Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

ORAL
NOEL = 100 mg/kg bw/day, NOAEL = 100 mg/kg bw/day; study performed in line with OECD Guideline 407; Morse (2004).
NOAEL = 175 mg/kg bw/day male and female; study performed in line with OECD 421; Knapp (2005).

NOAEL relevant for human risk assessment is considered to be 500 mg/kg/day, the highest dosage level tested for both sexes of rats; study performed in line with OECD 408; Haas (2017).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 January 2004 to 26 March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study 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:
Animals and Animal Husbandry

Animal Receipt, Identification and Housing

A total of 48 male and 48 female Sprague Dawley Crl:CD®(SD)IGS BR rats were received in two shipments from a reputable supplier. Twenty males and twenty females were received on January 15, 2004, for the range-finding study; and twenty-eight males and twenty-eight females were received on February 10, 2004, for the main study. At the time of receipt, each animal was identified with a temporary identification number recorded on the cage card and in the data records. The animals were group housed (two or three males or females per cage) for a minimum of three days following receipt to allow the animals to adjust to the automatic watering system. During the remainder of the acclimation period and while on study, the animals were housed individually in suspended stainless steel cages. A metal ear tag displaying a unique identification number was used to permanently identify each animal assigned to the study. A cage card displaying the study, animal and group numbers, and sex was affixed to each cage. The cage cards were color-coded according to group number following randomization.

Environmental Conditions

The environmental controls in the animal room were set to maintain room temperature and relative humidity ranges of 65 to 79°F and 30 to 70%, respectively. Environmental control equipment was monitored and adjusted as necessary to minimize fluctuations in the animal room environment. Light timers were set to maintain a 12-hour light/12-hour dark cycle and the room ventilation was set to produce 10 to 15 air changes per hour. The room temperature and relative humidity were recorded daily and ranged from 65 to 74°F and 35 to 52%, respectively.

Diet and Drinking Water
PMI Nutrition International Certified Rodent Chow® #5002 and municipal tap water were provided to the animals ad libitum, except when feed was withheld overnight prior to blood collection for clinical pathology determinations. The feed was analysed by the supplier for nutritional components and environmental contaminants. The lot number and expiration date of each batch of feed used during the study were recorded. The tap water was purified by reverse osmosis and supplied to the animals by an automatic watering system. Water supplying the facility is analysed for contaminants according to the Testing Facility's Standard Operating Procedure. The results of the feed and water analyses are maintained at the Testing Facility. Within generally accepted limits, there were no contaminants in the diet or drinking water which would interfere with the conduct of the study.

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Treatment
The test article and vehicle control were administered by oral gavage, once daily, for 28 consecutive days. The control animals received the vehicle at a dosage volume comparable to that received by the test animals. Individual doses were based on the most recent body weight data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Preparation

For each test article group, an appropriate amount of the test material was weighed into a beaker. A specified amount of corn oil was added to the beaker and the mixture was hand stirred. A sufficient amount of corn oil was then added to achieve the desired concentration and a stir bar was added. The mixture was stirred for a minimum of 30 minutes, dispensed into amber glass containers and stored at ambient conditions. Each test article mixture was prepared daily during the range-finding study and every three or four days during the main study. The dosing mixtures were stirred continuously prior to and during dosing. The appearance of each dosing preparation was determined and documented. The vehicle control was a clear yellow liquid and the test article preparations were clear yellow solutions.

Analysis of Dose Preparations

Homogeneity

Concentrations of the test article which encompassed the low- and high-dose concentrations were evaluated for homogeneity on day 0 and once again during the main study by analysing duplicate 10 mL samples taken from the top, middle and bottom of the two mixtures.

Stability

Concentrations of the test article which encompassed the low- and high-dose concentrations in the main study were evaluated. The results of the day 0 homogeneity analysis from the top, middle and bottom for each concentration were averaged and utilised for the day 0 stability analysis. During the main study, test article stability in the vehicle was confirmed by analysing duplicate 10 mL samples on day 1 (room temperature storage), day 3 (room temperature and refrigerated storage), day 8 (room temperature storage) and day 9 (room temperature storage).

Concentration

Duplicate 10 mL samples of each test article mixture and the vehicle control were collected from the first preparations for weeks 0, 1, 2 and 3 of the main study and analysed for test article concentration to verify the dosing concentrations.

The analytical chemistry report is included in attached Appendix C.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0 mg/kg/day (0 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day (10 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day (50 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day (100 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose level including control
Control animals:
yes, concurrent vehicle
Details on study design:
Acclimation
The animals were examined upon receipt and following separation from group housing (day -5 for the range-finding study/day -7 for the main study) for overt physical and behavioral abnormalities (cage-side observations). General health/mortality and moribundity checks were performed twice daily, in the morning and afternoon. Individual body weights were recorded on day -5 for the range-finding study/day -7 for the main study and prior to randomization on day -1 for the range-finding and main studies. The animals were acclimated to the laboratory conditions for a period of nine days for the range-finding study/ten days for the main study prior to in-life initiation.

Randomization and Group Assignment

Prior to randomization (day -1), the animals were weighed and examined in detail for signs of physical disorder. Animals determined to be suitable test subjects were assigned to groups using a computer randomization program. The program ranked the animals according to day -1 body weights and randomly assigned the rats to study groups. Disposition of animals not assigned to study was documented in the study records. On day -1, the animals were approximately seven weeks of age in the range-finding and main studies. Body weights in the range-finding study ranged from 203 to 225 grams for males and 146 to 175 grams for females; and body weights in the main study ranged from 188 to 218 grams for males and 152 to 174 grams for females.

Justification of Dose Level Selection

Dosage levels were selected in an attempt to produce graded responses to the test article. The high-dose level was expected to produce toxic effects, but not excessive lethality. The mid-dose level was expected to produce no or minimal observable effects. The low-dose level was expected to produce no observable effects. Dosage levels were selected by the Sponsor based on data available from previous studies. A 14-day range-finding study was conducted prior to the 28-day main study in order to determine dosage levels for the main study (See attached Appendix A).
Positive control:
None
Observations and examinations performed and frequency:
Parameters Evaluated

Clinical Observations

General health/mortality and moribundity checks were performed twice daily, in the morning and afternoon. Detailed clinical observations were performed on day 0 prior to dosing and on days 6, 13, 20 and 27. Cage-side observations were performed between one-half hour and two hours following dosing. A final detailed clinical observation was performed prior to scheduled euthanasia on day 28.

Functional Observation Battery (FOB)

An abbreviated FOB assessment (home cage, removal from home cage and open field evaluations) was performed prior to dosing (day -1) and once weekly during the study (days 6, 13 and 20). A full FOB assessment (home cage, removal from home cage, open field, manipulative and motor activity evaluations) was performed during the fourth week of exposure (day 27). All FOB and abbreviated FOB assessments were performed blind (animals were not identified by group).

The following parameters were evaluated:

Home Cage Observations

Body posture
Clonic involuntary motor movements
Tonic voluntary motor movements
Vocalization

Removal from Home Cage Observations

Ease of removal
Reactivity to being handled
Ocular discharge
Eyelid closure
Salivation
Piloerection

Open Field Observations

Clonic involuntary motor movements
Tonic voluntary motor movements
Gait score
Gait abnormalities
Mobility score
Arousal
Stereotypy
Bizarre behavior
Urination
Defecation
Rearing

Manipulative Tests

Approach response
Touch response
Startle response
Tail pinch
Pupil response
Righting ability
Forelimb grip strength
Hindlimb grip strength
Landing foot splay
Body temperature

Motor Activity

Each animal was evaluated for changes in motor activity using an open field chamber with infrared beams (San Diego Instruments, San Diego, California). The motor activity measurements were performed in a designated behavioral testing area with red lighting and white noise to reduce background interference with the measurements. For each animal, the test consisted of a one-hour observation period in the open field chamber. Data included the total number of infrared beams broken during the testing interval.

Body Weights

Individual body weights were recorded prior to initiation of treatment on day -1 and on days 6, 13, 20 and 27. A final fasted body weight was recorded prior to scheduled euthanasia on day 28.

Food Consumption

Individual food consumption was recorded on days 0, 6, 13, 20 and 27. Food consumption was calculated and reported as grams/animal/day.

Clinical Pathology

Blood samples were collected from all main study animals on the day of scheduled euthanasia for evaluation of selected hematology, coagulation and clinical chemistry parameters. The blood samples for the hematology and clinical chemistry analyses were obtained via the orbital plexus while the animals were under light isoflurane anesthesia. The blood samples for the coagulation analysis were obtained via vena cava puncture while the animals were under carbon dioxide anesthesia. Feed was withheld overnight (approximately 15 hours) prior to blood collection; however, water was available.

