2-Propenoic acid, (1-methyl-1,2-ethanediyl) bis[oxy(methyl-2,1-ethanediyl)] ester, reaction products with diethylamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, (1-methyl-1,2-ethanediyl) bis[oxy(methyl-2,1-ethanediyl)] ester, reaction products with diethylamine

EC Number:

601-101-8

Cas Number:

111497-86-0

Molecular formula:

Molecular formula not available for this UVCB.

IUPAC Name:

2-Propenoic acid, (1-methyl-1,2-ethanediyl) bis[oxy(methyl-2,1-ethanediyl)] ester, reaction products with diethylamine

Constituent 2

Reference substance name:

EBECRYL (R) P 116 Radiation Curing Resins

IUPAC Name:

EBECRYL (R) P 116 Radiation Curing Resins

Constituent 3

Reference substance name:

2 Propenoic acid, 1,1’-[(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)]] ester, reaction products with diethylamine

IUPAC Name:

2 Propenoic acid, 1,1’-[(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)]] ester, reaction products with diethylamine

Details on test material:

- Name of test material (as cited in study report): EBECRYL® P 116 Radiation Curing Resins (also known as EBECRYL P 116 and EB P116)- Physical state: Clear, pale yellow liquid- Analytical purity: 99.0%- Lot/batch No.: JBGK0064T- Expiration date of the lot/batch: 25 July 2010- Supplier: Cytec Surface Specialties, Drogenbos, Belgium

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Each rat was uniquely identified by a Monel® metal ear tag (National Band and Tag Company, Newport, KY) displaying the animal number.TEST ANIMALS- Source: Charles River Laboratories, Inc., Raleigh, NC- Age at study initiation: approximately 10 weeks old at initiation of dosing - Weight at study initiation: Males 328 g to 415 g; Females 200 g to 257 g; female body weights ranged from 214 g to 289 g on gestation day 0. - Housing: Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. Following positive evidence of mating, the males were housed in suspended wire mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material.- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during the period of fasting prior to clinical pathology blood collection- Water: Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system ad libitum- Acclimation period: 14 days prior to the first day of treatment. ENVIRONMENTAL CONDITIONS- Temperature: Actual 21.2°C to 22.1°C- Humidity: 41.9% to 48.8% - Air changes: minumum of 10/hr- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod. IN-LIFE DATES: From: 23 February 2010 To: 10 June 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The vehicle and test substance formulations were administered orally by gavage, via a 16 gauge (2.0 x L-70 mm) flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan).PREPARATION OF DOSING SOLUTIONS: The test substance formulations were prepared every 7-11 days as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first dosing formulations were visually inspected by the Study Director and were found to be clear yellow, visibly homogeneous liquids and acceptable for administration.VEHICLEThe vehicle used in preparation of the test item formulations and for administration to the control group was corn oil .- Concentrations in vehicle: 20, 60 or 200 mg/mL- Dosage volume: 5mL/kg- Lot no. YF0793, exp. date: 22 May 2011- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ.

Details on mating procedure:

- M/F ratio per cage: 1:1- Length of cohabitation: until evidence of mating, or a maximum of 14 days- Proof of pregnancy: vaginal copulatory plug or the presence of sperm following a vaginal lavage. The day when evidence of mating was identified was termed gestation day 0. - After successful mating each pregnant female was caged (how): plastic maternity cages with nesting material- Other: The animals were mated following 14 days of treatment. Pairing was in the home cage of the male.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fourteen-day room temperature stability and resuspension homogeneity of the formulated test substance in the vehicle were established at concentrations of 10 mg/mL and 200 mg/mL in a previous study (Stump, Draft, WIL-738005)Prior to the initiation of dose administration, quadruplicate samples for homogeneity and concentration determinations were collected from the top, middle, and bottom strata of the 20, 60, and 200 mg/mL formulations prepared for the first week of dosing; samples for concentration analysis were also collected from the middle stratum of the vehicle control formulation. Thereafter, quadruplicate samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group). One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored frozen (approximately -70°C) as back-up. All analyses were conducted by the Analytical Chemistry Department at WIL Research Laboratories, LLC using a validated gas chromatography method using flame ionization detection.

