1,2,4-Oxadiazole, 3-phenyl-5-(2-thienyl)-

Endpoint summary

Description of key information

Oral:
Under the conditions of this 90 d dietary study, the NOAEL for systemic toxicity of the test substance in rat was considered to be 250 ppm (equivalent to 16 and 19 mg/kg bw/day for males and females, respectively).
Dermal:
In a four week repeated dose dermal toxicity study, NOAEL was considered to be 100 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 22, 2010 to December 16, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 408 and OPPTS Guideline 870.3100, in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: 163 g to 198 g (males); 144 g to 169 g (females)
- Housing: 2-3 per cage by sex for approximately one day. Thereafter, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet: Certified Rodent LabDiet #5002 (meal), fed ad libitum except during FOB and locomotor activity assessments and during the period of fasting prior to clinical pathology blood collection when food, but not water, was withheld.
- Water: Reverse osmosis treated drinking water, delivered by an automatic watering system were provided ad libitum.
- The results of the diet and water analyses revealed no contaminants at concentrations sufficient to interfere with the objectives of this study.
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature: 71 ± 5°F
- Humidity (%): 50 ± 20%
- Air changes (per h): 10 (air handling units were set to provide a minimum of 10 fresh air changes per h).
- Photoperiod: 12 h light/12 h dark. The 12 h light/12 h dark photoperiod was interrupted as necessary to allow for the performance of protocol specified activities. The light status (on or off) was recorded once every 15 min.

- Individual body weights and food consumption were recorded and detailed physical examinations were performed periodically during the pretest period. Ophthalmic examinations were performed prior to randomization. Functional observational battery and locomotor activity data were also recorded for pretest animals prior to the initiation of diet administration.

Route of administration:

oral: feed

Vehicle:

other: Normal feed

Details on oral exposure:

Continuous exposure via oral diet for 13 weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability of test diet admixtures at concentrations ranging from 3 ppm to 10,000 ppm was 10 d when stored at room temperature or frozen.

- Prior to the initiation of diet administration, samples were collected from 10 and 1,500 ppm diet preparations. Samples for concentration analysis were collected weekly from the middle stratum of each dosing formulation (including the control group) and stored frozen. One duplicate set from the dietary preparations collected during study weeks 0, 3, 7, and 12 was analysed and the remaining duplicate set was frozen and retained as backup samples. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method using flame ionization detection.

The analysed dietary formulations were homogeneous (i.e., met the protocol specified criteria of an RSD ≤10% and a mean concentrations within ±15% of the target concentrations). The analysed dietary formulations were found to contain 90.7% to 113% of the target test substance concentration in the diet which met the WIL Research SOP requirements (85% to 115%). The test substance was not detected in the analysed basal diet formulation that was administered to the control group (group 1).

Average test substance consumption (mg/kg bw/day), were determined based on target dietary concentrations of the test substance.

Duration of treatment / exposure:

90 d

Frequency of treatment:

Continuously in feed

Remarks:

Doses / Concentrations:
10, 50, 250, 750 and 1,500 ppm test substance (i.e., equivalent to 1, 3, 16, 47 and 91 mg a.i./kg bw/day for the males and 1, 4, 19, 55 and 113 mg a.i./kg bw/day for the females)
Basis:
nominal in diet

No. of animals per sex per dose:

10 males and 10 females per dose group.

Control animals:

yes, plain diet

Details on study design:

Rationale for animal assignment: Seven days prior to the initiation of diet administration, all available rats were weighed and examined in detail for physical abnormalities. The animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design. Individual body weights at randomization were within ±20% of the mean for each sex. Each group (groups 1-6) consisted of 10 males and 10 females. These animals were then randomized into five study replicates to allow for the reasonable conduct of the functional observational battery and locomotor activity assessments. Each group and sex were approximately equally represented within each study replicate.

Observations and examinations performed and frequency:

SURVIVAL:
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Animals found dead were examined macroscopically as soon as possible to ensure that tissues were not lost due to autolysis.

CLINICAL OBSERVATIONS:
Clinical examinations were performed once daily. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Observations included, but were not limited to, changes in the skin, fur, eyes and mucous membranes; respiratory, circulatory, autonomic, and central nervous systems function; somatomotor activity; and behaviour patterns. Once daily clinical observations were not performed on days when detailed physical examinations were conducted. Detailed physical examinations, including removal from home cage and placement in a standard arena for observations for changes in gait, posture, clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), and permanent or semi-permanent signs, such as skin lesions and hair loss, were conducted on all animals approximately weekly throughout the study, beginning at pretest initiation and including the day of randomization, and prior to the scheduled necropsy. A separate computer protocol was used to record any treatments administered.

BODY WEIGHTS:
Individual body weights were recorded at pretest initiation (presented as study week -2), at randomization (presented as study week -1), approximately weekly throughout the study, and on the day of the scheduled necropsy (fasted). During study week 12, body weights were recorded twice weekly. Mean body weights and mean body weight changes were calculated for the corresponding intervals.

FOOD AND TEST SUBSTANCE CONSUMPTION:
Individual food consumption was recorded for two weeks during pretest (study week -2 to -1 and study week -1 to 0) and approximately weekly during the dosing period. During study week 12, food consumption was recorded twice weekly. Food intake was calculated as g/animal/day and g/kg/day for the corresponding body weight intervals. Food efficiency (body weight gained as percent of feed consumed) was also calculated for each interval. The mean amounts of test substance consumed (mg/kg bw/day) by each sex per diet group were calculated from the mean food consumed (g/kg of mean body weight/day) and the appropriate target concentration of test substance in the food (mg/kg of diet).