The following parameters were evaluated:
Hematology
Erythrocyte count (RBC)
Hematocrit (Hct)
Hemoglobin concentration (Hb)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Mean corpuscular volume (MCV)
Platelet count
Reticulocyte count
Total and differential leukocyte counts (including RBC morphology) Note: Reticulocyte slides were prepared but examination was not deemed necessary by the Study Director and Veterinary Pathologist.
Coagulation
Activated partial thromboplastin time (APTT)
Prothrombin time (PT)

Clinical Chemistry

Alanine aminotransferase (ALT)
Albumin
Albumin/globulin ratio (calculated)
Alkaline phosphatase
Aspartate aminotransferase (AST)
Blood creatinine
Blood urea nitrogen (BUN)
Calcium
Cholesterol
Electrolytes (sodium, potassium and chloride)
Globulin (calculated)
Glucose
Phosphorus
Total bilirubin
Total serum protein

Sacrifice and pathology:
Gross Necropsy

All animals were subjected to a complete gross necropsy on the day of scheduled euthanasia (day 28). The necropsy examination included evaluation of the external surfaces of the body and all viscera. Surviving animals were euthanized (one animal died following blood collection on the day of scheduled euthanasia) by carbon dioxide inhalation followed by exsanguination. The animals were fasted overnight prior to scheduled euthanasia.

With the exception of the bone marrow smear, the following organs/tissues were preserved in 10% neutral buffered formalin:

Accessory genital organs (epididymides, seminal vesicles and prostate or uterus and vagina)
Adrenals
All gross lesions
Aorta
Bone marrow smear (femur)
Brain (including sections of medulla/pons, cerebellar cortex and cerebral cortex)
Cecum
Colon
Duodenum
Esophagus
Exorbital lachrymal glands
Eyes with optic nerve
Femur (including articular surface) and bone marrow
Heart
Ileum
Jejunum
Kidneys
Liver (three sections collected)
Lungs (infused with formalin) with bronchi
Mammary gland
Mediastinal lymph node
Mesenteric lymph node
Pancreas
Peripheral nerve (sciatic)
Pituitary
Rectum
Skeletal muscle (thigh)
Skin
Spinal cord (cervical, midthoracic and lumbar)
Spleen
Sternum with bone marrow
Stomach (glandular/nonglandular)
Submandibular lymph node
Submaxillary salivary gland
Testes/ovaries
Thymus
Thyroid/parathyroid
Tongue
Trachea
Urinary bladder

Note: Bone marrow smears were prepared at scheduled necropsies (and accidental death following blood collection on the day of scheduled euthanasia). Examination was not deemed necessary by the Veterinary Pathologist.

Histopathology
All tissues and organs collected at necropsy from all animals in the control and high-dose groups were processed for histopathological examination. In addition, the kidney, liver and thyroid were identified as target organs and were processed from the animals in groups 2 and 3 for histopathological examination. The tissues were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin.
Other examinations:
Fresh organ weights were obtained on the day of scheduled euthanasia for the adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, testes with epididymides, and thymus. Paired organs were weighed together.
Statistics:
STATISTICAL ANALYSES

Inferential statistical analyses for the main study were performed on the Compaq Alpha Server DS10. Body weights, body weight changes, food consumption and parametric and count FOB data were analyzed by One-Way Analysis of Variance (ANOVA) [1]. If significance was detected with ANOVA (p<0.05), pair-wise group comparisons proceeded using the Tukey-Kramer test [2]. Ranked FOB data were analyzed by Kruskal-Wallis non-parametric ANOVA [3], followed by Dunn’s test [4], when p<0.05. Descriptive (categorical) and quanta FOB data were analyzed by Fisher’s Exact test [5]. If significance was detected, a group by group comparison proceeded using the Chi- Square test [6]. Clinical pathology data and absolute and relative organ weights were analyzed for homogeneity of variance using Levene’s test [7]. If significance was detected with Levene’s test (p<0.01), multiple group comparisons proceeded using the Kruskal-Wallis non-parametric ANOVA, followed by Dunn’s test, when p<0.05. If significance was not detected with Levene’s test, parametric procedures were used to analyze the data, i.e., ANOVA followed by Tukey-Kramer test when p<0.05. Summary data tables present group mean and standard deviation (S.D.) values, where appropriate. Statistical significance was reported at three levels of alpha: p<0.05, p<0.01 and p<0.001. No inferential statistical analyses were performed for the range-finding study.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males and females: Salivation at 500 and 1000 mg/kg/day; Males: Struggling during dosing at 500 (1 animal ) and 1000 (2 animals) mg/kg/day; Females: Struggling during dosing at 1000 (4 animals) mg/kg/day
Mortality:
mortality observed, treatment-related
Description (incidence):
Males and females: Salivation at 500 and 1000 mg/kg/day; Males: Struggling during dosing at 500 (1 animal ) and 1000 (2 animals) mg/kg/day; Females: Struggling during dosing at 1000 (4 animals) mg/kg/day
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weights: increased at 500 amd 1000 mg/kg/day in males and females; Kidney weights: increased at 500 amd 1000 mg/kg/day in males and at 1000 mg/kg females
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
Minimal to mild eosinophilia of hepatocytes was observed in all male groups and in group 3 and 4 females. Minimal hypertrophy of the thyroid follicular epithelium was observed in a single group 3 male, in all group 4 male
Details on results:
Survival and Clinical Observations

see attached Table 1 (Summary Data)
see attached Appendix D (Individual Data)

All animals survived to the day of scheduled euthanasia. One group 3 (500 mg/kg/day) female died prior to necropsy as a result of being bled for evaluation of clinical pathology parameters. This was categorised as an accidental death; tissues were collected from this animal and organ weights were recorded.

Clinical observations that were likely related to test article administration in male rats included salivation prior to dosing (observed at least once in each of two 1000 mg/kg/day males), struggling during dosing (observed once in one 500 mg/kg/day male and at least once in each of three 1000 mg/kg/day males), and salivation at one-half hour to two hours post-dose (observed once in each of three 500 mg/kg/day males and at least once in all five 1000 mg/kg/day males). In females, these same clinical observations were generally observed in high-dose females (1000 mg/kg/day) with a greater frequency than in high-dose males. Salivation prior to dosing was observed a total of 19 times in a total of three 1000 mg/kg/day females, struggling during dosing was observed 12 times in a total of four 1000 mg/kg/day females, and salivation at one-half hour to two hours post-dose was observed twice in one 500 mg/kg/day female and a total of 16 times in four 1000 mg/kg/day females.

Functional Observation Battery

see attached Table 2 (Summary Data)
see attached Appendix E (Functional Observation Battery Codes)
see attached Appendix F (Functional Observation Battery Historical Control Data)
see attached Appendix G (Individual Data)

Home cage observation parameters that were evaluated included body posture, clonic involuntary motor movements, tonic involuntary motor movements, and vocalization. The body posture categories observed in vehicle-treated or test article-treated males during the course of the study included category 1 (sitting or standing), category 2 (rearing), and category 3 (asleep, lying on side, or curled up). Category 1 was observed most frequently in groups 1 through 4, with a low incidence of categories 2 and 3. A single statistically significant difference was observed in group 3 (500 mg/kg/day) males on day 20, in which the animals had a significantly higher incidence of category 3 body posture calls. However, no dose-response was evident, as group 4 males did not exhibit this finding on day 20, and group 3 males rarely exhibited this finding on other test days. Therefore, this difference appeared to be a sporadic difference of no toxicological significance. No differences in body posture were observed in females. Clonic involuntary motor movements, tonic involuntary motor movements, and vocalization were not noted in home cage observations for male or female rats during this study.

Removal from home cage parameters that were evaluated included ease of removal, reactivity to being handled, ocular discharge, eyelid closure, salivation, and piloerection. There were some modest increases in the ease of removal and the reactivity to being handled scores found for test article-treated males and females. These differences were not statistically significant and were within the range of historical control data. No differences in ocular discharge, eyelid closure, salivation, or piloerection were observed among male or female groups.

Open field observation parameters that were evaluated included clonic involuntary motor movements, tonic involuntary motor movements, gait score, gait abnormalities, mobility score, arousal, stereotypy, bizarre behaviour, urination, defecation, and rearing. There were no differences among male or female groups in clonic involuntary motor movements, tonic involuntary motor movements, gait scores, or mobility scores. Modest, albeit statistically and toxicologically insignificant differences, were observed in arousal scores in males and females during the course of the study. No differences in stereotypy and bizarre behaviour were noted in males or females. No statistically or toxicologically significant differences in urination, defecation, or rearing were noted in males or females. Manipulative tests that were evaluated on day 27 only included approach response, touch response, startle response, tail pinch, pupil response, righting ability, forelimb grip strength, hindlimb grip strength, landing foot splay, and body temperature. No toxicologically significant differences in these parameters were noted among groups of male or female rats.