Duration of treatment / exposure:

Males: Days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. Females: Females received 14 daily doses prior to pairing and were dosed through lactation day 4 for a total of 40-44 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39-52 doses.

Frequency of treatment:

Once daily, at approximately the same time each day.

Details on study schedule:

- Age at mating: approximately 12 weeksAll females were allowed to deliver naturally and rear their young to PND 4. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

Doses / concentrations

Remarks:

Doses / Concentrations:100, 300, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg (dosing forumlations were not adjusted for purity) Basis:

No. of animals per sex per dose:

12 males/12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of a previous dose range-finding 14 days toxicity study of the test substance in rats (Stump, Draft, WIL 738005) and were provided by the Sponsor after consultation with the Study Director. In the previous study there were no effects on survival, clinical signs, body weights, body weight gains, food consumption, and organ weights and no remarkable macroscopic findings at dosage levels of 100, 500, and 1000 mg/kg/day. Based on these results, dosage levels of 100, 300, and 1000 mg/kg/day were selected for evaluation in the current study.- Rationale for animal assignment: computer randomization procedure

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.CLINICAL OBSERVATIONS: A detailed physical examination was conducted weekly on each animal beginning approximately 1 week prior to the initiation of dose administration, including on the day of necropsy. These examinations were performed outside the home cage in an open field at approximately the same time each week and included, but were not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic, and central nervous system function, somatomotor activity, and behavior. Each male and female was also observed for signs of toxicity immediately following dosing and approximately 1 hour following dose administration.BODY WEIGHT- Male: Individual male body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until the day prior to scheduled euthanasia. - Female: Individual female body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until the day evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, 4, and 5.FOOD CONSUMPTION: Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for all males was measured on a weekly basis until the scheduled euthanasia. Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.FOB ASSESSMENTS: FOB assessments were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males) and lactation day 4 (females). FOB testing was performed without knowledge of the animal’s group assignment. All animals were observed for the following parameters:- Home Cage Observations: Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure.- Handling Observations: Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone.- Open Field Observations (2-min observation period): Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing.- Sensory Observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation.- Neuromuscular Observations: Hindlimb extensor strength, Hindlimb foot splay, Grip strength hind and forelimb, Rotarod performance.- Physiological Observations: Catalepsy, Body temperature, Body weight.LOCOMOTOR ACTIVITY: Locomotor activity counts were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment. Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer controlled system that utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the clear plastic boxes to decrease the potential for distraction from extraneous environmental stimuli or stimuli from biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The testing of treatment groups was done according to replicate sequence. Each animal was tested separately. Data were collected in twelve, 5 minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.CLINICAL PATHOLOGY: Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 6 animals/sex/group at the scheduled necropsies (study day 28 for males and lactation day 5 females); the same animals evaluated for FOB and locomotor activity were selected for clinical pathology assessment. All animals were food-fasted overnight prior to blood collection, with water available. Blood for serum chemistry (approximately 1.5 mL) and hematology (approximately 0.5 mL) was collected from the retro orbital sinus following isoflurane anesthesia. Blood for coagulation parameters (approximately 1.8 mL) was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry). - Hematology: Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Prothrombin time, Activated partial thromboplastin time, Reticulocyte count Percent / Absolute, Mean Platelet Volume, Red cell distribution width, Hemoglobin Distribution Width, Differential leukocyte count, Platelet estimate, Red cell morphology.- Clinical Chemistry: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio [by calculation], Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides, Bile acids.

Litter observations:

PARTURITION:On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.LITTER VIABILITY AND DEATHS: Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. CLINICAL OBERSERVATIONS: Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.BODY WEIGHTS: Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group.SEX DETERMINATION: Pups were individually sexed on PND 0 (if possible), 1, and 4.