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) ASSESSMENTS:
FOB assessments were recorded for all animals prior to the initiation of diet administration and during study week 12. Testing was performed by the same technicians, whenever possible, without knowledge of the animals group assignments.
All animals were observed for following.
HOME CAGE OBSERVATIONS: Posture, convulsions/tremors, faeces consistency, biting and 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 colour and muscle tone
OPEN FIELD OBSERVATIONS: Mobility, rearing, convulsions/tremors, grooming, bizarre/stereotypic behaviour, time to first step (seconds), gait, arousal, urination/defecation, gait score and backing
SENSORY OBSERVATIONS: Approach response, startle response, pupil response, forelimb extension, air righting reflex, touch response, tail pinch response, eye blink response, hind limb extension and olfactory orientation
NEUROMUSCULAR OBSERVATIONS: Hindlimb extensor strength, hindlimb foot splay, grip strength-hind and forelimb and rotarod performance
PHYSIOLOGICAL OBSERVATIONS: Catalepsy, body temperature and body weight

LOCOMOTOR ACTIVITY: Locomotor activity was assessed for all animals prior to the initiation of diet administration and during study week 12. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of two or more consecutive photobeams).

CLINICAL PATHOLOGY:
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected from all animals on the day of the scheduled necropsy (study week 13). Haematology and coagulation parameters evaluated were total leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration (MCHC), platelet count (PLATELET), prothrombin time (PT), activated partial thromboplastin time (APTT), reticulocyte count percent (RETIC), absolute (RETIC ABSOLUTE), mean platelet volume (MPV), differential leukocyte count, red cell distribution width (RDW), haemoglobin distribution width (HDW), platelet estimate, red cell morphology (RBC Morphology).

SERUM CHEMISTRY: Parameters evaluated were albumin, total protein, globulin [by calculation], albumin/globulin ratio (A/G Ratio), total bilirubin (total Bile), 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) and sorbitol dehydrogenase (SDH)
URINALYSIS: Parameters evaluated were specific gravity (SG), pH, urobilinogen (URO), total volume (TVOL), colour (COL), clarity (CLA), protein (PRO), glucose (GLU), ketones (KET), bilirubin (BIL), occult blood (BLD), leukocytes (LEU), nitrites (NIT) and microscopy of sediment.

OPHTHALMIC EXAMINATIONS:
Ocular examinations were conducted on all animals prior to randomization (study week 2) and near the end of the treatment period (study week 12). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

Sacrifice and pathology:

MACROSCOPIC EXAMINATION:
A complete necropsy was conducted on all animals. Animals surviving to the scheduled necropsy were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The tissues and organs were collected for analysis were adrenals, aorta, bone with marrow, femur, sternum, bone marrow smear, brain, cerebrum level, cerebrum level, cerebellum with medulla/pons, cervix, epididymides, eyes with optic nerve, gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, larynx, liver (sections of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph nodes, nasal cavity, ovaries with oviducts, pancreas, peripheral nerve (sciatic), peyer’s patches, pituitary, pharynx, prostate, salivary glands, seminal vesicles, skeletal muscle (rectus femoris), skin (with mammary gland), spinal cord (cervical, thoracic,lumbar), spleen, testes, thymus, thyroid (with parathyroids, if present), trachea, urinary bladder, uterus, vagina, gross lesions.

ORGAN WEIGHTS:
The organs weighed from all animals at the scheduled necropsy were adrenals, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus, thyroid with parathyroids and uterus.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION: Microscopic examination was performed on tissues listed under macroscopic examination from the 1,500 ppm group female found dead and all animals in the control and 1,500 ppm groups at the scheduled necropsy.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. All statistical tests were performed using WTDMS™. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 5% and 1%, comparing each test substance treated group to the control group by sex.

All repeated measures analysis of variance (RANOVA) statistical analyses for total and ambulatory locomotor activity counts recorded during pretest and after dosing were conducted by BioSTAT Consultants, Inc., Portage, MI, using SAS version 9.1 software (SAS Institute, Inc., 2002-2003).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis 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:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

SURVIVAL:
There was no test substance related effect on mortality. One female from the 1,500 ppm group was found dead on study day 77. Haemorrhage within the soft tissue and bone peripheral to the nasal cavity were suggestive of trauma. Haemorrhage was also a component of extensive nasal exudation that likely resulted in impaired respiration, aerophagia, and the gas filled gastrointestinal tract noted at necropsy. In the absence of other noteworthy findings, the cause of death was attributed to traumatic injury unrelated to the test substance.

CLINICAL OBSERVATIONS:
There were no test substance related clinical observations. Low incidences of brown material around the anogenital area were noted in the 1,500 ppm group males at the time of detailed physical examinations and during the daily clinical observations, noted sporadically between study day 63 and 91. All other clinical findings in the test substance treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHTS:
Test substance related lower body weight gains were noted in the 750 ppm group females and 1,500 ppm group males and females. Test substance related lower mean cumulative body weight gains were noted throughout the study in the 750 ppm group females and 1,500 ppm group males and females (sometimes statistically significant) when compared to the control group. During the study, mean body weights were up to 10.0% lower in the 750 ppm group females and up to 7.5% and 10.5% lower in the 1,500 ppm group males and females, respectively, when compared to the control group. At end of the study (study week 12), mean body weights were 10.0% lower in the 750 and 1,500 ppm group females and 3.0% lower in the 1,500 ppm group males when compared to the control group. There were no test substance related effects noted on body weights in the 10, 50, or 250 ppm groups or 750 ppm group males. Lower mean body weights were noted in the 50 ppm group males and females during the study; however, these differences lacked a dose response trend and were preceded by significantly lower mean body weight gain, compared to the control group, during study week -1 to 0. Some additional statistically significant differences were noted when the test substance treated and control groups were compared; however, these differences were incidental and not considered test substance related.