Body Weights

see attached Tables 3 and 4 (Summary Data)
see attached Appendices H and I (Individual Data)

There were no statistically significant differences in body weights or body weight changes among groups of male or female rats during the course of the study. At day 27, test article-treated male and female groups actually weighed more than controls (group 1, 0 mg/kg/day), although these differences were neither statistically nor toxicologically significant.

Food Consumption

see attached Table 5 (Summary Data)
see attached Appendix J (Individual Data)

No statistically or toxicologically significant differences in food consumption were observed in this study for male or female rats.

Clinical Pathology

see attached Appendix K (Clinical Pathology Methods)
see attached Appendix L (Clinical Pathology Historical Control Data)

Hematology and Coagulation

see attached Table 6 (Summary Data)
see attached Appendix M (Individual Data)

The platelet count in group 2 (100 mg/kg/day) males was statistically less than that of control males. However, no such differences were observed in group 3 (500 mg/kg/day) or group 4 (1000 mg/kg/day) males, no dose response was evident, and the aberrant value was well within the range of historical control data, indicating that this difference is of no toxicological significance. Segmented neutrophils were statistically elevated in group 4 (1000 mg/kg/day) males and females indicating a potential test article-related effect, although the altered values were well within the range of historical control data. In group 4 (1000 mg/kg/day) males, monocytes were statistically decreased and eosinophils were statistically increased compared to group 1 (0 mg/kg/day) males; these changes were of no toxicological significance. No changes in coagulation parameters (prothrombin time and activated partial thromboplastin time) were observed in test article-treated males; test article-treated females had statistically lower prothrombin times compared to group 1 (0 mg/kg/day) females. These altered prothrombin times in test article-treated females were well within the range of historical control data and are therefore of no toxicological significance. No differences in active partial thromboplastin time were observed in test article-treated females. There were no differences in red cell morphology in test article-treated males or females compared to controls on day 28.

Clinical Chemistry

see attached Table 7 (Summary Data)
see attached Appendix N (Individual Data)

Group 4 (1000 mg/kg/day) males had statistically lower levels of aspartate aminotransferase (AST) and statistically higher levels of total bilirubin, globulin, and total protein compared to group 1 (0 mg/kg/day) males. Males in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) had statistically elevated levels of potassium, calcium, and phosphorus compared to group 1 (0 mg/kg/day) males. No statistically significant differences were observed in group 2 (100 mg/kg/day) males.

Group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) females had statistically elevated levels of total bilirubin, total protein, and albumin and statistically decreased levels of chloride compared to group 1 (0 mg/kg/day) females. All test article-treated female groups had statistically higher levels of calcium relative to controls. Group 4 (1000 mg/kg/day) females had statistically elevated levels of phosphorus relative to controls.

With the exception of the differences in total bilirubin in males and females and the male group 4 (1000 mg/kg/day) calcium, the magnitude of the differences observed in test article-treated males and females was 65% or less and the aberrant values were well within the range of historical control values, indicating that these differences were of no toxicological significance, although they may be test article related.

Gross Necropsy Observations

see attached Table 8 (Summary Data)
see attached Appendix O (Individual Data)

The only gross necropsy finding for the group 3 (500 mg/kg/day) female that died as a result of blood collection for evaluation of clinical pathology parameters was hair loss. In males, there were no gross necropsy observations that could be conclusively ascribed to treatment with test article. Malalignment of the incisor(s) was observed in one group 3 (500 mg/kg/day) male, ocular lesions due to blood collection were observed in one group 3 (500 mg/kg/day) male and in one group 4 (1000 mg/kg/day) male, hairloss was observed in one male in each of groups 1 through 3, dark material on the haircoat was observed in one group 3 (500 mg/kg/day) male, wet matting of the haircoat was observed in one group 4 (1000 mg/kg/day) male, abnormal content of the ileum and jejunum was observed in one group 4 (1000 mg/kg/day) male, dilated renal pelvis was observed in one group 4
(1000 mg/kg/day) male, renal cysts were observed in one group 2 (100 mg/kg/day) male, abnormal stomach content was observed in one group 4 (1000 mg/kg/day) male, and thickened stomach was observed in one group 2 (100 mg/kg/day) male. As in males, there were no gross necropsy observations that could be categorically determined to be due to treatment with test article in females that survived to scheduled euthanasia. Discolouration of the tail was observed in one group 3 (500 mg/kg/day) female, ocular lesion due to blood collection was observed in one group 1 (0 mg/kg/day) female, hairloss was observed in one group 1 (0 mg/kg/day) female and in one group 2 (100 mg/kg/day) female, dark material on the haircoat was observed in one group 1 (0 mg/kg/day) female and in one group 4 (1000 mg/kg/day) female, wet matting of the haircoat was observed in two group 4 (1000 mg/kg/day) females, pitted kidney was observed in one group 1 (0 mg/kg/day) female, scabbing of the skin was observed in one group 3 (500 mg/kg/day) female, spleen adhesion was observed in one group 4 (1000 mg/kg/day) female, roughened surface of the spleen was observed in one group 3 (500 mg/kg/day) female, and reddened thymus was noted in one group 1 (0 mg/kg/day) female and in one group 4 (1000 mg/kg/day) female.

Organ Weights

see attached Tables 9 and 10 (Summary Data)
see attached Appendix P (Individual Data)

Mean liver weight was statistically greater in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) males and females and the increase in liver weight exhibited a dose-response relationship in both sexes. The magnitude of the increased hepatic weight was 52% in group 4 (1000 mg/kg/day) males and 77% in group 4 (1000 mg/kg/day) females compared to controls, differences of clear toxicological significance. Kidney weights were also statistically greater in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) males and in group 4 (1000 mg/kg/day) females. As with the increase in liver weight, a dose-response relationship was evident for the increased kidney weights in both sexes. The magnitude of the increase was modest; group 4 (1000 mg/kg/day) males had a mean kidney weight 25% greater than controls and group 4 (1000 mg/kg/day) females had a mean kidney weight that was just 20% greater than controls. No other differences in organ weights were observed in test article-treated males or females. The liver-to-body weight and kidney-to-body weight ratios were correspondingly statistically greater in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) males, and the liver-to-body weight ratio was statistically greater in group 3 (500 mg/kg/day) females and in group 4 (1000 mg/kg/day) females. No other differences in organ-to-body weight ratios were observed in test article-treated males or females.

Histopathology

see attached Appendix Q (Histopathology Report)

Minimal to mild eosinophilia was observed in all test article-treated male groups and in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) females. This finding was characterized by the hepatocytes having a more homogeneous, amorphous, eosinophilic cytoplasm and at times appearing to be enlarged relative to those of control (group 1) animals. Minimal hypertrophy of the thyroid follicular epithelium characterized by a diffuse increase in follicular epithelial cell size was observed in one group 3 (500 mg/kg/day) male, in all group 4 (1000 mg/kg/day) males, and in one group 4 (1000 mg/kg/day) female. An increase in the incidence and/or severity of renal hyaline droplets characterized by increases in tubular epithelial cells with cytoplasmic protein droplets and increases in cytoplasmic protein droplets per tubular epithelial cell was observed in all test article-treated male groups. This finding was not observed in female rats.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Target organs identified were the liver and thyroid in females
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Target organs identified were the liver, thyroid and kidneys in males
Critical effects observed:
not specified
Conclusions:
The target organs identified for toxic effects of the test substance in this study were the liver and thyroid in males and females and the kidney in males. Because of the microscopic histopathologic findings in all test article-treated male groups, a no-observed-effect level (NOEL) could not be identified for males in this study. The 100 mg/kg/day dose was considered to be a no-observed-adverse effect level (NOAEL). The NOEL for females in this study was 100 mg/kg/day.
Executive summary:

The purpose of this study was to evaluate the potential toxicity of the test material when administered orally, by gavage, to rats for 28 consecutive days. For this purpose, Sprague Dawley rats were randomly assigned to four study groups as outlined in the table below:

Group

No. Animals

Dosage

Material

Dosage

Level

(mg/kg/day)

Dosage

Conc.