Postmortem examinations (parental animals):

SACRIFICE: All F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females were euthanized on lactation day 5; the numbers of former implantation sites and corpora lutea were recorded. Gross necropsies were conducted on all animals. GROSS NECROPSYNecropsies included examination of the external surface, all orifices, the external surface of the brain, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The following tissues and organs were placed in 10% neutral buffered formalin (except as noted): Adrenal glands, Aorta, Bone with marrow (sternebrae), Bone marrow smear(a), Brain (Cerebrum level, Cerebrum level, Cerebellum with medulla/pons), Coagulating gland, Eyes with optic nerve(b), Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative) Lymph nodes (Axillary, Mesenteric, Mandibular), Ovaries and oviducts(c), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Mandibular salivary glands, Seminal vesicles, Skeletal muscle (rectus femoris), Skin with mammary gland(d), Spinal cord (cervical), Spleen, Testes with epididymides(e), Thymus gland, Thyroids [with parathyroids, if present], Trachea, Urinary bladder, Uterus (f) with cervix and vagina, All gross lesions.ORGAN WEIGHTS: The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Spleen, Testes, Thymus gland, Thyroids with parathyroids.HISTOPATHOLOGY: Microscopic examination was performed on all tissues from all animals in the control and 1000 mg/kg/day groups. In addition, gross lesions from animals at all dosage levels were examined microscopically. The evaluation of the testes and epididymides was sufficient to identify possible treatment-related effects such as retained spermatids, missing cell layers or types, micronucleated giant cells, or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymides included the caput, corpus, and cauda, in addition to evaluation of leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. Microscopic examination of the ovary was sufficient to detect any relevant changes in the follicular development, follicular atresia, and corpora lutea formation.

Postmortem examinations (offspring):

SACRIFICE: Following a detailed physical examination on PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without internal examination. Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). The carcass of each pup was then discarded.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit.

Reproductive indices:

The following reproductive indices were calculated: Male Mating Index, Female Mating Index, Male Fertility Index, Female Fertility Index, Male Copulation Index, Female Conception Index, Pre-Coital Interval (days).

Offspring viability indices:

The following parameters were calculated: Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), Postnatal Survival for All Other Intervals (% Per Litter).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Red and clear material around the mouth was noted for the 1,000 mg/kg bw/day group males and females at approximately 1 h post-dosing. However, because the number of occurrences per animal was low and generally sporadic throughout the study and severity was generally slight, these clinical findings were not considered adverse. Moreover, the increased incidence of clear material around the mouth was likely a sign of taste aversion. A low incidence of rales was also observed for the 1,000 mg/kg bw/day group females at the time of and approximately 1 h following dose administration. This finding was likely related to aspiration of the test substance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mild hypertrophy of thyroid follicular epithelial cells was observed in 2 of 12 females at 1000 mg/kg bw/day. This change is not uncommon in rats and was of minimal severity. Hypertrophy of the thyroid follicular epithelial cells often occurs secondary to drug metabolizing enzyme induction in the liver, ultimately leading to thyroid gland hypertrophy. In the present study however, there was no evidence of hepatic enzyme induction in the females; therefore, this finding was treatment-related but considered non-adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance administration at all dosage levels.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)All animals survived to the scheduled necropsy on study day 28 (males) or lactation day 5 (females). Increased incidences of red and clear material around the mouth were observed for the 1000 mg/kg/day group males and females at approximately 1 hour post dosing. However, because the number of occurrences per animal was low and generally sporadic throughout the study, and the severity of these findings was generally slight, these clinical findings were not considered adverse. Moreover, the increased incidence of clear material around the mouth was likely a sign of taste aversion. The clinical finding of rales was also noted on 1-2 occasions for 4 females in the 1000 mg/kg/day group at the time of and/or approximately 1 hour following dose administration. This finding was likely related to aspiration of the test substance. All other clinical findings noted in the test substance-treated groups at the daily examinations and at the time of or approximately 1 hour following dose administration, including hair loss, scabbing, and yellow material on various body surfaces, occurred infrequently, in single animals, at similar frequencies in the control group, and/or in a manner that was not dose-related.BODY WEIGHT Males: Mean male body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.Females: Mean female body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were similar to the control group during the pre-mating treatment period and gestation period. No test substance related effects on mean body weights and body weight gains were noted in the 100, 300, and 1000 mg/kg/day groups during lactation. FOOD CONSUMPTIONMales: Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 1000 mg/kg/day group males was similar to that in the control group throughout the study.Females: Mean food consumption in the 100, 300, 1000 mg/kg/day group females was similar to that in the control group throughout the pre-mating period, gestation, and lactation.FUNCTIONAL OBSERVATIONAL BATTERYFOB parameters were unaffected by test substance administration at all dosage levels.LOCOMOTOR ACTIVITYLocomotor activity patterns (total activity as well as ambulatory activity counts) were unaffected by test substance administration at all dosage levels when evaluated on study day 27 (males) and lactation day 4 (females).CLINICAL PATHOLOGYThere were no test substance-related alterations in hematology and coagulation parameters. Mean platelet volume for the 100 and 1000 mg/kg/day group males was 8.8% and 8.4% lower, respectively, than the control group. The differences at 100 and 1000 mg/kg/day were statistically significant (p<0.05) but were considered isolated findings with no correlating hematologic, serum chemistry, or microscopic findings, or alterations in platelet numbers. The mean platelet volume at 300 mg/kg/day was 4.7% lower than the control group, which was not statistically significant. The differences at 100 and 1000 mg/kg/day were considered spurious and unrelated to the test substance. There were no test substance-related effects on serum chemistry. A higher (5.0%; 10.6 mg/dL) mean calcium value was noted for the 1000 mg/kg/day group males when compared to the control group (10.1 mg/dL); the difference was statistically significant (p<0.01). Since this value was within the historical control range (9.9-11.6 mg/dL; version 3.1), and may be explained by a slight and not statistically significantly higher albumin value in the 1000 mg/kg/day group males, the difference was considered spurious and unrelated to test substance administration. In addition, a higher (14.0%; 5.14 mEq/L) mean potassium level was noted for the 100 mg/kg/day group females. Although the difference was statistically significant (p<0.05), it was within the range of values in the WIL historical control database (4.07-5.98 mEq/L; version 3.1) and was not observed in a dose-dependent manner. Therefore, no relationship to the test substance was apparent.REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. The mean numbers of days between pairing and coitus in the test substance treated groups were similar to the control group value.GESTATION LENGTH and PARTURITIONMean gestation lengths in the 100, 300, and 1000 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups. The mean numbers of corpora lutea, unaccounted-for sites, and implantation sites in the 100, 300, 1000 mg/kg/day groups were similar to the control group values. GROSS PATHOLOGY (PARENTAL ANIMALS)There were no test substance-related macroscopic findings observed at any dosage level. White areas on the liver were noted for 3 males in the 1000 mg/kg/day group. Because there were no microscopic correlations for these findings, they were not considered to be test substance-related. Other macroscopic findings observed in the test substance treated groups occurred in single animals and/or in a manner that was not dose-related, and were therefore not considered test substance related.ORGAN WEIGHTSHigher mean absolute and relative (to final body and brain weights) liver and thyroid/parathyroid weights were noted for the 1000 mg/kg/day group males and females, respectively. Because there were no microscopic correlations for these findings, the increased organ weights were considered to be test substance-related but nonadverse. HISTOPATHOGYThere were no test substance-related histopathologic alterations in males and females. Mild hypertrophy of thyroid follicular epithelial cells was observed in 2 of 12 females of the 1000 mg/kg/group. This change is not uncommon in rats and was of minimal severity in the affected females. Hypertrophy of the thyroid follicular epithelial cells often occurs secondary to drug metabolizing enzyme induction in the liver, ultimately leading to thyroid gland hypertrophy. In the present study, however, there was no evidence of hepatic enzyme induction in the females; therefore, this finding was test substance related but considered nonadverse and toxicologically unimportant. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at the highest level tested.

2

Key result

Dose descriptor:

NOAEL

Remarks:

(Reproductive toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at the highest level tested.

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration. GENERAL PHYSICAL CONDITION (OFFSPRING)The general physical condition of all F1 pups in this study were unaffected by test substance administration.BODY WEIGHT (OFFSPRING)Mean male and female pup body weights and body weight changes in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration during PND 1 4. No statistically significant differences from the control group were noted.GROSS PATHOLOGY (OFFSPRING FOUND DEAD)The numbers of pups (litters) found dead during PND 0-4 numbered 3(2), 2(2), 1(1), and 1(1) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions, no test substance-related effects on reproductive performance, gestation length, parturition, reproductive organs, or neurobehavioral parameters were noted at any dosage level. Based on these results, a dosage level of 1000 mg/kg bw/day was considered to be the NOAEL for reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. Due to the absence of adverse clinical findings and no test substance-related effects on mean body weights, body weight changes, and food consumption at all dosage levels, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day. In the absence of effects on the general physical condition of the F1pups, the NOAEL for neonatal toxicity was 1000 mg/kg bw/day.