FOOD AND TEST SUBSTANCE CONSUMPTION:
Test substance related lower food consumption was noted in the 1,500 ppm group males and 750 and 1,500 ppm group females generally throughout the study. Test substance related lower food efficiency was noted in the 1,500 ppm group males and females during study week 0 to 1. Lower mean food consumption values (g/animal/day and g/kg/day) were noted beginning at study week 0 and generally throughout the study, when compared to the control group. Slightly lower mean food consumption values (g/animal/day and g/kg/day) were also noted, to a lesser extent, in the 750 and 1,500 ppm group females beginning at study week 4 and intermittently throughout the study. Statistically significantly lower mean food efficiency values were noted from study week 0 to 1 in the 1,500 ppm group males and females and from study week 1 to 2 in the 750 ppm group females when compared to the control group. Food efficiency values thereafter were generally similar to control group values. The correlating effects in food consumption and food efficiency in males during study week 0 to 1, along with a lack of effect in food consumption in females suggests toxicity, rather than palatability issues.
There were no test substance related effects noted in food consumption or food efficiency values in the 10, 50, 250, or 750 ppm group males or females. Some statistically significant differences were noted when the test substance-treated and control groups were compared; however, these differences were not considered test substance related.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
HOME CAGE OBSERVATIONS: Home cage observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated group males and females were compared to the control group at the study week 12 evaluation.
HANDLING OBSERVATIONS: Handling observations were unaffected by test substance administration. There were no statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
OPEN FIELD OBSERVATIONS: Open field observations were unaffected by test substance administration. There were no statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
SENSORY OBSERVATIONS: Sensory observations were unaffected by test substance administration. There were no statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
NEUROMUSCULAR OBSERVATIONS: Neuromuscular observations were unaffected by test substance administration. Statistically significantly lower mean rotarod performances were noted in the 50, 250, and 750 ppm group females when compared to the control group at the study week 12 evaluation; however, because mean rotarod performance for females in all test substance treated groups was generally equivalent to or higher than during pretest, and because the control group mean was higher than the WIL Research historical control reference range, these differences were not considered test substance related. There were no other statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
PHYSIOLOGICAL OBSERVATIONS: Test substance related lower body weights were noted in the 750 ppm group females and 1,500 ppm group males and females at study week 12 when compared to the control group.
All other physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated group males and females were compared to the control group at the study week 12 evaluation.
LOCOMOTOR ACTIVITY: Locomotor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. At study week 12, statistically significantly, non-dose-related higher ambulatory motor activity counts were noted over the 60-min test session in the 10 ppm group males, when compared to the control group. In addition, lower total motor activity counts were noted from 21-30 min at the study week 12 evaluation in the 750 and 1,500 ppm group females, when compared to the control group. Due to inconsistent trends and the lack of concurrent differences between ambulatory and total motor activity counts, the statistically significant differences noted at study week 12 were not considered test substance-related. There were no other statistically significant changes for the test substance-treated group males and females when compared to the control group at the study week 12 evaluation. With the exception of those differences listed above, values obtained from the six epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 min, 31-40 min, 41-50 min, and 51-60 min) and the overall 60-minute test session were comparable to the concurrent control values. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on study week 12.

HEMATOLOGY AND COAGULATION:
Test substance-related lower red blood cell counts (RBC), haemoglobin (HGB) levels, and haematocrit (HCT) levels were noted in the 1,500 ppm group females. Test substance-related alterations in haematology parameters were limited to statistically significantly lower mean RBC counts, HGB levels, and HCT levels in the 1,500 ppm group females compared to the control group mean. Although the magnitudes of change were small and, with the exception of two females with haemoglobin concentrations slightly lower than the WIL Research historical control reference range, all other values were within the historical control reference ranges. A similar effect was not observed in the test substance-treated male groups. Although not statistically significantly different from the control group, a higher mean total white blood cell count was noted in the 1,500 ppm group males; this variation also was due primarily to a higher mean absolute lymphocyte count. The variations for both parameters were attributed to high individual values for one male in the 1,500 ppm group, with total white blood cell and lymphocyte counts that exceeded the WIL Research historical control reference ranges. With the exclusion of these individual values, a dose response would not have been apparent in the 1,500 ppm group males, and therefore, the higher mean white blood cell and lymphocyte counts were considered to be incidental and unrelated to test substance administration. There were no test substance-related or statistically significant alterations in coagulation parameters. There were no test substance-related effects on haematology parameters noted in the 10, 50, 250, or 750 ppm group males or females. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. These findings included higher mean white blood cell and absolute lymphocyte counts in the 750 ppm group females. In the absence of a dose response, these variations were considered to be incidental and unrelated to test substance administration.

SERUM CHEMISTRY:
Test substance-related higher cholesterol levels were noted in the 1,500 ppm group males and females. Test substance-related alterations in serum chemistry parameters were limited to high mean cholesterol levels in the 1,500 ppm group males and females. The variation was statistically significant in the 1,500 ppm group females when compared to the control group. An effect of test substance administration on cholesterol levels in the 1,500 ppm group males was suggested by the magnitude of change and dose response similar to that noted in the females. With the exception of one male in the 1,500 ppm group, all individual animal cholesterol levels were within the historical control reference range. There were no test substance-related effects noted on serum chemistry parameters in the 10, 50, 250, or 750 ppm group males or females.

