Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/ Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals 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
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.
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
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).
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
Description (incidence and severity):
Higher mean absolute and relative (to final body and brain weights) liver and thyroid/parathyroid weights were noted at 1,000 mg/kg bw/day. Because there were no microscopic correlations for these findings, the increased organ weights were considered to be treatment-related but non-adverse.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
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: 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.
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.
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.
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.
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
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
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no
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. 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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, in compliance with GLP.

The substance was administered 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 1,000 mg/kg bw/day (5 mL/kg bw). 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 test start. Males received 14 daily doses prior to mating then 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. Clinical observations, bodyweight and food consumption were recorded at appropriate intervals. Functional observation battery (FOB) and locomotor activity data were recorded for 6 males/group following approximately 28 days of dose administration and for 6 females/group on Lactation Day 4. All F0 females 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 bodyweights were recorded on Postnatal Day 1 and 4. Pups were euthanized and discarded on Postnatal Day 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on 6 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period and F0 females were euthanized on Lactation Day 5. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals 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 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. Mean bodyweight, bodyweight change and food consumption were unaffected compared to controls at 100, 300, and 1000 mg/kg bw/day. No treatment-related effects were noted during the FOB 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 at 1,000 mg/kg bw/day. Because there were no microscopic correlations for these findings, the increased organ weights were considered to be treatment-related but non-adverse. 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. There were no treatment-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 ofcorpora lutea, unaccounted-for sites, 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 bodyweight and bodyweight gain at all dosage levels were unaffected by dose administration. No treatment-related clinical or macroscopic findings for F1 pups that were found dead were noted at any dosage level.

Under the conditions of this screening study, no treatment-related effects on reproductive performance, gestation length, parturition, reproductive organs or neurobehavioral parameters were noted at any dose level. Based on these results, 1,000 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 treatment-related effects on mean bodyweight, bodyweight change and food consumption at all dose levels, the NOAEL for systemic toxicity was considered to be 1,000 mg/kg bw/day. In the absence of effects on the general physical condition of the F1 pups, the NOAEL for neonatal toxicity was 1,000 mg/kg bw/day (Stump, 2011).

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 October 2015 - 09 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: JBLE0012T- Expiration date of the lot/batch: May 2016STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Controlled room temperature protected from lightTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: None
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS - Source: Charles River Laboratories, Sulzfeld, Germany - Age at study initiation: 11 weeks old at mating - Weight at study initiation: 197-233 g - Fasting period before study: - Housing: - Diet (e.g. ad libitum): ssniff SM R/M "Autoclavable Complete Feed for Rats and Mice - Breeding and Maintenance" - Water (e.g. ad libitum): tap water - Acclimation period: 5 days ENVIRONMENTAL CONDITIONS - Temperature (°C): 20.0 - 23.8°C - Humidity (%): 39-70% - Air changes (per hr): 15-20/hour - Photoperiod (hrs dark / hrs light):12 / 12 IN-LIFE DATES: From: 17 November 2015 To: 07 December 2015
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: VEHICLE - Justification for use and choice of vehicle (if other than water): corn oil - Lot/batch no. (if required): MKBQ9948V / MKBS6944V- 3 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test substance for concentration and/or homogeneity by validated GC method.Duplicate samples (top, middle and bottom samples) taken from the test formulation two times during the study (second and last weeks of treatment). One duplicate sample taken on each occasion from vehicle control for concentration measurement.
Details on mating procedure:
- Impregnation procedure: cohoused - If cohoused: - M/F ratio per cage: 1:1 - Length of cohabitation: 2 hours - Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 6 to GD 19
Frequency of treatment:
Daily
Duration of test:
Until GD 20
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 mated females/group
Control animals:
yes
Details on study design:
- Dose selection rationale: based on available data, including an OECD 422 study
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: Twice daily each day DETAILED CLINICAL OBSERVATIONS: Yes / No / No data - Time schedule: at the onset of treatment (GD 6) then weekly BODY WEIGHT: Yes - Time schedule for examinations: GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20 FOOD CONSUMPTION: Yes - Time schedule: GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20 POST-MORTEM EXAMINATIONS: Yes - Sacrifice on gestation day 20 - Organs examined: viscera, ovaries, uterus, OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes Examinations included: - Gravid uterus weight: Yes - Number of corpora lutea: Yes - Number of implantations: Yes - Number of early resorptions: Yes - Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter - Soft tissue examinations: Yes: half per litter - Skeletal examinations: Yes: half per litter - Head examinations: Yes:half per litter
Statistics:
SPSS PC+4.0
Historical control data:
Yes
Clinical signs:
effects observed, non-treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly decreased bodyweight and bodyweight gain were seen in the high dose group on several days/periods. However, the difference in terminal bodyweight and bodyweight gain during the study (GD 0 - 20) was less than 10%. Furthermore, the bodyweight loss duing the treatment period (GD 6 - 20) was not considered statistically significant. Therefore, these findings were not considered as clear treatment-related adverse effects. A similar but stronger effect was detected for the corrected terminal bodyweight, corrected bodyweight gain and corrected net bodyweight gain. The corrected bodyweight gain and corrected net bodyweight gain values were significantly lower compared to the control group (by 22 and 29%, respectively). These adverse effects were considered related to administration of the test substance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A slight effect on food consumption was observed for all test groups. Statistically significant decreased food consumption was seen for the high dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Remarks on result:
other: Embryotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Remarks on result:
other: Foetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Remarks on result:
other: Teratogenicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Executive summary:

A study was conducted to assess the effect of amine synergist on the embryonic and fetal development of Hannover Wistar rats in their first pregnancy according to OECD Guideline 414, in compliance with GLP. Groups of 24 pregnant females were administered the substance diluted in corn oil (3 mL/kg) at 0, 100, 300 and 1000 mg/kg/day from Gestation Day 6 to 20. Cesarian sections, necrospy of dams and examinations of uterine content were performed on GD 20.

Dose formulations were analysed for test substance concentration twice during the treatment period using a validated GC method. Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumtion. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed ande xamined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. The number of confirmed pregnant, evaluated dams in the dose groups treated was 24 in the control group and 23, 24 and 24 in the low, mid and high dose groups, respectively.

All formulations were within the range of 92 to 107% of nominal concentration an were found to be homogenous. No test item was detected in the control samples. Based on these results, test item formulations were considered suitable for the study purposes. There was no unscheduled mortality or treatment related clinical signs in the study. Statistically significantly decreased body weight or body weight gain values were observed for the High dose group (100 mg/kg bw/day) on several days / periods. However, the difference in the terminal body weight and the body weight gain during the study (GD0-20) was less than 10%. Furthermore the body weight loss during the treatment period GD6-20) was not considered statistically significant. Therefore these findings were not considered as a clear test item related adverse effect. A similar but stronger effect was detected for the corrected terminal body weight, corrected body weight gain and corrected net body weight gain. The corrected body weight gain and corrected net body weight gain values were significantly lower compared to the control group (by 22% and 29% respectively). These adverse effects were considered related to the administration of the test item. A slight effect on the food consumtpion was observed in the study for all test groups. Additionally, statistically significant decreased food consumption was observed for the high dose group. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined in the conditions of this study. The number of corpora lutea, implantation sites and pre-implantation loss mean values were comparable to the control for the test item treated groups at all dose levels. There were no effects on the early and late embryonic loss, post-implantation (total resorption, including the early and late embryonic loss) or total intrauterine mortality in the test item-related dams. No remarkable internal or external observations were recorded for any pregnant animals during necropsy. No abnormalities were observed on the placentas in any examined groups. The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control.

The weight of foetuses in the low, mid and high dose groups did not differ significantly from the control mean, when evaluated by both litter mean and group mean.

The number of body weight retarded foetuses in the test item treated groups was lower than in the control group. All abnormalities observed at external, visceral or skeletal examination in this study were considered as incidental findings, which were also observed in the concurrent study control group. The findinds lauid within historical control (HC) data or were considered to be spontaneous events unrelated to treatment.

In conclusion, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD 6 to 19, amine synergist caused a slight maternal body weight and food intake effect in the high dose group, demonstrating an adequate but relatively minor maternal toxicity. There was no evidence of any adverse maternal effects in the mid and low dose groups. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined in the conditions of this study. No foetal effects were seen at external, visceral and/or skeletal examination of foetuses in the study which could be related to the test item administration. The following NOAELs were derived (Hargitai, 2016):