(mg/mL)

Dosage

Volume

(mL/kg)

Males

Females

1

5

5

Corn Oil

0

0

10

2

5

5

 Test material

100

10

10

3

5

5

Test Material

500

50

10

4

5

5

Test material

1000

100

10

The test article and vehicle control were administered by oral gavage, once daily, for 28 consecutive days. Detailed clinical observations were performed on day 0 prior to dosing and on days 6, 13, 20 and 27. Cage-side observations were performed between one-half hour and two hours following dosing. A final detailed clinical observation was performed prior to scheduled euthanasia on day 28. A functional observation battery assessment was performed prior to dosing and once weekly during the study to evaluate the animals for gross neurological changes. Individual body weights were recorded weekly (non-fasted). A final body weight (fasted) was recorded on the day of scheduled euthanasia. Food consumption was recorded on days 0, 6, 13, 20 and 27. Blood samples were obtained from all animals on the day of scheduled euthanasia (day 28) for evaluation of selected clinical pathology parameters. All rats were subjected to a complete gross necropsy examination on the day of scheduled euthanasia. Fresh organ weights were obtained for all animals and selected tissues were preserved from all rats. All tissues collected at necropsy. From the control and high-dose animals and the kidney, liver and thyroid (target organs) from the low- and mid-dose animals were examined microscopically.

Results

All animals survived to scheduled euthanasia with the exception of one group 3 (500 mg/kg/day) female that died on the day of necropsy as a result of being bled for evaluation of clinical pathology parameters. Clinical observations that were likely related to test article administration included salivation prior to dosing, struggling during dosing, and salivation at one-half hour to two hours post-dose and were observed primarily in group 4 (1000 mg/kg/day) animals.

No toxicologically relevant differences among male or female groups were observed in home cage observation parameters, removal from home cage parameters, open field observation parameters, and manipulative tests that were conducted as part of the functional observation battery tests for this study. No statistically or toxicologically significant differences in body weights, body weight changes, or food consumption were observed among groups of male or female rats during the course of the study. No toxicologically relevant differences in hematologic, coagulation, red blood cell morphology, or clinical chemistry parameters were observed in test article-treated males or females in this study. At necropsy, there were no findings that could be categorically ascribed to test article treatment in male or female rats. Mean liver weights were elevated in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) males and females and mean kidney weights were elevated in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) males and in group 4 (1000 mg/kg/day) females. There were microscopic histopathologic findings in the liver, thyroid, and kidneys of test article-treated animals. Minimal to mild eosinophilia of hepatocytes was observed in all test article-treated male groups and in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) females. Minimal hypertrophy of the thyroid follicular epithelium was observed in a single group 3 (500 mg/kg/day) male, in all group 4 (1000 mg/kg/day) males, and in one group 4 (1000 mg/kg/day) female. An increase in the incidence and/or severity of renal tubular epithelial cytoplasmic hyaline droplets was observed in all test article-treated male groups but not in females.

Conclusion

The target organs identified for toxic effects of the test material in this study were the liver and thyroid in males and females and the kidney in males. Because of the microscopic histopathologic findings in all test article-treated male groups, a no-observed-effect level (NOEL) could not be identified for males in this study. The 100 mg/kg/day dose was considered to be a no-observed-adverse effect level (NOAEL). The NOEL for females in this study was 100 mg/kg/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
3 October 2005 to 7 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
This study was designed to investigate the potential adverse effects of the test article on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition and lactation of the F0 generation and the development of offspring from conception through day 4 of postnatal life. Although the test is designed to examine the potential reproductive and developmental toxicity of the test substance, the observations performed in the parental generation after repeated exposure of the test material are appropriate for the purposes of determining the subchronic toxicity of the test material using a suitable mammalian model.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMAL RECEIPT AND ACCLIMATION

Fifty-five male and 55 female Crl:CD(SD) rats were received in good health from a reputable supplier on 22 November 2005. The animals were 59 days old upon receipt. Each animal was examined by a qualified technician on the day of receipt and weighed the following day. Each rat was uniquely identified by a Monel® metal eartag displaying the animal number. The animals were housed for an acclimation period of 13 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior.

ANIMAL HOUSING

Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The nesting material is periodically analyzed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The dams were housed in these cages until euthanasia on lactation day 4. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

DIET, DRINKING WATER AND MAINTENANCE

The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer. Feed lots used during the study are documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. The results of the diet and water analyses are maintained at WIL Research Laboratories, LLC. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet was provided ad libitum throughout the acclimation period and during the study.

ENVIRONMENTAL CONDITIONS

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain daily averages of 71 °F ± 5 °F (22 °C ± 3 °C) and 50 % ± 20 % relative humidity. Room temperature and relative humidity were controlled and monitored using the Metasys® DDC Electronic Environmental control system. These data were recorded approximately hourly and are summarized in attached Appendix D. Actual mean daily temperature ranged from 70.3 °F to 71.2 °F (21.3 °C to 21.8 °C) and mean daily relative humidity ranged from 30.2% to 43.2% during the study. Light timers were calibrated to provide a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS

For the control group (Group 1), a sufficient amount of corn oil was added to a glass container. The vehicle was stirred continuously throughout sampling and dispensation. Aliquots of the vehicle were prepared for daily dispensation and stored at room temperature. The vehicle was stirred continuously throughout dose administration.

The appropriate amount of the test article for each group was weighed into a tarred, calibrated container. Vehicle was added to each container to bring the formulations nearly to the calibration mark. The formulations were mixed until uniform using a magnetic stirrer. Vehicle was added to each container to bring the formulations to the calibration mark, and the formulations were stirred until uniform using a magnetic stirrer. The test article formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored at room temperature. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

The test article formulations were visually inspected, and were found to be visibly homogeneous and acceptable for dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSES
Prior to the initiation of dose administration, duplicate samples (1 mL each) for homogeneity determination were collected on 21 November 2005 from the top, middle and bottom strata of each dosing formulation, including the vehicle for the control group. In addition, duplicate samples (1 mL each) for stability and resuspension homogeneity determinations were collected on 1 December 2005 from the top and bottom strata of aliquots of these same dosing suspensions following storage at room temperature for 10 days; the aliquots were mixed using a magnetic stirrer for a minimum of 10 minutes prior to sample collection. Samples (1 mL each) for concentration analysis were collected weekly from the middle stratum of each dosing formulation (including the vehicle for the control group); only the samples from the first, fourth and last weekly formulations were analyzed. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC. The methodology and results of these analyses are presented in attached Appendix B.
Duration of treatment / exposure:
The vehicle and test article formulations were administered orally by gastric intubation, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily. The males were dosed during study days 0-55 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 56 doses. The females were dosed from study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-53 doses. The female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The dosage volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0 mg/kg/day (0 mg/mL)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg/day (10 mg/mL)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
175 mg/kg/day (35 mg/mL)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg/day (120 mg/mL)
Basis:
actual ingested
No. of animals per sex per dose:
12 animals per sex per dose level including control
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 representative after consultation with the study director.


ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS

Near the end 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 (300 g to 500 g for males and 200 g to 300 g for females) was selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into the WIL Toxicology Data Management System (WTDMS™). A printout containing the animal numbers, corresponding body weights and individual group assignments was generated using a computer program which randomised the animals based on stratification of body weights in a block design. The animals then were arranged into groups according to the printout. Individual body weights at randomization were within ± 20% of the mean for each sex. The experimental design for WIL-186044 consisted of 3 test article-treated groups and 1 control group composed of 12 rats/sex/group. At the initiation of dose administration (study day 0), the males and females were approximately 10 weeks old. Body weights ranged from 334 g to 399 g for males and from 215 g to 271 g for females on study day 0. The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 230 g to 301 g on gestation day 0.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded weekly (prior to test article administration during the treatment period). Each male and female was also observed for signs of toxicity at the time of dose administration and approximately 1-2 hours following dose administration. All significant findings were recorded.

BODY WEIGHTS
Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire generation (males only). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4. Mean gestation body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation interval (days 0-20). For individual data, the time periods a given animal was not weighed were designated as “NA” (Not Applicable).

FOOD CONSUMPTION
Individual food consumption was recorded weekly until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4. Following mating, food consumption for all males was measured on a weekly basis until the scheduled euthanasia. Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.
Sacrifice and pathology:
MACROSCOPIC EXAMINATIONS

UNSCHEDULED DEATHS
A complete necropsy was performed on the male that was found dead. This included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain and the thoracic, abdominal and pelvic cavities, including viscera.

SCHEDULED EUTHANASIA
All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the female necropsies. Females that delivered were euthanized on lactation day 4. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating). Necropsy included examination of the external surface, all orifices and the cranial cavity, external surfaces of the brain, and the 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):

Cervix,
Coagulating glands,
Mammary gland,
Ovaries and oviducts (2),
Pituitary gland,
Prostate gland,
Seminal vesicles (2),
Testes with epididymides (a) and vas deferens (2),
Thyroids [with parathyroids if present (2)],
Uterus (b) with vagina,
All gross lesions (c).