URINALYSIS:
Test substance-related lower urine pH was noted in the 1,500 ppm group males and variable urine colour (yellow, dark yellow, and/or red) was noted in the 1,500 ppm group males and females and 750 ppm group females. Test substance-related lower mean urine pH values were statistically significantly in the 1,500 ppm group males when compared to the control group. In addition, macroscopic evaluation of the urine revealed test substance-related variations in urine colour (yellow, dark yellow, and red) in the 1,500 ppm group males and females and in the 750 ppm group females. There were no test substance-related effects on urinalysis parameters in the 10, 50, or 250 ppm group males or females or the 750 ppm group males.

OPHTHALMIC EXAMINATIONS:
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

MACROSCOPIC EXAMINATION:
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.

ORGAN WEIGHTS:
Test substance-related alterations in mean liver weights were noted in the 1,500 ppm groups. Statistically significantly higher mean liver relative to final body weights were noted in the 1,500 ppm group males and females. There were no test substance-related differences in organ weights noted in the 10, 50, 250, or 750 ppm group males or females. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Mean kidney relative to final body weights and absolute testes weights were higher in the 1,500 ppm group males. The higher kidney relative to final body weight was a function of a slightly higher mean absolute kidney weight and slightly lower mean final body weight resulting in a ratio statistically significantly different from that noted in the control group males. Although statistically significantly different from the control group mean, mean absolute testes weights were only 7.8% higher in the 1,500 ppm group. All but two of 10 individual absolute testes weights for the 1,500 ppm group were within two standard deviations of the WIL Research historical control mean. Therefore, due to the small magnitudes of change and absence of histologic correlations for the organ weight differences, all of the statistically significant variations noted in the 1,500 ppm group males were considered to be incidental and unrelated to test substance administration. As was noted in the males, there were multiple statistically significantly higher mean relative organ weight variations due to slightly higher absolute organ weights in conjunction with the 10.5% lower final body weight noted in the 1,500 ppm group females. These higher organ relative to final body weight ratios were noted with the brain, kidney, and liver in the 1,500 ppm group females. Due to the small magnitudes of change and absence of histologic correlations for the organ weight differences, all of the statistically significant variations noted in the 1,500 ppm group females were considered to be incidental and unrelated to test substance administration. A higher mean thymus relative to final body weight ratio was noted in the 750 ppm group females and a lower mean absolute thyroid/parathyroid weight was noted in the 50 ppm group females. In the absence of a dose response, these variations were also considered to be incidental and unrelated to test substance administration.

MICROSCOPIC EXAMINATION:
Test substance related histopathologic findings were noted in the femoral bone (metaphyseal hyperostosis) at ≥750 ppm and kidneys (foreign material, tubular hyperplasia, and brown pigment) at ≥250 ppm group males and females. In the femur, hyperostosis was characterized by increased amounts of metaphyseal trabeculae filling the marrow space. This change was most obvious in the 1,500 ppm group males. In the control group males, the metaphyseal trabeculae typically did not extend into the diaphysis, the trabeculae were oriented parallel to the bone with minimal lateral anastomoses or branching, and this bone occupied <50% of the marrow cavity within the metaphysis. Hyperostosis in the males was characterized by the extension of trabeculae into the diaphysis with increased amounts of ‘lateral branching’ and occupation of >50% of the marrow cavity. Normal baseline morphology of the femur in the control group females was similar to minimal to mild hyperostosis in the males with trabeculae extending well into the diaphysis, lateral branching was common and trabeculae occupied up to approximately 75% of the marrow cavity in the metaphysis. Minimal hyperostosis was diagnosed in the females when this lateral branching was so prominent as to nearly span the diameter of the metaphysis. Hyperostosis was diagnosed in one female each from the control, 10, and 50 ppm groups which was attributed to the arbitrary subjective assessment of ‘normal’ and some variations in tissue morphology due to sectioning. Hyperostosis noted in the 750 ppm group males and females was considered to represent a test substance-related effect. In the kidney, there was a foreign material within the tubular lumina in the cortex (minimal), and rarely, the outer stripe of the outer medulla (mild). This material was brown to gray, granular, and formed small aggregates; the surrounding tubular epithelium appeared normal. The composition of this material was unknown but was inconsistent with tubular cell or inflammatory cell debris and was noted at dietary concentrations ≥250 ppm in both sexes. These findings associated with the foreign material were not considered adverse and the foreign material was not in the same location as the renal tubular hyperplasia noted. Renal tubular hyperplasia was characterized by small aggregates of basophilic tubules with epithelial cells that were enlarged and often piled upon each other. These tubules were consistently in the outer stripe of the outer medulla and were morphologically distinct from chronic progressive nephropathy (CPN) and basophilic tubules considered to be the precursors of CPN. This finding was noted only in the 1,500 ppm group males and females.
Two males in the 1,500 ppm group and one male from the 750 ppm group had small amounts of a coarsely granular brown pigment within tubular epithelial cells. Although this material was considered to be consistent with lipofuscin, a special staining procedure (AFIP method for lipofuscin) was negative. Therefore, the identity and toxicological importance of this pigment were undetermined. There were no other definitive test substance related histologic changes. The female in the 1,500 ppm group that died on study day 77 had minimal multifocal and bilateral renal tubular necrosis of the cortex. It was unclear if this finding was secondary to the animal’s moribund condition prior to death or was related to test substance administration.

Dose descriptor:

NOAEL

Effect level:

16 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Metaphyseal hyperostosis noted microscopically in the femoral bone

Dose descriptor:

NOAEL

Effect level:

19 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Metaphyseal hyperostosis noted microscopically in the femoral bone

Critical effects observed:

not specified

Conclusions:

Under the conditions of this 90 d dietary study, the NOAEL for systemic toxicity of the test substance in rat was considered to be 250 ppm (equivalent to 16 and 19 mg/kg bw/day for males and females, respectively).

