and P2 (migrated information)

Results: F1 generation

Details on results (F1)

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.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at this concentration.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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.