(a) = Testes and epididymides fixed in Bouin’s solution.
(b) = Not retained if placed in ammonium sulfide solution.
(c) = Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Brain
Epididymides (a)
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Testes (a)
Thyroids with parathyroids (b)

(a) = These paired organs were weighed separately.
(b) = Fixed in 10% neutral-buffered formalin prior to weighing.

Except as noted, paired organs were weighed together. Absolute weights, organ to final body weight and organ to brain weight ratios were reported.

MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed according to standard operating procedures 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 following exceptions. PAS and hematoxylin staining were used for the right and left testes and epididymides. The testes were fixed in Bouin’s solution and embedded in paraffin. Sections of 2 to 4 microns were made for the testis (transverse) and the epididymis (longitudinal). Special emphasis was placed on the stages of spermatogenesis and histopathology of interstitial testicular cell structure. Microscopic examination was performed on all tissues from the male that was found dead and all animals in the control and 600 mg/kg/day groups at the scheduled necropsies; gross lesions from all dosage groups were also examined. Because of test article-related findings in the 600 mg/kg/day group males, the thyroid glands were also examined from all males in the 50 and 175 mg/kg/day groups. 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.
Statistics:
STATISTICAL ANALYSES
All statistical tests were performed using appropriate computing devices or programs. For full details please see 'Any other information on materials and methods incl. tables'.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MALES

CLINICAL OBSERVATIONS AND SURVIVAL
Summary Data: see attached Tables 1, 2, 3
Individual Data: see attached Tables 49, 50, 51

Test article-related clinical findings were noted in the 175 and 600 mg/kg/day group males at the time of and 1-2 hours following dose administration. Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. During the week prior to death, this male had an unkempt appearance, rales and red material around the eyes, nose and mouth, lower body weight gain (relative to its gain during the first week of dose administration) during study days 7-21 and a body weight loss during study days 21-27. Fractured nasal bones were noted at necropsy for this animal. The death of male no. 11517 was attributed to complications from the fractures (presumed to be the result of a mechanical injury) and not to the test article. All other males survived to the scheduled necropsy. No test article-related clinical findings were observed in the males at the weekly detailed physical examinations. Test article-related increased incidences of salivation or evidence thereof (clear material around the mouth) were noted in the 175 and 600 mg/kg/day males at the time of and/or 1-2 hours following dose administration; the severity of these findings was primarily slight or moderate. Salivation was noted in a total of 9 and 12 males in the 175 and 600 mg/kg/day groups, respectively; the onset of this finding occurred as early as study day 15 in the 175 mg/kg/day group and study day 2 in the 600 mg/kg/day group. Clear material around the mouth was first observed on study day 15 and study day 3 in a total of 10 and 12 males in the same respective groups. These findings were generally noted throughout the study. However, because the onset of salivation and clear material around the mouth occurred immediately following daily dose administration, these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate) was also noted for 6 males in the 600 mg/kg/day group 1-2 hours following dose administration beginning on study day 15 and continued sporadically throughout the study. This finding was considered test article-related. Other clinical findings, including hair loss, red material and/or yellow material on various body surfaces, were observed infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

BODY WEIGHTS
Summary Data: see attached Tables 4, 5
Individual Data: see attached Tables 52, 53

Transient, test article-related lower mean body weight gains were noted in the 600 mg/kg/day group males relative to the control group. The reductions were not of sufficient magnitude to be considered adverse. Test article-related lower mean body weight gain was noted in the 600 mg/kg/day group males during the pre-mating period (study days 0-13) due to lower mean body weight gain observed study days 7-13; the difference from the control group was statistically significant (p<0.05) during study days 7-13. Mean body weight gain in this group was similar to that in the control group during the mating and post-mating periods, with the following exception. Lower (not statistically significant) mean body weight gain in this group was noted during study days 21-27 compared to the control group value. The reductions observed in the 600 mg/kg/day group resulted in a slightly lower mean body weight gain when the entire generation (study days 0-56) was evaluated; the difference from the control group was not statistically significant. However, because the reductions were transient and not of sufficient magnitude to result in substantially lower mean body weights, the lower mean body weight gains in this group were not considered adverse. Mean body weights and body weight gains in the 50 and 175 mg/kg/day group males were unaffected by test article administration. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 6, 7
Individual Data: see attached Tables 54, 55

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group males was unaffected by test article administration. The values in the test article-treated groups were generally similar to the control group values throughout the treatment period. None of the differences from the control group were statistically significant.

ANATOMIC PATHOLOGY
For organ weight results please see 'Any other information on results incl. tables'.

MACROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 11, 12
Individual Data: see attached Tables 59, 60

Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. Macroscopic findings for this male consisted of fractured nasal bones and dark red contents in the stomach, duodenum, jejunum, ileum and cecum. This death was attributed to complications from the fractures (presumed to be a mechanical injury) and not to the test article. At the scheduled F0 male necropsies, no test article-related internal findings were
observed at any dosage level. Macroscopic findings observed in the test article-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

MICROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 13, 14
Individual Data: see attached Tables 59, 60
Pathology Report: see attached Appendix E

Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. No test article-related microscopic findings were noted in the organs that were examined for this male. Microscopic findings in the bone, related to the macroscopic finding (fracture), consisted of fracture, hemorrhage, inflammation and fibrosis. A cause of death could not be determined. Test article-related microscopic findings were observed in the thyroid glands of the 600 mg/kg/day group males. Follicular cell hypertrophy was noted in 9 of 11 males in the 600 mg/kg/day group compared to no males in the control group; the difference was statistically significant (p<0.05). This finding was characterized by an increase in cell size, cytoplasm and basophilia. Occasionally, follicular cell hyperplasia was observed as well, and colloid depletion was identified in a single male in this group. These findings correlated with higher mean thyroid gland weights in the 600 mg/kg/day group males. Thyroid gland alterations were below the threshold of detection by routine light microscopy in the 50 and 175 mg/kg/day group males. The only other statistically significant (p<0.05) difference from the control group was an increase in the incidence of ultimobranchial cysts in the thyroid gland in the 600 mg/kg/day group males (10/11 males affected versus 2/12 males in the control group). However, ultimobranchial cysts are common congenital anomalies of the thyroid gland arising during early development of the organ (Hardisty and Boorman, 1990). Because the rats in this study were adults at the time of test article exposure, this finding was considered to be spurious rather than test article-related. In addition, there were no ultimobranchial cysts noted in the thyroid glands from the 50 and 175 mg/kg/day group males. No other test article-related findings were noted in the males in the 50, 175 and 600 mg/kg/day group. Remaining histologic changes were considered to be incidental findings, manifestations of spontaneous disease or related to some aspect of experimental manipulation other than exposure to the test article. There were no test article-related alterations in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.


FEMALES

CLINICAL OBSERVATIONS AND SURVIVAL
Summary Data: see attached Tables 16, 17, 18
Individual Data: see attached Tables 61, 62, 63

Test article-related clinical findings were noted in the 175 and 600 mg/kg/day group females. However, the findings were not considered adverse.
No test article-related clinical findings were observed in the females at the weekly detailed physical examinations. Test article-related increased incidences of salivation or evidence thereof (clear material around the mouth) were noted in the 175 and 600 mg/kg/day group females at the time of and/or 1-2 hours following dose administration. Salivation was noted in a total of 4 and 11 females, and clear material around the mouth was observed in a total of 8 and 12 females in the 175 and 600 mg/kg/day groups, respectively. The severity of these findings was primarily slight or moderate. A slight increase in the incidence of red material around the mouth (slight) was observed in a total of 8 females in the 600 mg/kg/day group 1 hour following dose administration. This finding was noted as early as study day 4, and was considered test article-related based on the occurrence in the 600 mg/kg/day group males. Salivation was first noted on study day 16 and study day 7 in the 175 and 600 mg/kg/day groups, respectively, and clear material around the mouth was noted beginning on study day 12 and study day 1 in the same respective groups. However, because the onset of these findings was immediately following dose administration, salivation and clear material around the mouth were not considered indicative of systemic toxicity. Other clinical findings, including hair loss on various body surfaces and/or red material around the nose, were observed infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

PRE-MATING PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 19, 20
Individual Data: see attached Tables 64, 65

There were no test-article-related effects on mean female body weights or body weight gains at any dosage level during the pre-mating period. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 21, 22
Individual Data: see attached Tables 66, 67

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group females was unaffected by test article administration during the pre-mating period. None of the differences from the control group were statistically significant.