Executive summary:

A 90 d dietary study in Sprague-Dawley rats was conducted at concentrations of 0, 10, 50, 250, 750 and 1,500 ppm test substance (i. e., equivalent to 0, 1, 3, 16, 47 and 91 mg a.i./kg bw/day for males and 0, 1, 4, 19, 55 and 113 mg a.i./kg bw/day for females) according to OECD Guideline 408 and OPPTS 870.3100, in compliance with GLP. This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity assessments. Ten males and 10 females per dose group were used. Adverse effects included haematological differences noted at 1,500 ppm (females only), tubular hyperplasia noted microscopically at 1,500 ppm, and metaphyseal hyperostosis noted microscopically in the femoral bone at ≥750 ppm. Under the conditions of this study, the NOAEL for systemic toxicity of the test substance in rat was considered to be 250 ppm (equivalent to 16 and 19 mg/kg bw/day for males and females, respectively) (Kirkpatrick JB, 2013).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

16 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality database.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 4, 2012 to January 11, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 410 and OPPTS Guideline 870.3200, in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh
- Age at study initiation: 44 d old
- Housing: Individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Racks were rotated within the animal room at least once every two weeks during the study to change the physical location from one area of the room to another to ensure similarly varied environmental exposure for all animals. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® #5002. Basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinicalpathology blood collection.
- Water: Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, provided ad libitum
- Acclimation period: 14 d. During acclimation period, each animal was observed twice daily for mortality and changes in general appearance or behavior.

The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.2°C - 22.0°C
- Humidity:34.1% - 48.3%
- Air changes (per h): 10
- Photoperiod: 12 h light/12 h dark cycle

Type of coverage:

other: Skin was covered with a wrap consisting of gauze secured in place with non-irritating tape

Vehicle:

water

Details on exposure:

On the day prior to dosing, and during the study as needed (after completion of the daily exposure), the hair was clipped from the back (down each side to the ventral surface) and flanks of each animal using an electric clipper; a different set of clippers was used for the control and test substance-treated groups to avoid potential cross contamination.

Test substance moistened with the vehicle, deionized water (0.6 or 0.7 mL), was administered by topical application to the shaved intact dorsal surface (covering approximately 10% of the body surface area) of three groups of rats once daily for up to 28 consecutive days. The test substance was allowed to remain in contact with the skin for 6 h (±30 min), during which time the treated skin was covered with a wrap consisting of gauze secured in place with non-irritating tape, and the rats wore Elizabethan collars. At the end of the 6 h (±30 min) exposure period, the collars and
dressings were removed and the test sites were wiped and cleaned using gauze soaked in deionized water to remove any remaining test substance from the skin. A concurrent control group received the vehicle on a comparable regimen.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 d

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
100, 300 and 1,000 mg/kg bw/day
Basis:

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on using the recommended limit test exposure level of 1,000 mg/kg/day as the Group 4 target exposure concentration (references in the OPPTS 870.3200 testing).

- Four days prior to the initiation of dose administration, all available rats were weighed and examined in detail for physical abnormalities. The animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design. Individual body weights at randomization were within ± 20% of the mean for each sex.

Observations and examinations performed and frequency:

SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

On Day 0 (first dose), one male and one female from 300 mg/kg bw/day dose group were found dead prior to completion of the 6 h (±30 min) dosing period and were replaced with other male and female replacement animals, prior to dosing on the following day; therefore, these animals were only dosed for 27 consecutive days. Also on Day 0, one female (300 mg/kg bw/day) died during dose application and was replaced with other replacement female. These early deaths were considered to be procedure-related and not considered test substance-related.

CLINICAL OBSERVATIONS
Clinical examinations were performed twice daily, prior to dose administration and 1 to 2 h following bandage removal. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals within four days of receipt, on the day of randomization, and weekly (± 1 d) during the dosing period, and on the day of the scheduled necropsy. Any observations noted outside of the above specified intervals were also recorded.

DERMAL OBSERVATIONS:
Dermal observations of the dosing site were conducted during the week prior to randomization (± two days), on the day of randomization, prior to dosing on study day 0, weekly (± 2 d; prior to dosing) during the dosing period, and on the day of the scheduled necropsy for erythema and oedema in accordance with a four step grading system, of very slight, slight, moderate, and severe. Other remarkable dermal findings, if present, were recorded. The presence or absence of findings was recorded for individual animals.

BODY WEIGHTS
Individual body weights were recorded within four days of receipt, on the day of randomization, on study Day -1, weekly (± one day) throughout the study, and on the day prior to the scheduled necropsy (non-fasted). Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION
Individual food consumption was calculated beginning one week prior to randomization (± two days), on the day of randomization, and weekly (± one day) throughout the study. Food intake was calculated as g/animal/day and g/kg/day for the corresponding body weight intervals. In addition, food efficiency (body weight gained as percent of feed consumed) was calculated.

CLINICAL PATHOLOGY:
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected from all animals at the scheduled necropsy (study Day 28). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for haematology and serum chemistry evaluation via the retro-orbital sinus of animals anesthetized by inhalation of isoflurane. The site of collection, including left or right, was documented for each animal. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide. Blood was collected into tubes containing potassium EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

HEMATOLOGY AND COAGULATION PARAMETERS: Total leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration (MCHC), platelet count (PLATELET), prothrombin time (PT), activated partial thromboplastin time, reticulocyte count percent (RETIC), absolute (RETIC ABSOLUTE), differential leukocyte count, percent a and absolute, haemoglobin distribution width, platelet estimate, and red cell morphology (RBC Morphology).

