Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose toxicity with reproduction / developmental screening test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 Jan, 2010 to 19 May, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Few minor deviation were present. However, these deviations did not negatively impact the quality or integrity of the data nor the outcome of the study.
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
Deviations:
yes
Remarks:
Few minor deviation were present. However, these deviations did not negatively impact the quality or integrity of the data nor the outcome of the study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC on 12 January 2010
- Age at study initiation: 63 d old
- Weight at study initiation: Male body weights ranged from 354 g to 452 g and female body weights ranged from 198 g to 284 g on study Day 0
- Housing: Housed individually in clean, stainless steel wire-mesh cages suspended above cage-board.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: Municipal water, ad libitum
- Acclimation period: 17 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22ºC ± 3ºC
- Humidity: 50% ± 20%
- Air changes: 10 fresh air changes/h
- Photoperiod: 12-h light (0600 h to 1800 h)/12-h dark photoperiod

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
For the control group (Group 1), a sufficient amount of vehicle was transferred into a glass container. The vehicle was divided into aliquots for daily dispensation and stored at room temperature. The vehicle was mixed throughout preparation, sampling, and dose administration procedures. Dosing formulations were prepared at the test substance concentrations of 0, 5, 15 and 40 mg/L. The test substance formulations were prepared approx weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The test substance formulations were not adjusted for purity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
15 d room temperature resuspension homogeneity and stability of the test substance formulated in the vehicle at concentrations of 10 and 200 mg/mL were established in a previous study (Stump, Draft, WIL-738003). Therefore, resuspension homogeneity and stability were not assessed for the 15 and 40 mg/mL formulations prepared for the current study. Prior to the initiation of dose administration, quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the 5 mg/mL non-dosing formulation. However, because the analytical results of the initial samples as well as the back-up samples did not meet the WIL Research Laboratories, LLC’s SOP requirements, a new 5 mg/mL non-dosing formulation was prepared. Quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the new 5 mg/mL non-dosing formulation. In addition, quadruplicate samples for resuspension homogeneity and stability determinations were collected from aliquots prepared from this same non-dosing formulation following room temperature storage for 5 and 13 d; the aliquots were stirred for at least 60 min prior to sampling. Quadruplicate samples for homogeneity and concentration analyses were collected from the top, middle, and bottom of the test substance formulations prepared for the first week of dose administration; samples were also collected from the middle stratum of the vehicle control formulation. Additionally, quadruplicate samples for concentration analysis were collected from the middle stratum of each test substance formulation and vehicle control formulation prepared for the remainder of the study. One set of duplicate samples from each collection was subjected to the appropriate analyses. The remaining set of duplicate samples was stored frozen (approx -70°C ± 5°C) as back-up. All analyses were conducted by the Analytical Chemistry Department at WIL Research Laboratories, LLC using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
Duration of treatment / exposure:
Males: Days 0-27 (14 d prior to pairing through 1 d 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 40-47 doses.
Females that failed to deliver were dosed through the day prior to euthanasia (post-mating Day 25) for a total of 41 doses.
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
25, 75, and 200 mg/kg bw/day administered at a dosage volume of 5 mL/kg (dosing formulations were not adjusted for purity)
Basis:

No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of previous studies and were provided by the Sponsor after consultation with the Study Director. In a previous 14 d toxicity study in Crl:CD(SD) rats (Stump, Draft, WIL-738003), the maximum tolerated dose was exceeded at 1000 mg/kg bw/day. At 300 mg/kg bw/day, lower mean body weight gains, reduced food consumption, adverse clinical signs, and/or changes in mean organ weights were noted for males and females. Based on these results, dosage levels of 25, 75, and 200 mg/kg bw/day were selected to be evaluated in the current study.
- Rationale for animal assignment (if not random): At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS™. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated based on body weight stratification randomized in a block design. The animals then were arranged into groups according to the printout. Animals not assigned to study were euthanized by carbon dioxide inhalation and discarded.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Approx 2 weeks prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until the day prior to scheduled euthanasia

FOOD CONSUMPTION AND COMPOUND INTAKE:
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 the female with no evidence of mating was measured on a weekly basis until parturition and is presented on the individual report tables. 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.

BREEDING PROCEDURES:
The animals were paired on a 1:1 basis within each treatment group following 14 d of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation Day 0. If evidence of copulation was not detected after 14 d of pairing, any females that had not shown evidence of mating were placed in plastic maternity cage. For the purpose of calculating pre-coital intervals, rats paired over a 12-h dark cycle were considered to have been paired for 1 d.

PARTURITION
All females were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. 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.