GESTATION PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 23, 24
Individual Data: see attached Tables 68, 69

No test article-related effects on mean maternal body weights or body weight gains were noted during gestation at any dosage level. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 25, 26
Individual Data: see attached Tables 70, 71

Higher mean maternal food consumption, (primarily g/animal/day) was noted in the 600 mg/kg/day group females during gestation days 0-4, 4-7 and 11-14, resulting in higher mean food consumption when the entire gestation period (gestation days 0-20) was evaluated. The differences from the control group were statistically significant (p<0.05 or p<0.01). The increases in food consumption in this group were considered test article-related, but not adverse, because the increases were slight (2-3 g/animal/day) compared to the control group, and increases in food consumption of this magnitude are not considered adverse. No test article-related effects on food consumption were noted in the 50 and 175 mg/kg/day groups during gestation. No statistically significant differences from the control group were observed.

LACTATION PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 27, 28
Individual Data: see attached Tables 72, 73

Mean body weights and body weight gains in the 50, 175 and 600 mg/kg/day groups were similar to the control group values during lactation days 1-4. No statistically significant differences were noted.

FOOD CONSUMPTION
Summary Data: see attached Tables 29, 30
Individual Data: see attached Tables 74, 75

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group females was unaffected by test article administration during the early lactation period. The values in the test article-treated groups were similar to the control group values throughout the lactation period. No statistically significant differences were observed.

ANATOMIC PATHOLOGY
For organ weight results please see 'Any other information on results incl. tables'.

MACROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 37, 38, 39
Individual Data: see attached Tables 82, 83

At the scheduled F0 female necropsies, no test article-related internal findings were observed at any dosage level. Macroscopic findings observed in the test article-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

MICROSCOPIC EXAMINATIONS
Summary Data: see attached Table 40
Individual Data: see attached Table 83
Pathology Report: see attached Appendix E

No test article-related findings were noted in the 600 mg/kg/day group females. Histologic changes were considered to be incidental findings, manifestations of spontaneous disease or related to some aspect of experimental manipulation other than exposure to the test article. Ultimobranchial cysts were noted in the thyroid gland of 6/12 and 4/12 females in the control and 600 mg/kg/day groups, respectively, which was the reverse of the trend observed in the males; there were no ultimobranchial cysts noted in the thyroid glands from the 50 and 175 mg/kg/day group males.There were no test article-related alterations in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Increased organ weights and microscopic changes in 600 mg/kg/day males.
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Increased liver weights in 600 mg/kg/day females.
Critical effects observed:
not specified

ANATOMIC PATHOLOGY

ORGAN WEIGHTS

Males

Summary Data: see attached Tables 8, 9, 10

Individual Data: see attached Tables 56, 57, 58

Test article-related effects on organ weights were noted in the 50, 175 and 600 mg/kg/day group males and are summarised in the following table:

Table 1: Test Article-Related Effects on Organ Weights in Male Rats

 

50 mg/kg/day

175 mg/kg/day

600 mg/kg/day

Thyroid gland

Increased (27.7%)(1)

Increased (30.8%)(1)

Increased (71.3%)(1)

Liver

NC

Increased (17.5%)(1)

Increased (27.8%)(1)

Kidney

NC

NC

Increased (15.3%)(1)

(1) = Change occurred for absolute, relative to final body weight and relative to brain weight; % difference from control group is shown for absolute weight.

NC = No change

Test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights occurred in a dose-related manner in the 50, 175 and 600 mg/kg/day group males when compared to the control group; the differences were statistically significant (p<0.05 or p<0.01). In addition, test article-related higher mean absolute and relative (to final body weight and to brain weight) liver weights occurred in a dose-related manner in the 175 and 600 mg/kg/day group males, and test article-related higher mean absolute and relative (to final body weight and to brain weight) kidney weights occurred in the 600 mg/kg/day group males. The differences from the control group values were statistically significant (p<0.05 or p<0.01). These increases were considered test article-related. Based on the lack of correlating microscopic findings and the lack of toxicological effects on male reproduction, which suggested that the alterations in the glandular weights were not adverse, the increase in mean thyroid gland weights in the 175 mg/kg/day group males was not considered adverse.

No other test article-related differences in organ weights were noted. Statistically significantly (p<0.05) higher mean absolute and/or relative (to final body weight) right epididymis weights were observed in the 50 and 600 mg/kg/day group males. These increases were not attributed to the test article because there was no dose-response relationship, and a similar increase was not noted in the contralateral organ in these groups.

ORGAN WEIGHTS

Females

Summary Data: see attached Tables 31, 32, 33, 34, 35, 36

Individual Data: see attached Tables 76, 77, 78, 79, 80, 81

Test article-related effects on organ weights were noted in the 175 and 600 mg/kg/day group females. The findings are summarised in the following table:

Table 2: Test Article-Related Effects on Organ Weights in Female Rats

 

50 mg/kg/day

175 mg/kg/day

600 mg/kg/day

Liver

NC

NC

Increased (34.4%)(1)

Kidney

NC

Increased (15.0%)(1)

Increased (21.4%)(1)

(1) = Change occurred for absolute, relative to final body weight and relative to brain weight; % difference from control group is shown for absolute weight.

NC = No change

Test article-related higher mean absolute and relative (to final body weight and to brain weight) liver weights occurred in the 600 mg/kg/day group females; when compared to the control group, the differences were statistically significant (p<0.01). Test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights occurred in the 175 and 600 mg/kg/day group females when compared to the control group values. Although, the higher mean thyroid gland weights in the 175 and 600 mg/kg/day group females were considered potentially test article-related, the differences from the control group were not statistically significant and there were no correlating microscopic findings in the 600 mg/kg/day group. Therefore, the higher mean thyroid gland weights in the 175 and 600 mg/kg/day group females were not considered adverse. No other test article-related effects on organ weights were noted. Differences from the control group were slight and not statistically significant.

Conclusions:
CONCLUSIONS
Under the conditions of this study, the Study Director concluded that the NOAEL for male systemic toxicity was 50 mg/kg/day based on increased organ weights in the 175 and 600 mg/kg/day groups and microscopic findings in the 600 mg/kg/day group. However, after re-examining the study results, the NOAEL of 50 mg/kg/day for F0 male systemic effects seemed too conservative and 175 mg/kg/day was proposed to replace that value for the following reasons (See Appendix F Re-evaluation of Results):
1) Thyroid weight increases observed at 175 mg/kg/day was not considered to be “adverse” as microscopic examinations of histological structure of the thyroid glands did not reveal significant changes, and there were no toxicological effects on male reproduction.
2) Increased liver weights recorded at necropsy in male rats treated with 175 mg/kg/day was also not considered to be “adverse”. Due to lack of effects from clinical pathology analyses, macroscopic and microscopic examination, as well as liver function, the liver enlargement was considered to be consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction.
The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group.
Executive summary:

Objective

This study was designed to investigate the potential adverse effects of the test article on male and female reproduction within the scope of a screening study. Inclusive of this study F0 males and females were dosed repeatedly with the test article and observed for mortality and moribundity, clinical observations, body weights and food consumption. Post mortem observations included macro and microscopic examination of selected tissues.

Study design

The test article, thiophene, [3-(decyloxy) tetrahydro-1, 1-dioxide], in the vehicle, corn oil was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 12 males and 12 females. Dosage levels were 50, 175 and 600 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle on a comparable regimen. Males and females received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia (for a total of 56 doses). Females were dosed through lactation day 3 for a total of 39-53 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. All F0 females were allowed to deliver and rear their pups until lactation day 4. F0 females were euthanized on lactation day 4. F0 males were euthanized following completion of the F0 female necropsies. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dosage groups; thyroid glands were also examined from the F0 males in the 50 and 175 mg/kg/day groups.