SERUM CHEMISTRY: 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, calcium, chloride, phosphorus, potassium, sodium, sorbitol dehydrogenase (SDH), triglycerides (Triglyceride), haemolysis, lipemia and Icterus.

URINALYSIS: Parameters evaluated were specific gravity (SG), pH, urobilinogen (URO), total volume (TVOL), colour (COL), clarity (CLA), protein (PRO), glucose (GLU), ketones (KET), bilirubin (BIL), occult blood (BLD), leukocytes (LEU), nitrites (NIT) and microscopy of sediment.

OPHTHALMIC EXAMINATIONS
Ocular examinations were conducted on all animals prior to randomization (study Day -8) and near the end of the dosing period (study Day 27). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

Sacrifice and pathology:

MACROSCOPIC EXAMINATION:
A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The tissues and organs were collected for analysis were adrenals, aorta, bone with marrow, femur, sternum, bone marrow smear, brain, cerebrum level, cerebrum level, cerebellum with medulla/pons, cervix, epididymides, eyes with optic nerve, gastrointestinal tract, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, larynx, liver (sections of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph nodes, nasal cavity, ovaries with oviducts, pancreas, peripheral nerve (sciatic), peyer’s patches, pituitary, pharynx, prostate, salivary glands, seminal vesicles, skeletal muscle (rectus femoris), skin (with mammary gland), spinal cord (cervical, thoracic, lumbar), spleen, testes, thymus, thyroid (with parathyroid, if present), trachea, urinary bladder, uterus, vagina, gross lesions.

ORGAN WEIGHTS:
The organs weighed from all animals at the scheduled necropsy were adrenals, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus, thyroid with parathyroids and uterus.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION:
Microscopic examination was performed on all tissues listed under macroscopic examination from all animals in the control and 1,000 mg/kg bw/day groups at the scheduled necropsy. In addition, gross lesions were examined from all animals and correlated to microscopic findings, if possible. Adrenal glands, liver, and skin (treated and untreated) were examined from the 100 and 300 mg/kg bw/day groups. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations 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.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. All statistical tests were performed using the WIL Toxicology Data Management System (WTDMS™). Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance treated group to the control group by sex.
Microscopic findings were compared using a one-tailed Fisher’s exact test for significance levels of 5% and 1%.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(300 and 1,000 mg/kg bw/day group males and females)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

SURVIVAL:
There were no test substance related deaths; however, on study day 0, one 300 mg/kg bw/day group female died during dose application and one male and one female were found dead prior to completion of the 6 h (± 30 min) dosing period. These deaths were not considered test substance-related, but instead a result of the dose application and wrapping procedures. Due to the timing of the early deaths (all occurring on study Day 0), these animals were replaced.

CLINICAL OBSERVATIONS:
Test substance-related clinical observations of yellow material around the urogenital area was noted in the 1,000 mg/kg bw/day group males and 300 and 1,000 mg/kg bw/day group females and yellow material around the anogenital area was noted in the 1,000 mg/kg bw/day group females. The effects were noted mainly during the first week of test substance administration, in a variable frequency, and in no more than three of the 10 animals in a group. Only in one 1,000 mg/kg bw/day group female were these effects observed after study Day 15. All other clinical findings in the test substance treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and were common findings for laboratory rats of this age and strain.

DERMAL OBSERVATIONS:
There was no dermal irritation observed for any animal in the control or test substance treated groups.

BODY WEIGHTS:
Test substance related lower body weights were noted in the 300 and 1,000 mg/kg bw/day group males and females. Lower mean body weight gains were generally noted in the 300 and 1,000 mg/kg bw/day group males and females; these values were statistically significantly lower for intervals 13 to 20 (300 and 1,000 mg/kg bw/day group males) and 20 to 27 (1,000 mg/kg bw/day group males) when compared to the control group. As a result of the lower mean body weight gains, lower mean cumulative body weight gains (occasionally statistically significant) were noted. On study Day 27, mean body weights in the 300 and 1,000 mg/kg bw/day group males were 5.1% and 8.5% lower than the control group, respectively, and mean body weights in the 300 and 1,000 mg/kg bw/day group females were 5.5% and 4.3% lower than the control group, respectively. The mean cumulative body weight gains from study Days -1 to 27 were lower than control by 15% and 28% in the males and by 21% and 19% in the females for the 300 and 1,000 mg/kg bw/day groups, respectively. There were no test substance related effects on body weight in the 100 mg/kg bw/day group males or females. Lower mean body weight gains were noted in the 100 mg/kg bw/day group females from study Days 13 to 20; however, the difference was not considered test substance related due to the transient or inconsistent pattern throughout the study.

FOOD CONSUMPTION:
Test substance-related lower food consumption and food efficiency was noted in the
1000 mg/kg/day group males. Statistically lower mean food consumption values were noted in the 1,000 mg/kg bw/day group males from study Day -1 to 6 (g/animal/day and g/kg/day) and 13 to 20 (g/animal/day) when compared to the control group. Additionally, a lower mean food efficiency value was noted in the 1,000 mg/kg bw/day group males from study Day 20 to 27 when compared to the control group. Although the food consumption and food efficiency differences were minimal, the slightly lower values likely contributed to the lower cumulative body weight gains and lower mean body weight values in the 1,000 mg/kg bw/day group males. There were no other test substance related effects on food consumption or food efficiency. Some statistically significant food consumption and/or food efficiency differences were noted in the 100 and 300 mg/kg bw/day group males; however, these differences were not considered treatment-related due to the small magnitude of the changes, changes occurring in the opposite direction from that generally associated with an adverse effect, and/or the presence of a transient or inconsistent pattern throughout the study. There were no effects noted in the females.