FOB ASSESSMENTS
FOB assessments were recorded for 6 animals/sex/group prior to dose administration and prior to fasting for clinical pathology sampling on study Day 27 (males) and lactation day 4 (females). The FOB used at WIL Research Laboratories, LLC is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al., 1991; and O’Donoghue, 1989). FOB testing was performed without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters as described below:
- 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: 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. The locomotor activity sessions were performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. Locomotor activity, recorded at approx the same time each test day 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 min epochs and the test session duration was 60 min. These data were compiled as six, 10 min subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

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 for 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 (approx 1.5 mL) and hematology (approx 0.5 mL) was collected from the retro-orbital sinus following isoflurane anesthesia just prior to euthanasia. Blood (approximately 1.8 mL) for coagulation parameters 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). The following parameters were evaluated:
- HEMATOLOGY AND COAGULATION: Total leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), reticulocyte count, mean platelet volume (MPV), red cell distribution width (RDW), hemoglobin distribution width (HDW), differential leukocyte count, platelet estimate, red cell morphology

CLINICAL CHEMISTRY
Albumin, total protein, globulin, albumin/globulin ratio (A/G Ratio), total bilirubin (Total Bili), urea nitrogen, creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT) , aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), glucose, total cholesterol (Cholesterol), calcium, chloride, phosphorus, potassium, sodium ,triglycerides (triglyceride), bile acids.
Sacrifice and pathology:
ANATOMIC PATHOLOGY
-MACROSCOPIC EXAMINATIONS
All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation Day 5; the numbers of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-mating Day 25; when observed, the number of macroscopic implantations was recorded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). Gross necropsies were conducted on all animals including the males and females that were found dead or euthanized (by carbon dioxide inhalation) in extremis; the numbers of corpora lutea and implantation sites were recorded for the female that was found dead during gestation. Necropsies included examination of the external surface, all orifices, the external surface of the brain, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted): Adrenal glands, lymph node, aorta, axillary, bone with marrow (sternebrae), mesenteric, bone marrow smeara, mandibular, brain, ovaries and oviducts, cerebrum, pancreas, cerebrum, peripheral nerve (sciatic), cerebellum with medulla/pons, pituitary gland, coagulating gland, prostate gland, eyes with optic nerve, mandibular salivary glands, gastrointestinal tract, seminal vesicles, esophagus, skeletal muscle (rectus femoris), stomach, skin with mammary gland, duodenum, spinal cord (cervical), jejunum, spleen, ileum, testes with epididymides, cecum, thymus gland, colon, thyroids, rectum, heart, trachea, kidneys, urinary bladder, liver (sections of 2 lobes), uterus with cervix and vagina, lungs (including bronchi), all gross lesions (all groups)
ORGAN WEIGHTS: The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, ovaries with oviducts, brain, spleen, epididymides, testes, heart, thymus gland, kidneys, thyroids with parathyroids
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and organ to brain weight ratios were reported.

-MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed according to WIL Research Laboratories, LLC’s SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin with the exception of the tests and epididymides which were stained with PAS to allow detailed histopathological examination. Microscopic examination was performed on all tissues from all animals in the control and 200 mg/kg bw/day groups at the scheduled necropsies and from all animals that were found dead or euthanized in extremis. In addition, the adrenal cortex, the non-glandular portion of the stomach, and all gross lesions from all animals at all dosage levels and the thymus from all females 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. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.
Other examinations:
F1 LITTER PARAMETERS
-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. Intact offspring dying 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.
-CLINICAL OBSERVATIONS
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. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data.

-SEX DETERMINATION
Pups were individually sexed on PND 0 (if possible), 1, and 4.