Results

One male in the 600 mg/kg/day group was found dead on study day 32 due to complications from fractured nasal bones; the death was not attributed to the test article. All other animals survived to the scheduled necropsy. Salivation and clear material around the mouth were noted in the 175 and 600 mg/kg/day group males and females at the time of and/or 1-2 hours following dose administration. Salivation was observed on at least 1 occasion each in 9 and 4 males and females, respectively, in the 175 mg/kg/day group and in 12 and 11 males and females, respectively, in the 600 mg/kg/day group. Clear material around the mouth was also observed on at least 1 occasion each in 10 and 8 males and females, respectively, in the 175 mg/kg/day group and in all males and females in the 600 mg/kg/day group. The severity of salivation and clear material around the mouth was primarily slight or moderate in both groups. These findings were attributed to the test article, but were not considered adverse because the onset occurred immediately following dose administration; thus these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate in severity) was also noted 1-2 hours following dose administration in 6 and 10 males and females, respectively, in the 600 mg/kg/day group. Slightly lower mean body weight gains were noted in the 600 mg/kg/day group males during the pre-mating period and when the entire treatment period was evaluated. The reductions in the males in this group were due to transient lower mean body weight gains during study days 7-13 and 21-27. However, the reductions in mean body weight gain were not of sufficient magnitude to result in substantially lower mean body weights in the 600 mg/kg/day group males; therefore, the lower mean body weight gains observed in these males was not considered adverse. No test article-related effects were observed on mean body weights or body weight gains during the pre-mating, gestation or lactation periods in the 50, 175 or 600 mg/kg/day group females. Food consumption in the males and females was not adversely affected by test article administration at any dosage level. Higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted in the 50, 175 and 600 mg/kg/day group males. Potentially test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted the 175 and 600 mg/kg/day group females. The increases occurred in a dose-related manner and were more pronounced in the males. However, the increases in the females and the 50 mg/kg/day group males were not considered adverse based on the lack of correlating microscopic findings in the 50 mg/kg/day group males and the 600 mg/kg/day group females and no statistical significance. Mean absolute and relative (to final body weight and to brain weight) liver weights were higher in the 175 mg/kg/day group males and the 600 mg/kg/day group males and females. Mean absolute and relative (to final body weight and to brain weight) kidney weights were also higher in the 600 mg/kg/day group males. There were no test article-related macroscopic findings in the males and females at the scheduled necropsy. At the microscopic evaluation, follicular cell hypertrophy of the thyroid gland in the 600 mg/kg/day group males correlated with higher mean thyroid gland weights. No test article-related microscopic findings were noted in the thyroid glands of the 50 and 175 mg/kg/day group males. There were no test article-related microscopic findings observed in the 600 mg/kg/day group females.

Conclusion

Under the conditions of this study, the Study Director concluded that the NOAEL for male systemic toxicity was 50 mg/kg/day based on increased organ weights in the 175 and 600 mg/kg/day groups and microscopic findings in the 600 mg/kg/day group. However, after re-examining the study results, the NOAEL of 50 mg/kg/day for F0 male systemic effects seemed too conservative and 175 mg/kg/day was proposed to replace that value for the following reasons (See Appendix F Re-evaluation of Results): 1) Thyroid weight increases observed at 175 mg/kg/day was not considered to be “adverse” as microscopic examinations of histological structure of the thyroid glands did not reveal significant changes, and there were no toxicological effects on male reproduction. 2) Increased liver weights recorded at necropsy in male rats treated with 175 mg/kg/day was also not considered to be “adverse”. Due to lack of effects from clinical pathology analyses, macroscopic and microscopic examination, as well as liver function, the liver enlargement was considered to be consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction. The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
175 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Three Klimisch 1 studies performed in line with standardized OECD guidelines and conducted to GLP are available to address this endpoint, the quality of the dataset is therefore considered to be high.

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

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

ORAL

Key study

The objectives of this study were to evaluate the potential toxicity of the test item when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days, as well as to evaluate the recovery, persistence, or progression of any effects following a minimum of a 28-day recovery period. The protocol was designed in general accordance with OECD Guideline for the testing of Chemicals 408 (September 1998).

 

Test item in the vehicle (corn oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to 5 toxicology groups (Groups 2, 3, 4, 5, and 6) of Crl:CD(SD) rats. Dosage levels were 5, 25, 125, 250, and 500 mg/kg/day for Groups 2, 3, 4, 5, and 6, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 6 each consisted of 15 animals/sex and Groups 2–5 each consisted of 10 animals/sex. Following a minimum of 90 consecutive days of dose administration, 9–10 animals/sex/group were euthanised; the remaining 5 animals/sex in the control and high-dose groups were euthanised following a 28/29-day nondosing (recovery) period. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights and cage food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and motor activity data were recorded for all animals during Study Week 12. Ophthalmic examinations were performed during Study Weeks -2 and 12. Clinical pathology parameters (haematology, coagulation, serum chemistry, and urinalysis) were analysed for all animals assigned to the primary (Study Week 12/13) and recovery (Study Week 16/17) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

 

One female (No. 8487) in the 25 mg/kg/day group was found dead prior to dosing on Study Day 82. This animal was noted with a ruptured mass at the gross necropsy examination and was considered to be the likely cause of death.

 

One male (No. 8381) in the 125 mg/kg/day group was found dead prior to dosing on Study Day 88. There were no clinical observations noted and no macroscopic findings noted at the gross necropsy. The cause of death was undetermined.

 

All other animals survived to the scheduled necropsy. There were no test substance-related effects on haematology, serum chemistry, urinalysis, functional observational battery parameters, or motor activity. There were no test substance-related ophthalmic or macroscopic findings. Higher prothrombin time and activated partial thromboplastin time noted in the 500 mg/kg/day group males at Study Week 12/13 were considered to be potentially related to test material administration.

 

Test substance-related clinical observations noted in the 250 and 500 mg/kg group males and/or females were clear and/or yellow material around the mouth. Salivation, yellow material around the mouth, and yellow material around the urogenital and/or anogenital areas were noted in the 250 mg/kg/day group females and/or the 500 mg/kg/day group males and/or females. In addition, clear material around the mouth was noted in the 125 mg/kg/day group males. There were no clinical observations noted during the recovery period.

 

Test substance-related, nonadverse higher body weights and correlating higher food consumption were noted for the 250 and 500 mg/kg/day group females throughout the dosing period. The effects on body weights and food consumption did not persist during the recovery period for the 250 mg/kg/day group females. Test substance-related higher food consumption persisted during the recovery period for the 500 mg/kg/day group females.

 

Test substance-related changes in organ weights were noted in the 125, 250, and/or 500 mg/kg/day group males and females at the primary necropsy. Higher kidney weights were noted in the 125 mg/kg/day group males and the 250 and 500 mg/kg/day group males and females. There were no microscopic correlates or a definitive dose-response noted. Higher mean liver weights were noted in the 125 mg/kg/day group females and the 250 and 500 mg/kg/day group males and females. Higher weights correlated microscopically with hepatocellular hypertrophy and were considered to be an adaptive response to test item administration. Higher mean thyroid/parathyroid gland weights were noted in the 500 mg/kg/day group males and females, correlated with follicular cell hypertrophy, and were considered to be an adaptive response. At the recovery necropsy, higher mean kidney, liver, and thyroid/parathyroid gland weights persisted in the 500 mg/kg/day group females, with no microscopic correlates. Kidney, liver, and thyroid/parathyroid gland weights in the 500 mg/kg/day group males were similar to the control group, consistent with recovery.

 

At the primary necropsy, test item-related microscopic findings noted in the kidneys of the 125, 250, and/or 500 mg/kg/day group males consisted of increased alpha-2u globulin-immunopositive hyaline droplet accumulation, single cell necrosis within proximal convoluted tubule (PCT) epithelial cells, granular casts, and a higher incidence and/or severity of chronic progressive nephropathy (CPN); hyaline droplet accumulation was additionally noted in the 5 and 25 mg/kg/day group males. Hepatocellular hypertrophy was noted in the liver of the 125 mg/kg/day females and the 250 and 500 mg/kg/day group males and females and correlated with higher liver weights. Thyroid follicular cell hypertrophy was noted in the 500 mg/kg/day group males and females and correlated with higher thyroid/parathyroid gland weights. At recovery, renal findings noted in the 500 mg/kg/day group males consisted of increased alpha-2u globulin immunopositivity, granular casts, and higher incidence/severity of CPN. Hepatocellular and thyroid follicular cell hypertrophy were not noted in the 500 mg/kg/day group males and females at the recovery necropsy.

 

Based on the results of this study, oral administration of test item to Crl:CD(SD) rats at dosage levels of 5, 25, 125, 250, and 500 mg/kg/day for a minimum of 90 days was generally well tolerated at all dosages. Adverse microscopic renal changes related to a well-known male rat-specific alpha-2u globulin accumulation were noted in male rats at ≥ 125 mg/kg/day. All other findings were considered to be nonadverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 25 mg/kg/day for male rats and 500 mg/kg/day for female rats tested in this study. However, because the renal changes observed in the male rats in this study were in association with alpha-2u globulin accumulation, which is a well-known male rat-specific effect, not occurring in female rats or other species, including humans, and thus are of no human relevance, this effect should be excluded from any subsequent human risk assessment. Thus, in the absence of the alpha 2u globulin accumulation effect, the NOAEL relevant for human risk assessment is considered to be 500 mg/kg/day, the highest dosage level tested for both sexes of rats in this study.

Two supporting studies were conducted that evaluate the systemic toxicity of this substance, Morse (2004) and Knapp (2005).