NOAELmaternal toxicity:300 mg/kg bw/day
NOAEL
embryotoxicity:1000 mg/kg bw/day
NOAEL foetotoxicity
: 1000 mg/kg bw/day
NOAELteratogenicity
: 1000 mg/kg bw/day
Endpoint:
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/ Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals 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
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.
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.
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.
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.
Duration of test:
Females received 14 daily doses prior to pairing and were dosed through gestation and until lactation day 4 for a total of 40-44 doses.
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-day 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
Maternal 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.
Fetal examinations:
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.
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.
Indices:
Uterine Data: the numbers of former implantation sites and corpora lutea were recorded.Offspring Viability: 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).
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
Description (incidence and severity):
Higher mean absolute and relative (to final body and brain weights) liver and thyroid/parathyroid weights were noted at 1,000 mg/kg bw/day. Because there were no microscopic correlations for these findings, the increased organ weights were considered to be treatment-related but non-adverse.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
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
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
necropsy findings
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects: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. 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.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
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 development 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 bw. 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 for 6 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 bw/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 bw/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 no-observed-adverse-effect level (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 (Stump, 2011).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

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, in compliance with GLP. The study is described above (see ‘Effects on fertility’). In the absence of effects on the general physical condition of the F1 pups, the NOAEL for neonatal toxicity in this study was 1,000 mg/kg bw/day (Stump, 2011).

A study was conducted to determine the potential developmental toxicity of amine synergist to Hannover Wistar rats in accordance with OECD Guideline 414, in compliance with GLP. Groups of 24 pregnant females were administered the substance by gavage at 0, 100, 300 and 1000 mg/kg/day from Gestation Day (GD) 6 to 20.

Dose formulations were analysed for test item concentration twice during the treatment period using a validated GC method. Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumtion. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

The number of confirmed pregnant, evaluated dams in the dose groups treated was 24 in the control group and 23, 24 and 24 in the low, mid and high dose groups, respectively.

All formulations were within the range of 92 to 107% of nominal concentration an were found to be homogenous. No test item was detected in the control samples. Based on these results, test item formulations were considered suitable for the study purposes. There was no unscheduled mortality or treatment related clinical signs in the study. Statistically significantly decreased body weight or body weight gain values were observed for the high dose group on several days / periods. However, the difference in the terminal body weight and the body weight gain during the study (GD0-20) was less than 10%. Furthermore the body weight loss during the treatment period GD6-20) was not considered statistically significant. Therefore these findings were not considered as a clear test item related adverse effect. A similar but stronger effect was detected for the corrected terminal body weight, corrected body weight gain and corrected net body weight gain. The corrected body weight gain and corrected net body weight gain values were significantly lower compared to the control group (by 22 and 29% respectively). These adverse effects were considered related to the administration of the test item. A slight effect on the food consumtpion was observed in the study for all test groups. Additionally, statistically significant decreased food consumption was observed for the High dose group. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined in the conditions of this study. The number of corpora lutea, implantation sites and pre-implantation loss mean values were comparable to the control for the test item treated roups at all dose levels. There were no effects on the early and late embryonic loss, post-implantation (total resorption, including the early and late embryonic loss) or total intrauterine mortality in the test item-related dams. No remarkable internal or external observations were recorded for any pregnant animals during necropsy. No abnormalities were observed on the placentas in any examined groups. The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control.

The weight of foetuses in the low, mid and high dose groups did not differ significantly from the control mean, when evaluated by both litter mean and group mean. The number of body weight retarded foetuses in the test item treated groups was lower than in the control group. All abnormalities observed at external, visceral or skeletal examination in this study were considered as incidental findings, which were also observed in the concurrent study control group. The findings were within historical control (HC) data or were considered to be spontaneous events unrelated to treatment.

In conclusion, amine synergist, when administered daily by oral gavage to pregnant Hannover Wistar rats from GD 6 to 19, caused a slight maternal body weight and food intake effect in the high dose group, demonstrating an adequate but relatively minor maternal toxicity. There was no evidence of any adverse maternal effects in the mid and low dose groups. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined in the conditions of this study.

No foetal effects were seen at external, visceral and/or skeletal examination of foetuses in the study which could be related to the test item administration. The following NOAELs were derived (Hargitai, 2016):

NOAEL maternal toxicity: 300 mg/kg bw/day
NOEL embryo toxicity: 1000 mg/kg bw/day
NOEL foetotoxicity: 1000 mg/kg bw/day
NOEL teratogenicity: 1000 mg/kg bw/day

Justification for classification or non-classification

Based on the NOAELs for developmental toxicity of 1,000 mg/kg/day from an OECD Guideline 422 and an OECD Guideline 414 study in which no treatment-related effects were observed, it can be concluded that amine synergist does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.