CALCULATION OF LITTER PARAMETERS
Litter parameters were defined as follows:
Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0
Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) / No. of Litters Per Group *100
Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/ No. of Litters Per Group *100
Where N = PND 0-1 and 1-4
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. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
F0 GENERATION
-CLINICAL OBSERVATIONS AND SURVIVAL: Test substance-related mortality and/or moribundity were noted in the 75 mg/kg bw/day group (2 males) and the 200 mg/kg bw/day group (2 males and 3 females). In the 200 mg/kg bw/day group, female no. 64665 was found dead shortly after dose administration study Day 3 and male no. 64682 and female nos. 64628 and 64672 were found dead prior to dose administration on Day 15, 16, and gestation Day 13, respectively. In addition, male nos. 64729 and 64677 in the 75 mg/kg bw/day group were euthanized in extremis on study Days 10 and 13, respectively, and male no. 64694 in the 200 mg/kg bw/day group was euthanized in extremis on study Day 26; all 3 males euthanized in extremis were noted with body weight losses (9 g to 49 g) during the period prior to euthanasia. Clinical findings noted for the majority of the animals that did not survive to the scheduled necropsy included gasping, rales, limbs cool to touch, cool body, labored respiration, mucoid feces, salivation, and red, yellow, and clear material primarily around the mouth; single animals were also noted with wiping mouth in the bedding, splayed limbs, and tonic convulsions at the post-dosing observations. Although the cause of death of death/moribundity of these animals was not determined upon microscopic examination, all animals were noted with lesions in the stomach and lymphoid depletion or lymphoid necrosis in the thymus and generally in the spleen. All other animals survived to the scheduled necropsies. For animals that survived to the scheduled necropsy, salivation-related findings (salivation prior to or at time of dosing and clear material around the mouth and nose) were noted for the majority of the animals in the 75 and 200 mg/kg bw/day groups primarily at the time of dose administration or approx 1 h following dose administration; these findings were also occasionally noted for the 25 mg/kg bw/day group animals and were likely signs of taste aversion related to the irritating properties of the compound that resulted in stomach findings and were not considered adverse. Furthermore, the majority of the females in the 25, 75, and 200 mg/kg bw/day groups were noted to be wiping their mouth in the bedding following dose administration due to the irritative nature of the test substance formulations. Incidences of yellow and red material primarily around the mouth were also noted sporadically across all dosage levels during the treatment period, and incidences of rales were noted for a few animals primarily in the 75 and 200 mg/kg bw/day groups at the time of dose administration and/or approx 1 h following dose administration. These findings were likely related to aspiration of the test substance formulations. Other clinical findings noted at the daily examinations, at the time of dose administration, or approx 1 h following dose administration, including hair loss on various body surfaces, occurred infrequently and/or at similar frequencies in the control group, and were not attributed to test substance administration.

-BODY WEIGHTS
• MALES : Test substance-related lower mean body weight gains were noted in the 200 mg/kg bw/day group throughout the treatment period (study Days 0-27); the difference from the control group achieved significance (p<0.01) during Days 7-13. As a result, mean body weight gains in this group were significantly (p<0.01) lower when the overall pre-mating period (Days 0-13) and treatment period (Days 0-27) were evaluated and mean body weights were 7.1% to 8.8% lower during study Days 13-27 compared to the control group; differences in mean body weights were significant (p<0.05) on Days 21 and 27. In the 75 mg/kg bw/day group males, slightly (not statistically significant) lower mean body weight gains were noted during study Days 0-21. During study Days 21-27, mean body weight gain in this group was markedly (not statistically significant) lower primarily due to 2 animals (male nos. 64703 and 64725) noted with body weight losses of 29 g to 34 g during this interval. However, the differences in mean body weight gains were not of a sufficient magnitude to result in statistically significant differences when the overall pre-mating (study Days 0-13) and treatment (study Days 0-27) periods were evaluated and mean body weights in this group were similar to the control group values throughout the treatment period. Mean male body weights and body weight gains in the 25 mg/kg bw/day group were similar to the control group throughout the treatment period. Differences from the control group were slight and not statistically significant.
• FEMALES: During pre-mating . mean female body weights and body weight gains in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group during the pre-mating period (study Days 0-13). Differences from the control group were slight and not statistically significant. During gestation, mean body weights and body weight gains in the 25, 75, and 200 mg/kg bw/day groups were unaffected by test substance administration during gestation. A significantly (p<0.05) lower mean body weight gain was noted in the 200 mg/kg bw/day group during gestation Days 7-11 compared to the control group. However, this difference was transient and therefore, not attributed to test substance administration. In the 75 mg/kg bw/day group, mean body weight gain was significantly (p<0.05) lower when the overall gestation period (gestation Days 0-20) was evaluated. In the absence of a dose-related trend, this difference was not attributed to test substance administration. All other differences in mean body weight gains during gestation were slight and not statistically significant when the test substance-treated groups were compared to the control group. During lactation, mean body weights and body weight gains in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group during lactation Days 1-4. Differences from the control group were slight and not statistically significant.

-FOOD CONSUMPTION
• MALES : Mean male food consumption, evaluated as g/animal/day and g/kg bw/day, in the 200 mg/kg bw/day group, was slightly reduced during the pre-mating period (Days 0-13). Although differences from the control group did not achieve statistical significance, the lower mean food consumption in this group corresponded to reductions in mean body weight gains during this period. Mean male food consumption in the 25 and 75 mg/kg bw/day groups was similar to the control group during the pre-mating period (Days 0-13). Differences from the control group were slight and not statistically significant.
• FEMALES : During pre-mating , mean female food consumption in the 25, 75, and 200 mg/kg bw/day groups was similar to the control group throughout the pre-mating period (study Days 0-13). During gestation, mean food consumption in the 25, 75, and 200 mg/kg bw/day groups was unaffected by test substance administration during gestation. Significantly (p<0.05) lower mean food consumption was noted in the 200 mg/kg bw/day group during gestation Days 14-17 (g/animal/day and g/kg bw/day) and when the overall gestation period (gestation Days 0-20; g/kg/day only) was evaluated. During lactation, mean food consumption in the 25, 75, and 200 mg/kg bw/day groups was unaffected by test substance administration during lactation Days 1-4.

-FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
• HOME CAGE OBSERVATIONS : Home cage parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group on study Day 27 (males) or lactation Day 4 (females).
• HANDLING OBSERVATIONS : Handling parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Day 27 (males) or lactation day 4 (females).
• OPEN FIELD OBSERVATIONS : Open field parameters were unaffected by test substance administration at all dosage levels. A significant (p<0.01) increase in mean defecation counts was noted in the 25 mg/kg bw/day group females (1.7 counts) compared to the control group (0.0 counts) on lactation Day 4. However, in the absence of a dose-related response, this difference was not attributed to test substance administration. There were no other statistically significant differences for the test substance-treated groups when compared to the control group on study Day 27 (males) or lactation Day 4 (females).
• SENSORY OBSERVATIONS : Sensory parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group during on Day 27 (males) or lactation Day 4 (females).
• NEUROMUSCULAR OBSERVATIONS: Neuromuscular parameters were unaffected by test substance administration at all dosage levels. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Day 27 (males) or lactation Day 4 (females).
• PHYSIOLOGICAL OBSERVATIONS: A lower (10.3%) mean body weight was noted for the 200 mg/kg bw/day group males compared to the control group at the physiological observations on Day 27. Although this difference did not achieve statistical significance, the lower mean body weight corresponded to the lower weekly mean body weights recorded for this group during the treatment period. No other differences in physiological parameters were attributed to test substance administration at any dosage level. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Day 27 (males) or lactation Day 4 (females).
-LOCOMOTOR ACTIVITY : Locomotor 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). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 min) and the overall 60-min test session values were comparable to the concurrent control values and the WIL historical control data (Version 1.3). Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL historical control data ranges, and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups.

-CLINICAL PATHOLOGY
• HEMATOLOGY AND COAGULATION: Higher mean absolute neutrophil counts were noted in the 75 and 200 mg/kg bw/day group males (approximately 149% and 185%, respectively) and females (approx 46% and 118%, respectively). The difference for the 200 mg/kg bw/day group females achieved significance (p<0.01). These higher neutrophil counts were consistent with the test substance-related ulcerations and associated inflammation that was present in the non-glandular stomach. There were no other test substance-related effects on hematology or coagulation parameters. Significant (p<0.05 or p<0.01) differences from the control group included lower mean red blood cell counts and hemoglobin values in the 25 and 200 mg/kg bw/day group males and a lower hematocrit in the 25 mg/kg bw/day group males. These group mean differences were not considered to be test substance-related because the values did not show a dose-related response. Mean absolute reticulocyte count for the 200 mg/kg bw/day group males was higher (not statistically significant) than the 0 mg/kg/day group males but the value was within the range of historical control data (version 3.1). Significant (p<0.05 or p<0.01) findings that involved percentage reticulocyte or leukocyte differential counts were not itemized above and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
• SERUM CHEMISTRY : There were no test substance-related effects on serum chemistry parameters. There were significantly (p<0.05 or p<0.01) lower mean alkaline phosphatase values in the 75 (101 ± 7.5 U/L) and 200 mg/kg bw/day (92 ± 6.6 U/L) group F0 males but values were within the range of historical controls (version 3.1). In addition, a significantly (p<0.01) higher mean cholesterol value was noted for the 200 mg/kg bw/day group males (65 ± 3.1 mg/dL). These group mean differences were not considered to be test substance-related.

-REPRODUCTIVE PERFORMANCE: 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/exposed groups. Males that did not sire a litter numbered 1, 1, 0, and 0 in the control, 25, 75, and 200 mg/kg bw/day groups, respectively. Females that had evidence of mating but did not deliver numbered 1, 1, 0, and 0 in the same respective groups. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. None of these differences were statistically significant.

-GESTATION LENGTH AND PARTURITION : Mean gestation lengths in the 25, 75, and 200 mg/kg bw/day groups were unaffected by test substance administration. A significantly (p<0.01) longer gestation length was noted for 75 mg/kg bw/day group (22.0 d) compared to the concurrent control group (21.4 d). However, the magnitude of difference was small, there was no dose-related response, and the value for the 75 mg/kg bw/day group was within the range of the historical control data (21.5-22.3 d). Therefore, this difference was not attributed to test substance administration.