HEMATOLOGY AND COAGULATION:
There were no test substance-related alterations in haematology and coagulation parameters. However, a statistically significant difference was observed when the control and test substance treated groups were compared. A lower mean corpuscular haemoglobin (MCH) value was noted in the 300 mg/kg bw/day group females; however, it was not considered test substance-related due to the lack of a dose response.

SERUM CHEMISTRY:
Test substance related alterations in serum chemistry parameters include higher bilirubin in the 1,000 mg/kg bw/day group males, higher cholesterol in the 300 and 1,000 mg/kg bw/day group females, and lower alanine aminotransferase (ALT) in the 1,000 mg/kg bw/day group females. Statistically significantly higher mean bilirubin value was noted in the 1, 000 mg/kg bw/day group males when compared to the control group. The elevated mean total bilirubin value in the 1,000 mg/kg bw/day group males was associated with minimal changes in the urine.
Higher mean cholesterol values were noted in the 300 and 1,000 mg/kg bw/day group females when compared to the control group and occurred in a dose responsive manner. A lower mean ALT value in the 1,000 mg/kg bw/day group females was considered test substance related; however, this change was not toxicologically relevant. There were no other test substance related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test substance treated groups were compared. Higher mean total protein was noted in the 100 mg/kg bw/day group males but was not considered test substance related due to the lack of a dose response. Higher mean albumin values in the 300 and 1,000 mg/kg bw/day group females were not test substance related, as the change was minimal and due to biologic variation.

URINALYSIS:
Test substance related alterations in urinalysis parameters were limited to lower pH values in the 300 and 1,000 mg/kg bw/day group males, a higher mean urobilinogen value in the 1,000 mg/kg bw/day group males, and red urinary discoloration in one 100 mg/kg bw/day male and in the 300 and 1,000 mg/kg bw/day group males and females. Lower mean pH values were noted in the 300 and 1,000 mg/kg bw/day group males (statistically significant in the 1,000 mg/kg bw/day group). The lower mean pH values were not associated with microscopic kidney lesions. A statistically significantly higher mean urobilinogen value was noted in the 1,000 mg/kg bw/day group males when compared to the control group and was associated with a higher mean total bilirubin value. Red discoloration of the urine was noted at 100, 300, and 1,000 mg/kg bw/day dose levels in the males and at 300 and 1,000 mg/kg bw/day dose levels in the females. While the cause of this red discoloration of the urine is not confirmed, it is most likely a urinary excretion of the test substance or a metabolite and not indicative of renal or systemic toxicity. Occasionally the urines from these animals were also noted positive for nitrite.

OPHTHALMIC EXAMINATIONS:
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

MACROSCOPIC EXAMINATION:
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.

ORGAN WEIGHTS:
Test substance related changes in organ weights in the 1,000 mg/kg bw/day group included higher heart weights and lower thymus weights in males; higher kidney, liver, and thyroid/parathyroid weights in females; and higher lung weights in males and females. In the 300 mg/kg bw/day group, test substance related organ weight changes were limited to lower thymus weights in males. Statistically significantly higher mean lung weights (absolute and relative to final body weight) were noted in the 1,000 mg/kg bw/day group males and females when compared to the control group. The higher lung weights were not associated with microscopic lung changes. A statistically significantly higher mean heart weight (relative to final body weight) was noted in the 1,000 mg/kg bw/day group males when compared to the control group; however, this was not associated with microscopic heart findings. Statistically significantly lower mean thymus weights (absolute and relative to final body weight) were noted in the 300 and 1,000 mg/kg bw/day group males but were not associated with microscopic changes. Although not associated with microscopic findings, statistically significantly higher mean kidney weight (relative to final body weight) and thyroid/parathyroid weights (absolute [not statistically significant] and relative to final body weight) were noted in the 1,000 mg/kg bw/day group females. Higher mean liver weights (absolute [not statistically significant] and relative to final body weight [statistically significant]) were noted in the 1,000 mg/kg bw/day group females and correlated to microscopic findings but were not associated with any liver enzyme elevations suggestive of toxicity. The relative liver weight in the 1,000 mg/kg bw/day females was 12% higher than that in the control group. All test substance related organ weight changes were minimal and were not considered adverse. A statistically significantly lower mean absolute adrenal gland weight was noted in the 1,000 mg/kg bw/day group females when compared to the control group; however, this change was considered to be a result of an effect on final body weight. There were no other test substance-related effects on organ weights.