Executive summary:

The toxicity to reproduction of amine synergist was tested in a repeated dose and reproduction / developmental toxicity screening study (OECD 422 and OPPTS 870.3650) in rats.

The test substance in the vehicle, corn oil, was administered orally by gavage once daily to 3 groups of Sprague Dawley [Crl:CD(SD)] rats, each group consisting of 12 males and 12 females. Dosage levels were 100, 300, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle (corn oil) on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 4 for a total of 40-44 doses. 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for6 males/group following approximately 28 days of dose administration and for 6 females/group on lactation day 4. All F₀females were allowed to deliver and rear their pups until lactation day 4. Each litter was examined daily for survival and all deaths were recorded. F₁ clinical observations and body weights were recorded on postnatal day (PND) 1 and 4. Pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on 6 F₀animals/sex/group at necropsy. F₀males were euthanized following completion of the mating period and F₀females were euthanized on lactation day 5. Complete necropsies were conducted on all F₀animals, and selected organs were weighed. Selected tissues were examined microscopically from all F₀animals in the control and high-dose groups; gross lesions from all animals in all dosage groups were also examined microscopically.

All animals survived to the scheduled necropsies. Red and clear material around the mouth were noted for the 1000 mg/kg/day group males and females at approximately 1 hour post-dosing. However, because the number of occurrences per animal was low and generally sporadic throughout the study, and the severity of these findings was generally slight, these clinical findings were not considered adverse. Moreover, the increased incidence of clear material around the mouth was likely a sign of taste aversion. A low incidence of rales was also observed for the 1000 mg/kg/day group females at the time of and approximately 1 hour following dose administration. This finding was likely related to aspiration of the test substance.

Mean body weights, body weight changes, and food consumption were unaffected by test substance administration in the 100, 300, and 1000 mg/kg/day group males and females throughout the study. No test substance-related effects were noted during the FOB assessments or locomotor activity evaluations at any dosage level. Higher mean absolute and relative (to final body and brain weights) liver and thyroid/parathyroid weights were noted for the 1000 mg/kg bw/day group males and females, respectively. Because there were no microscopic correlations for these findings, the increased organ weights were considered to be test substance-related but nonadverse. Mild hypertrophy of thyroid follicular epithelial cells was observed in 2 of 12 females of the 1000 mg/kg bw/day group. This change is not uncommon in rats and was of minimal severity in the affected females. Hypertrophy of the thyroid follicular epithelial cells often occurs secondary to drug metabolizing enzyme induction in the liver, ultimately leading to thyroid gland hypertrophy. In the present study, however, there was no evidence of hepatic enzyme induction in the females; therefore, this finding was test substance-related but considered nonadverse and toxicologically unimportant. There were no test substance‑related changes in clinical pathology parameters or gross necropsy observations at any dosage level.  Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance administration at all dosage levels. Mean numbers of corpora lutea, unaccounted-for sites, and implantation sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 100, 300, and 1000 mg/kg bw/day groups were similar to the control group values. Mean pup body weights and body weight gains at all dosage levels were unaffected by dose administration. No test substance-related clinical findings or macroscopic findings for F₁pups that were found dead were noted at any dosage level.

Under the conditions of this screening study, no test substance-related effects on reproductive performance, gestation length, parturition, reproductive organs, or neurobehavioral parameters were noted at any dosage level. Based on these results, a dosage level of 1000 mg/kg bw/day was considered to be the NOAEL for reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. Due to the absence of adverse clinical findings and no test substance-related effects on mean body weights, body weight changes, and food consumption at all dosage levels, the NOAEL for systemic toxicity was considered to be 1000 mg/kg bw/day. In the absence of effects on the general physical condition of the F₁pups, the NOAEL for neonatal toxicity was 1000 mg/kg bw/day.