-ANATOMIC PATHOLOGY
• MACROSCOPIC EXAMINATIONS : In the 200 mg/kg bw/day group, male no. 64682 was found dead on study 15 and female nos. 64665, 64628, and 64672 were found dead on study Day 3, study Day 16, and gestation Day 13, respectively. In addition, male nos. 64729 and 64677 in the 75 mg/kg bw/day group were euthanized in extremis on study Days 10 and 13, respectively, and male no. 64694 in the 200 mg/kg bw/day group was euthanized in extremis on Day 26. The majority of these animals in the 200 mg/kg bw/day group were noted with macroscopic findings in the gastrointestinal tract (stomach, cecum, colon, duodenum, ileum, and/or jejunum was distended, eroded, thickened, and/or had dark red areas), indicating irritation from test substance administration. All other animals survived to the scheduled necropsies. At the scheduled necropsy, test substance-related incidences of thickened stomach were noted for the 75 and 200 mg/kg bw/day group males and females and adhesions on the liver and spleen were noted for males in the 200 mg/kg bw/day group. All other macroscopic findings occurred in single animals and/or in a manner that was not dose-related. The mean numbers of corpora lutea, unaccounted-for sites, and implantation sites in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group values.
• ORGAN WEIGHTS : Mean final body weight and organ weight alterations presented in the following table were considered to be associated with administration of the test substance. Also, the mean brain weight relative to final body weight was significantly (p<0.05) higher in the 200 mg/kg bw/day group males. This difference was considered secondary to the lower final body weight in this group. No other differences were statistically significant when the test substance-treated groups were compared to the control group.
• MICROSCOPIC EXAMINATIONS: Test substance-related histopathologic alterations in both males and females included hypertrophy of the zona fasciculata of the adrenal cortex and ulceration, epithelial hyperplasia and hyperkeratosis, and chronic-active inflammation in the non-glandular stomach. In the 200 mg/kg bw/day group males, there was also cytoplasmic vacuolation of the zona fasciculata of the adrenal cortex, and in the 200 mg/kg bw/day group females, there was lymphoid depletion in the thymus. Test substance-related histologic alterations are presented in the following table: Ulceration was the focal, or typically multifocal, loss of the full-thickness of mucosal epithelium. Erosion, epithelial, was the focal or multifocal loss of a partial thickness of the mucosal epithelium. Erosion was not diagnosed when ulceration was present. Hyperplasia of the squamous epithelial mucosa of the non-glandular stomach was an increased thickness and irregular basal margin of the layers of the epithelium and it was invariably accompanied by hyperkeratosis, and increased thickness of the luminal layers of keratin which was not separately diagnosed. Chronic active inflammation was attributed to neutrophils as well as mononuclear inflammatory cells along with variably increased connective tissue. Inflammation was invariable at sites of ulceration but was not diagnosed separately unless it was also observed at other sites. Inflammation was invariable in the submucosa but also extended upward into the mucosa or downward into the gastric wall. In some instances, inflammation was transmural, extending throughout the gastric muscular wall to the outer serosa, and resulted in fibrous connective tissue adhesions of the gastric serosa to either the liver or the spleen. Hypertrophy of the adrenal cortex was an increase in the size of cells in the zona fasciculata. Cytoplasmic vacuolation of the adrenal cortex also occurred in the zona fasciculata. Lymphoid depletion in the thymus was a decreased number of lymphocytes in the cortex which made the distinction between the cortex and medulla less distinct. There were no other test substance-related histologic changes. 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.

F1 LITTER DATA
The mean number of pups born, live litter size, and the percentage of males at birth in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group values. Postnatal survival in the 25, 75, and 200 mg/kg bw/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant. The general physical condition of all F1 pups in this study were unaffected by test substance administration. Pups (litters) that were found dead numbered 15(4), 3(2), 4(3), and 4(4) in the control, 25, 75, and 200 mg/kg bw/day groups, respectively. Four (1), 0(0), 0(0), and 2(1) pups (litters) in these same respective groups were missing and presumed to have been cannibalized. Mean male and female pup body weights and body weight changes in the 25, 75, and 200 mg/kg bw/day groups were unaffected by test substance administration during PND 1-4. Significantly (p<0.05 or p<0.01) higher mean male and female pup body weights were noted on PND 1 for the 75 mg/kg bw/day group. However, in the absence of a dose-related trend, these differences were not attributed to maternal test substance administration. No other statistically significant differences from the control group were noted. The numbers of pups (litters) found dead during PND 0-4 numbered 15(4), 3(2), 4(3), and 4(4) in the control, 25, 75, and 200 mg/kg bw/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
Remarks:
(for systemic toxicity)
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
not specified

Analytical chemistry:

The analyzed dosing formulations were within protocol-specified range (100% ± 7%), were homogeneous, and were stable for up to 13 days when stored at room temperature. Based on these results, the protocol-specified dosages of test substance were administered to the animals. The test substance was not detected in the vehicle formulations that were administered to the control group animals (Group 1).