MICROSCOPIC EXAMINATION:
Test substance related microscopic findings included epidermal hyperplasia in the treated skin (100, 300, and 1,000 mg/kg bw/day group males and females), cytoplasmic alteration of hepatocytes (300 and 1,000 mg/kg bw/day group males and 100, 300, and 1,000 mg/kg bw/day group females), and vacuolation of the adrenal cortex (100, 300, and 1,000 mg/kg bw/day group males and 1,000 mg/kg bw/day group females).
Treated skin: Minimal to mild epidermal hyperplasia, characterized by thickening of the stratum spinosum and granulosum and hypertrophy of the basal cell layer, was noted in the 100, 300, and 1,000 mg/kg bw/day group males and females. A similar change was noted in a low number (4/10) of control group males but was not observed in control group females. The incidence of epidermal hyperplasia was statistically significant in the 100, 300, and 1,000 mg/kg bw/day group males and females when compared to the control group.
Liver: Minimal to mild cytoplasmic alteration of the hepatocytes was noted in the 300 and 1,000 mg/kg bw/day group males and mild cytoplasmic alteration of the hepatocytes was noted in the 100, 300, and 1,000 mg/kg bw/day group females. Cytoplasmic alteration was characterized by tinctoral variations in staining characteristics of centrilobular hepatocytes (pale pink) when compared to periportal and midzonal hepatocytes. Periportal and midzonal hepatocytes were slightly smaller and more eosinophilic than centrilobular hepatocytes; however, hypertrophy of centrilobular hepatocytes was not detected. The incidence of cytoplasmic alteration was statistically significant in the 1,000 mg/kg bw/day group females when compared to the control group. The higher liver weights and corresponding histologic correlate are considered to be likely a compensatory response possibly related to liver enzyme induction and a nonadverse effect of test substance administration. Adrenal cortex: Minimal to moderate vacuolation, characterized by multiple fine vacuoles and/or larger distinct vacuoles in the zona fasciculata and/or zona reticularis, was noted in the 100, 300, and 1,000 mg/kg bw/day group males and 1,000 mg/kg bw/day group females. Two females in the 1,000 mg/kg bw/day group had multifocal, minimal brown pigment in cortical cells of the zona fasciculata. Adrenal cortical vacuolation was also observed in control group males (5/10) and females (3/10). The incidence of adrenal cortical cytoplasmic vacuolation in the 300 and 1,000 mg/kg bw/day group males and 1,000 mg/kg bw/day group females was statistically significant when compared to the control group. There were no other test substance-related histologic changes. One male in the 1,000 mg/kg bw/day group had a focal area of tubular necrosis in the cortex of the right kidney. This was not considered to be test substance-related given the focal nature of the lesion. One female in the 1,000 mg/kg bw/day group had minimal acute pyelonephritis of the right kidney. This was not considered test substance related given the background nature of the lesion. Remaining histologic changes were considered to be incidental findings or related to some aspect 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.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on lower mean cumulative body weight gains in the 300 and 1,000 mg/kg bw/day group males and females, and adrenal cortical cytoplasmic vacuolation in the 300 mg/kg bw/day group males and 1,000 mg/kg bw/day group males and females

Critical effects observed:

not specified

Conclusions:

In a four week repeated dose dermal toxicity study, NOAEL was considered to be 100 mg/kg bw/day.

Executive summary:

A four week dermal toxicity study in Sprague-Dawley rats was conducted at concentrations of 0, 100, 300, and 1,000 mg/kg bw/day test substance according to OECD Guideline 410 and OPPTS 870.3200, in compliance with GLP. Ten males and ten females per dose group were used. Test substance moistened with the vehicle (deionized water) was applied topically under occlusive conditions to the shaved intact dorsal surface of three groups of rats once daily for 6 h for up to 28 consecutive days. A concurrent control group received the vehicle on a comparable regimen. There were no test substance-related effects on survival, dermal observations, haematology, coagulation, ophthalmic examinations and macroscopic observations. Treatment-related lower mean cumulative body weight gains were observed in the 300 and 1,000 mg/kg bw/day group males and females. Also adrenal cortical cytoplasmic vacuolation were observed in the 300 mg/kg bw/day group males and 1,000 mg/kg bw/day group males and females. Hence, the NOAEL was considered to be 100 mg/kg bw/day (Mertens JJWM, 2014).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality database.

Additional information

Oral

A 90 d dietary study in Sprague-Dawley rats was conducted at concentrations of 0, 10, 50, 250, 750 and 1,500 ppm test substance (i.e., equivalent to 0, 1, 3, 16, 47 and 91 mg a.i./kg bw/day for males and 0, 1, 4, 19, 55 and 113 mg a.i./kg bw/day for females) according to OECD Guideline 408 and OPPTS 870.3100, in compliance with GLP. This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity assessments. Ten males and 10 females per dose group were used. Adverse effects included haematological differences noted at 1,500 ppm (females only), tubular hyperplasia noted microscopically at 1,500 ppm, and metaphyseal hyperostosis noted microscopically in the femoral bone at ≥750 ppm. Under the conditions of this study, the NOAEL for systemic toxicity of the test substance in rat was considered to be 250 ppm (equivalent to 16 and 19 mg/kg bw/day for males and females, respectively) (Kirkpatrick JB, 2013).

Dermal

A four week dermal toxicity study in Sprague-Dawley rats was conducted at concentrations of 0, 100, 300, and 1,000 mg/kg bw/day test substance according to OECD Guideline 410 and OPPTS 870.3200, in compliance with GLP. Ten males and ten females per dose group were used. Test substance moistened with the vehicle (deionized water) was applied topically under occlusive conditions to the shaved intact dorsal surface of three groups of rats once daily for 6 h for up to 28 consecutive days. A concurrent control group received the vehicle on a comparable regimen. There were no test substance-related effects on survival, dermal observations, haematology, coagulation, ophthalmic examinations and macroscopic observations. Treatment-related lower mean cumulative body weight gains were observed in the 300 and 1,000 mg/kg bw/day group males and females. Also adrenal cortical cytoplasmic vacuolation were observed in the 300 mg/kg bw/day group males and 1,000 mg/kg bw/day group males and females. Hence, the NOAEL was considered to be 100 mg/kg bw/day (Mertens JJWM, 2014).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study followed internationally accepted guidelines and was conducted in compliance with GLP.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The study followed internationally accepted guidelines and was conducted in compliance with GLP.

Justification for classification or non-classification

Oral route

Based on the results of an oral repeated dose toxicity study, the test substance is not classified for this endpoint according to CLP criteria (EC 1272/2008) but is classified as Xn; R48/21/22 under Directive 67/548/EEC.

Dermal route

Based on the results of the dermal repeated dose toxicity study, the test substance does not need to be classified for this endpoint according to CLP criteria (EC 1272/2008) but is classified as Xn; R48/21/22 under Directive 67/548/EEC.