Study 1:

In Morse (2004) Sprague-Dawley Crl:CD®(SD)IGS BR strain rats were treated by oral gavage at 1000, 500 and 100 mg/kg/day (5 rats/sex/group) for 28 consecutive days. Vehicle control animals received Corn oil at the same dose volume as administrated to other groups. Clinical observations were performed on all animals daily. A functional observation battery assessment was performed prior to dosing and once weekly during the study to evaluate the animals for gross neurological changes. Body weight and food consumption measurements were performed during the study. Hematology and clinical chemistry parameters were evaluated at the end of the treatment period. All animals were subjected to a gross necropsy examination and a comprehensive histopathological evaluation of selected tissues.

Clinical observations that were likely related to test article administration included salivation prior to dosing, struggling during dosing, and salivation at one-half hour to two hours post-dose and were observed primarily in group 4 (1000 mg/kg/day) animals. No toxicologically relevant differences among male or female groups were observed in the functional observation battery tests for this study. No statistically or toxicologically significant differences in body weights, body weight changes, or food consumption were observed, no toxicologically relevant differences in hematologic, coagulation, red blood cell morphology, or clinical chemistry parameters were observed in test article-treated males or females in this study. Mean liver weights were elevated in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) males and females and mean kidney weights were elevated in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) males and in group 4 (1000 mg/kg/day) females. There were microscopic histopathologic findings in the liver, thyroid, and kidneys of test article-treated animals. Minimal to mild eosinophilia of hepatocytes was observed in all test article-treated male groups and in group 3 (500 mg/kg/day) and group 4 (1000 mg/kg/day) females. Minimal hypertrophy of the thyroid follicular epithelium was observed in a single group 3 (500 mg/kg/day) male, in all group 4 (1000 mg/kg/day) males, and in one group 4 (1000 mg/kg/day) female. An increase in the incidence and/or severity of renal tubular epithelial cytoplasmic hyaline droplets was observed in all test article-treated male groups but not in females.

The effects detected at 500 mg/kg/day were confined to liver (male and female) and kidney (male) weight increases. The liver enlargement and hepatocyte hypertrophy are frequently observed in animals exposed to xenobiotic (Schulte-Hermann, 1979).The increase in absolute and relative weight (to body weight) without associated inflammatory or degenerative changes, it was considered as adaptive in nature and not as adverse (PSD duidance, Lewiset al,2002, Karbeet al, 2001). The renal responses in the males, for example, weight increase, renal tubular epithelial cytoplasmic hyaline droplet formations (without neoplasia), this nephropathy appeared unique to the male rat and were considered not to represent an adverse health effect (Royet al, 1983).The rare incidence of salivation prior to or after dosing is considered not as adverse at this dose groups.An assessment of the data found that that the NOAEL and NOEL for systemic toxicity for males and females was 100 mg/kg/day, respectively, based on the presence of thyroid follicular gland hypertrophy at 500 mg/kg/day (LOAEL).

Study 2:

The Knapp (2006) study consisted of a vehicle control and three treatment groups, with 12 males and 12 females in each group. CAS 18760-44-6 was dissolved in corn oil and administered at dosage levels of 15, 175 and 600 mg/kg/day, by once daily oral gavage, to F0 parental animals. All doses were given at a constant volume of 5 mL/kg. Control animals were administered corn oil under the same experimental conditions at an equivalent dose volume. Males and females received 14 daily doses prior to mating; F0 males were treated for 56 days in total, F0 females were treated for a total of 39 -53 doses. Both F0 parental animals and F1 offspring were closely examined for indications of toxicity. Experimental endpoints for F0 animals included clinical observations, body weights, selected organ weights, food consumption, mating, parturition, lactation, hematology determinations and necropsies; for F1 animals included clinical observations, body weights, growth and viability.

One male in the 600 mg/kg/day group was found dead on study day 32 due to complications from fractured nasal bones; the death was not attributed to the test article. All other animals survived to the scheduled necropsy. Salivation and clear material around the mouth were noted in the 175 and 600 mg/kg/day group males and females at the time of and/or 1-2 hours following dose administration. Salivation was observed on at least 1 occasion each in 9 and 4 males and females, respectively, in the 175 mg/kg/day group and in 12 and 11 males and females, respectively, in the 600 mg/kg/day group. Clear material around the mouth was also observed on at least 1 occasion each in 10 and 8 males and females, respectively, in the 175 mg/kg/day group and in all males and females in the 600 mg/kg/day group. The severity of salivation and clear material around the mouth was primarily slight or moderate in both groups. These findings were attributed to the test article, but were not considered adverse because the onset occurred immediately following dose administration; thus these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate in severity) was also noted 1-2 hours following dose administration in 6 and 10 males and females, respectively, in the 600 mg/kg/day group. Slightly lower mean body weight gains were noted in the 600 mg/kg/day group males during the pre-mating period and when the entire treatment period was evaluated. The reductions in the males in this group were due to transient lower mean body weight gains during study days 7-13 and 21-27. However, the reductions in mean body weight gain were not of sufficient magnitude to result in substantially lower mean body weights in the 600 mg/kg/day group males; therefore, the lower mean body weight gains observed in these males was not considered adverse. No test article-related effects were observed on mean body weights or body weight gains during the pre-mating, gestation or lactation periods in the 50, 175 or 600 mg/kg/day group females. Food consumption in the males and females was not adversely affected by test article administration at any dosage level. Higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted in the 50, 175 and 600 mg/kg/day group males. Potentially test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted the 175 and 600 mg/kg/day group females. The increases occurred in a dose-related manner and were more pronounced in the males. However, the increases in the females and the 50 mg/kg/day group males were not considered adverse based on the lack of correlating microscopic findings in the 50 mg/kg/day group males and the 600 mg/kg/day group females and no statistical significance. Mean absolute and relative (to final body weight and to brain weight) liver weights were higher in the 175 mg/kg/day group males and the 600 mg/kg/day group males and females. Mean absolute and relative (to final body weight and to brain weight) kidney weights were also higher in the 600 mg/kg/day group males. There were no test article-related macroscopic findings in the males and females at the scheduled necropsy. At the microscopic evaluation, follicular cell hypertrophy of the thyroid gland in the 600 mg/kg/day group males correlated with higher mean thyroid gland weights. No test article-related microscopic findings were noted in the thyroid glands of the 50 and 175 mg/kg/day group males. There were no test article-related microscopic findings observed in the 600 mg/kg/day group females.

Under the conditions of this study, the Study Director concluded that the NOAEL for male systemic toxicity was 50 mg/kg/day based on increased organ weights in the 175 and 600 mg/kg/day groups and microscopic findings in the 600 mg/kg/day group. However, after re-examining the study results,the observed effects were re-evaluated, and interpretations of adverse and non-adverse effects in this study were based on a review article by Lewiset al. The NOAEL of 50 mg/kg/day for F0 male systemic effects seemed too conservative and 175 mg/kg/day was proposed to replace that value for the following reasons:

1) Thyroid weight increases observed at 175 mg/kg/day was not considered to be “adverse” as microscopic examinations of histological structure of the thyroid glands did not reveal significant changes, and there were no toxicological effects on male reproduction.

2) Increased liver weights recorded at necropsy in male rats treated with 175 mg/kg/day was also not considered to be “adverse”. Due to lack of effects from clinical pathology analyses, macroscopic and microscopic examination, as well as liver function, the liver enlargement was considered to be consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction.

An assessment of the data found that the NOAEL for both males and females was 175 mg/kg/day based on thyroid follicular gland hypertrophy at 600 mg/kg/day (LOAEL).

INHALATION

Integrated testing strategies for acute toxicity state that the determination of the most likely route of exposure needs to take into account not only the substance is manufactured and handled but also the physicochemical properties of the substance. The uses and physico-chemical properties of the substance suggest that it is unlikely that the inhalatory route of exposure will be of concern and appropriate risk management measures demonstrate safe use of the substance for this route of exposure.

DERMAL

Available dermal absorption, dermal toxicity and skin irritation data suggest that the substance has low toxicity via the dermal route of exposure and that the dermal absorption of the substance will also be low such that systemic exposure is limited. Exposure assessment of the uses of the substance show that suitable operating conditions and risk management measures are in place to ensure that any dermal exposure from the use of the substance will not result in any harm to workers or the general population. On this basis it is considered that there is no scientific justification for performing a repeated dose toxicity waiver.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: The 90 -day study takes precedence over two studies provided in a weight of evidence basis, where one cannot be selected over the other.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; glandular: thyroids; urogenital: kidneys

Justification for classification or non-classification

The substance does not require classification in accordance with the criteria specified in either Regulation 1272/2008 or subsequent amendments.