Results of the analyses of dosing formulations are summarized below:

Text Table 1. Results of Homogeneity, Resuspension Homogeneity, and Stability Analyses (Non-Dosing Formulations)

 

Group 2 (5 mg/mL)

Homogeneity assessment of the 20 January 2010 formulations*

Mean Concentration (mg/mL)

3.53

RSD (%)

35

Mean % of Target

70.6

Homogeneity Assessment of the 20 January 2010 Formulations (Back-Up Samples)*

Mean Concentration (mg/mL)

3.19

RSD (%)

10

Mean % of Target

63.7

Homogeneity Assessment of the 22 January 2010 Formulations*

Mean Concentration (mg/mL)

5.06

RSD (%)

2.0

Mean % of Target

101

Resuspension homogeneity/stability assessment of the 22 January 2010 formulations following 5-d room temperature storage

Mean Concentration (mg/mL)

4.97

RSD (%)

2.7

Mean % of Target

99.5

Mean % of Time Zero

98.3

Resuspension homogeneity/stability assessment of the 22 January 2010 formulations following 13-d room temperature storage

Mean Concentration (mg/mL)

4.96

RSD (%)

1.9

Mean % of Target

99.2

Mean % of Time Zero

98.0

* = Analytical results were outside the acceptable criteria; new pre-initiation formulations were prepared on 22 January 2010 for analyses.

Text Table 2. Results of Homogeneity and Concentration Analyses (Dosing Formulations)

 

Group 2

(5 mg/mL)

 

Group 3

(15 mg/mL)

 

Group 4

(40 mg/mL)

 

Homogeneity Assessment of the 27 January 2010 Formulations

Mean Concentration (mg/mL)

5.33

16.1

42.0

RSD (%)

0.81

1.1

1.2

Mean % of Target

107

107

105

Concentration Assessment of the 4 February 2010 Formulations

Mean Concentration (mg/mL)

5.16

15.3

40.9

RSD (%)

0.058

1.4

0.25

Mean % of Target

103

102

102

Concentration Assessment of the 12 February 2010 Formulations

Mean Concentration (mg/mL)

5.18

15.8

42.4

RSD (%)

0.10

0.49

0.84

Mean % of Target

104

105

106

Concentration Assessment of the 23 February 2010 Formulations

Mean Concentration (mg/mL)

5.23

15.7

41.0

RSD (%)

3.5

1.9

0.022

Mean % of Target

105

105

102

Concentration Assessment of the 4 March 2010 Formulations

Mean Concentration (mg/mL)

5.14

15.4

40.5

RSD (%)

0.79

0.19

1.3

Mean % of Target

103

103

101

 

Conclusions:
Under the study conditions, the NOAEL of the test substance for systemic toxicity was considered to be 75 mg/kg bw/day.
Executive summary:

A study was conducted to determine the combined repeated dose and reproduction/developmental toxicity of the test substance PETIA to rat in a screening study conducted according to OECD Guideline 422 and EPA Guideline OPPTS 870.3650, in compliance with GLP. The test substance was administered orally in corn oil by gavage once daily to 3 groups of 12 male and 12 female Crl:CD(SD) rats. Dosage levels were 25, 75 and 200 mg/kg bw/day, administered at 5 mL/kg. Test substance-related mortality and/or moribundity occured at 75 and 200 mg/kg bw/day. Clinical findings were recorded at all dose levels and included salivation or evidence thereof, yellow and red material primarily around the mouth and wiping of mouth in the bedding (females only). Incidences of rales were also noted sporadically at 75 and 200 mg/kg bw/day. All findings were primarily noted at the time of dose administration or shortly thereafter and were attributed to the irritating properties of the substance. In the 200 mg/kg bw/day group males, lower mean body weight gains were noted during the pre-mating (Days 0-13) and treatment (Days 0-27) periods, with correspondingly lower mean food consumption during the pre-mating period. As a result, mean male body weight in this group was 8.4% lower than controls on Day 27. For the 75 mg/kg bw/day group males, slightly lower mean body weight gains were noted throughout the treatment period, with the most severe reduction occurring during Days 21-27. However, the magnitude of these differences was not sufficient to affect mean body weights when compared to controls and mean food consumption was similar to that of controls during the pre-mating period (Days 0-13). The effects were therefore not considered to be treatment-related. Mean body weights, body weight gains and food consumption were unaffected by test substance administration in the 25 mg/kg bw/day group males throughout the study and in the 25, 75, and 200 mg/kg bw/day group females during the pre-mating, gestation and lactation periods. No test substance-related effects were noted during the functional observation battery (FOB) or locomotor activity evaluations at any dosage level. At the scheduled necropsy, there were higher mean absolute neutrophil counts at 75 and 200 mg/kg bw/day, consistent with the test substance-related ulceration and associated inflammation in the non-glandular stomach. Gross observations consisted of thickened non-glandular stomach in the 75 and 200 mg/kg bw/day group males and females and adhesions to the liver or spleen in males at 200 mg/kg bw/day. Test substance-related histopathologic alterations included ulceration, epithelial hyperplasia and hyperkeratosis, and/or chronic-active inflammation in the non-glandular stomach at 75 and 200 mg/kg bw/day. Organ weight changes included higher mean adrenal gland weights in the 75 mg/kg bw/day group males and 200 mg/kg bw/day group males and females and lower mean thymus weight in the 200 mg/kg bw/day group females. Hypertrophy of the zona fasciculata of the adrenal cortex and lymphoid depletion was also noted in the 200 mg/kg bw/day group males and females, respectively. Under the study conditions, the NOAEL of the test substance for systemic toxicity was considered to be 75 mg/kg bw/day (Stump, 2011).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The information requirements for this tonnage band is sufficiently met with the available data.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 16, 1996 to December 18, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 6 weeks
- Housing: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed at least once per week, rotated every 2 weeks
- Bedding: Sani-Chip® hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed at least once per week. Bedding was irradiated
- Diet: NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 72°±3° F
- Humidity: 50±15%
- Air changes: 10/h
- Photoperiod: 12 h light/dark cycle
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST MATERIAL
- Amount(s) applied: 0.5 mL/kg

VEHICLE
- Lot/batch no.: KP206 and LS0051
- Purity: Greater than 99%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed at the beginning, midpoint, and end of the study; animal room samples of these dose formulations were also analysed. Of the dose formulations analyzed, all 15 were within 10% of the target concentration, with no value greater than 104% of the target concentration; 10 of 15 animal room samples were within 10% of the target concentration.
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week for 14 weeks
Remarks:
Doses / Concentrations:
0, 0.75, 1.5, 3, 6, or 12 mg/kg bw
Basis:
analytical per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Blood was collected from the retroorbital sinus on Days 4 and 23 and at the end of the studies for hematology and clinical chemistry.
Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials
Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids
Sacrifice and pathology:
Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Complete histopathologic examinations were performed on vehicle control and 12 mg/kg bw rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The skin at the site of application was also examined.
Other examinations:
At the end of the studies, sperm samples were collected from male animals in the vehicle control and 3, 6, and 12 mg/kg bw groups for sperm count and motility evaluations. The following parameters were evaluated: spermatid heads per testis or cauda and per gram testis or cauda and epididymal spermatozoal motility. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females in the vehicle control and 3, 6, and 12 mg/kg bw groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Dermal irritation:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Details on results:
All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats.

On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant.

Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls.

No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation. The incidence and severity of hyperplasia increased with increasing dose; most animals administered 3 mg/kg bw or greater were affected. Severity was minimal to mild and characterized by focally extensive to diffuse increased thickness of the epidermis, from the normal one to three cell layers thick to four to six layers thick. Hyperplasia was accompanied by minimal to mild increased thickness of the superficial keratin layer (hyperkeratosis). Minimal hyperkeratosis without accompanying hyperplasia was present in the 0.75 and 1.5 mg/kg bw groups. Degeneration was diagnosed in many animals treated with 1.5 mg/kg bw or greater.

Degeneration was a minimal focal change consisting of intraepidermal vacuolization, presumably due to intra or intercellular fluid accumulation. Vacuoles occasionally coalesced to form small vesicles that contained a few neutrophils. Epidermal necrosis was present in some males, although a dose-related response was not clear. Necrosis consisted of partial to full-thickness coagulative change of the epidermis and was likely a pathogenic sequela of degeneration. Intraepidermal infiltration of neutrophils (suppurative inflammation) often accompanied degeneration or necrosis of the epidermis. A mixed inflammatory cell infiltrate (chronic active inflammation) was present in the dermis of animals administered 1.5 mg/kg bw or greater, with dose dependent increases in incidences and severity. Sebaceous glands at the site of application were slightly enlarged and prominent in animals with the other changes described above.
Key result
Dose descriptor:
NOAEL
Effect level:
12 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
0.75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Irritation
Critical effects observed:
not specified
Executive summary:

A study was conducted to assess the repeated dose dermal toxicity of the read-across substance PETIA in male and female rats. Groups of 10 male and 10 female rats received dermal applications of 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw of test substance in acetone, 5 days per week for 14 weeks; the dosing volume was 0.5 mL/kg bw. Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Blood was collected from the retroorbital sinus on Days 4, 23 and at the end of the studies for hematology and clinical chemistry. All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats. On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant. Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls. No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation. The local NOAEL for this study could be considered to be 0.75 mg/kg bw/day (Hejtmancik, 2005).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
12 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral


A study was conducted to determine the combined repeated dose and reproduction/developmental toxicity of the read across substance PETIA to rat in a screening study conducted according to OECD Guideline 422 and EPA Guideline OPPTS 870.3650, in compliance with GLP. The read across substance was administered orally in corn oil by gavage once daily to 3 groups of 12 male and 12 female Crl:CD(SD) rats. Dosage levels were 25, 75 and 200 mg/kg bw/day, administered at 5 mL/kg. Read across substance-related mortality and/or moribundity occurred at 75 and 200 mg/kg bw/day. Clinical findings were recorded at all dose levels and included salivation or evidence thereof, yellow and red material primarily around the mouth and wiping of mouth in the bedding (females only). Incidences of rales were also noted sporadically at 75 and 200 mg/kg bw/day. All findings were primarily noted at the time of dose administration or shortly thereafter and were attributed to the irritating properties of the substance. In the 200 mg/kg bw/day group males, lower mean body weight gains were noted during the pre-mating (Days 0-13) and treatment (Days 0-27) periods, with correspondingly lower mean food consumption during the pre-mating period. As a result, mean male body weight in this group was 8.4% lower than controls on Day 27. For the 75 mg/kg bw/day group males, slightly lower mean body weight gains were noted throughout the treatment period, with the most severe reduction occurring during Days 21-27. However, the magnitude of these differences was not sufficient to affect mean body weights when compared to controls and mean food consumption was similar to that of controls during the pre-mating period (Days 0-13). The effects were therefore not considered to be treatment-related. Mean body weights, body weight gains and food consumption were unaffected by read across substance administration in the 25 mg/kg bw/day group males throughout the study and in the 25, 75, and 200 mg/kg bw/day group females during the pre-mating, gestation and lactation periods. No substance-related effects were noted during the functional observation battery (FOB) or locomotor activity evaluations at any dosage level. At the scheduled necropsy, there were higher mean absolute neutrophil counts at 75 and 200 mg/kg bw/day, consistent with the substance-related ulceration and associated inflammation in the non-glandular stomach. Gross observations consisted of thickened non-glandular stomach in the 75 and 200 mg/kg bw/day group males and females and adhesions to the liver or spleen in males at 200 mg/kg bw/day. Substance-related histopathologic alterations included ulceration, epithelial hyperplasia and hyperkeratosis, and/or chronic-active inflammation in the non-glandular stomach at 75 and 200 mg/kg bw/day. Organ weight changes included higher mean adrenal gland weights in the 75 mg/kg bw/day group males and 200 mg/kg bw/day group males and females and lower mean thymus weight in the 200 mg/kg bw/day group females. Hypertrophy of the zona fasciculata of the adrenal cortex and lymphoid depletion was also noted in the 200 mg/kg bw/day group males and females, respectively (Stump, 2011).Based on the results of the read across study, the NOAEL of the test substance for systemic toxicity is considered to be 75 mg/kg bw/day.


Dermal


A study was conducted to assess the repeated dose dermal toxicity of the read-across substance PETIA in male and female rats. Groups of 10 male and 10 female rats received dermal applications of 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw of test substance in acetone, 5 days per week for 14 weeks; the dosing volume was 0.5 mL/kg bw. Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly and at the end of the studies for core study animals. Necropsies were performed on all animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus. Blood was collected from the retroorbital sinus on Days 4, 23 and at the end of the studies for hematology and clinical chemistry. All rats survived to the end of the study. Final mean body weights and body weight gains of 12 mg/kg bw males were significantly less than those of the vehicle controls. Irritation at the site of application occurred in 12 mg/kg bw rats. On Day 23, an increase in segmented neutrophils occurred in 12 mg/kg bw males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg bw males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant. Thymus weights of males administered 3 mg/kg bw or greater were significantly less than those of the vehicle controls. There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls. No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg bw groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation. The local NOAEL for this study could be considered to be 0.75 mg/kg bw/day (Hejtmancik, 2005).

Justification for classification or non-classification

The NOAEL of 75 mg/kg bw/day from a repeated dose and reproductive/developmental toxicity screening study with the read-across substance was established on the basis of lower mean body weight. Other effects observed at 75 and 200 mg/kg bw/day relating to mortality/morbidity, adrenal gland weights, neutrophil counts, macroscopic and/or microscopic findings of the stomach, adrenal cortex and thymus were attributed to the irritative properties of the test substance and corresponding stress, rather than systemic toxicity. The effect on the body weight is considered not to support classification for specific target organ toxicity following repeated exposure. Hence, it can be concluded that test substance DPHA does not warrant classification according to CLP (EC 1272/2008) criteria.


In repeated dose dermal toxicity study conducted with rats, the effects observed were all related to the inflammation at the site of application caused by the irritating properties of the substance.