DL-hexane-1,2-diol

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from May 18, 2000 to May 13, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test following GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

DL-hexane-1,2-diol

EC Number:

230-029-6

EC Name:

DL-hexane-1,2-diol

Cas Number:

6920-22-5

Molecular formula:

C6H14O2

IUPAC Name:

hexane-1,2-diol

Constituent 2

Reference substance name:

BCS697-147

IUPAC Name:

BCS697-147

Test material form:

other: liquid

Details on test material:

CAS No.: 6920-22-5
EC No.: 230-029-6

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
1.Description. Identification and Housing
Young adult, male and female Sprague-Dawley rats were received at the testing facility on May 9, 2000 from Charles River Laboratories, Kingston, NY. Upon receipt, the animals were housed individually in hanging wire cages. Cage cards displaying the pre-randomization/cage number, sex, study number, receipt date and number, facility number, and supplier were affixed to each cage for identification during acclimation and prior to randomization. After randomization, metal ear tags displaying unique identification numbers were used to individually identify the animals, and the animal number, group number, dose level, and treatment group color code was added to each cage card. During the study, immediately prior to and during expanded clinical observation testing sessions, cage cards displaying the blind number only were affixed to each cage. All housing and care was based on the standards recommended by the current Guide for the Care and Use of Laboratory Animals
2. Environment
Environmental control equipment (Edstrom Industries, Environmental Watchdog System, Version 4) was used to monitor/record and adjust environmental conditions as necessary to minimize fluctuations in the animal room environment. The animal room was maintained at room temperature and relative humidity ranges of 74-70 °F and 69-45% t respectively, during the conduct of the study, with the exception of two approximately 30 minute periods on 8/9/00 when the humidity increased to up to 72%. This is a protocol deviation, but it does not affect the integrity of the study. A 12-hour light/12-hour dark cycle was maintained in the animal room during the course of the study, with the exception of occasional <=30 minute disruptions of the light/dark cycle which occurred primarily as a result of collar removal. These disruptions were documented in the facility Edstrom records. In addition, on May 17 and August 23, 2000 the light cycle was disrupted for approximately 2 hours for completion of ophthalmic examinations. Room ventilation was set to produce at least 10 air changes/hour.
Acclimation
Upon receipt, the animals were removed from the shipping cartons, weighed, and placed into individual cages. The animals were examined by qualified personnel and identified with cage cards. Animals were acclimated to the laboratory conditions for seven days. The animals were observed daily for overt physical or behavioral abnormalities, morbidity, and mortality.
Animal Selection
Only healthy animals meeting protocol specified body weight requirements and exhibiting normal behavior were chosen for study use. To avoid potential bias, the animals chosen for study use were randomly selected from all available healthy animals obtained specifically for this study. All animals received a detailed pretest observation period prior to dosing. Females were nulliparous and nonpregnant.
Animals not selected during randomization remained housed in the study room until study day 7. On study day 1, during pre-dosing observations, two of the study animals (G4M2726 and G2F2682) exhibited clinical and dermal, respectively, findings. As a result, these animals were replaced on study with animals not selected previously during randomization. Pretest animal #3 was used to replace G4M2726, and was ear tagged/identified as study animal # G4M2751, Pretest animal # 78 was used to replace G2F2682, and was ear tagged/identified as study animal # G2F2752. On study day 7, animals not assigned to study were physically separated from study animals via transfer to another study and room. The fate of all animals obtained for this study has been documented.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Vehicle:
Water, treated by reverse osmosis, was used as the vehicle of choice for delivery of the test article.
Dosing
Beginning on study day 1, and for 91-93 consecutive days, the dose solutions were applied daily as a single dermal dose to approximately 10% of the body surface area (BSA) of 15 rats/sex/dose group. For a typical 200-300 g rat, the test site was defined as an approximately 2” x 3" area within the clipped region. The test site was delineated within the clipped area using an indelible marker, and was remarked when necessary.
On study day -1, and as necessary throughout the study, the hair was removed from the dorsal trunk area of the animals using a small animal clipper. The clipped area was approximately 10% of the animal's body surface area, and this area included the scapular (shoulder) region to the wing region of the ileum (hipbone) and halfway down the flank on each side of the animal.
Approximately six hours after the last animal was dosed, the Elizabethan collars were removed from all the animals Dosing was performed sequentially starting with the vehicle control group and proceeding from the low to the high dose groups.
Elizabethan collars were applied to the animal immediately prior to or after application of the dose solution to the test site.The dose solution was administered at a dose volume of 2 ml/Kg using an appropriately sized plastic syringe fitted with a stainless steel ball-tipped. The dose was delivered slowly, spreading evenly over the entire test site during application, using the ball-tipped needle surface and with the aide of a glass rod. Individual doses were initially calculated based on the animal’s day 1 body weight, and doses were adjusted weekly using the most current body weight. Doses administered were based on body weight.
At collar removal, clinical signs observed which were related to the stress of collaring were not documented. Animals scheduled for necropsy on August 29, 30, and 31, 2000 received 91, 92, and 93 doses, respectively. On the last day of dosing for each animal, the Elizabethan collars were removed approximately four hours after the last animal had been dosed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulation Analyses
Dose solutions were prepared weekly, and in study weeks 1 through 6, 8 and 13, duplicate samples (5 ml each) were taken from the middle of each concentration of dose solution for analytical purposes. Verification of concentration analysis was performed on samples for use in study weeks 1, 2, 4, 5, 6, 8’ and 13 by Primedica Corporation, Worcester, MA. Samples were taken and sent for analysis from the week 5 dose solution preparation because the mean of the analytical results for one solution prepared week 4 were not within acceptable range (±10% nominal concentration). All samples were sent for concentration analyses on the day of preparation.
Stability and Homogeneity of Dose Formulations
On study day 1, duplicate samples (15 ml each) were taken for stability determination from the middle of both the lowest and highest concentration (175 and 500 mg/ml, respectively) dose solutions prepared. The samples were sent for concentration analyses, to the contact and address listed above, on the day of preparation. Concentration analyses to establish stability of the test article formulations was performed on study days 3 and 9,
On study day 1, duplicate samples (5 ml each) were taken for homogeneity determination from the top, middle, and bottom of each of the lowest and highest concentration (175 and 500 mg/ml, respectively) dose solutions prepared. These samples were shipped as described above. Analyses to establish homogeneity of the test article formulations were performed on study day 3.

Duration of treatment / exposure:

91-93 consecutive days

Frequency of treatment:

once per day

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 350, 700, or 1000 mg/kg
Basis:
nominal per unit body weight

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

Disposition of Test Article
After completion of the proposed studies, remaining test article was returned to the Sponsor Representative. Disposal of all unused portions of the test article solutions was by drain- wasting, according to facility SOPs.
Dose Solution Formulations
The test article was prepared weekly. The test article was prepared as four separate w/v solutions in vehicle, at the following target concentrations for each dose level: 500 mg/ml for the 1000 mg/kg/day level, 350 mg/ml for the 700 mg/kg/day level, 175 mg/ml for the 350 mg/kg/day level, and 0 mg/ml for the vehicle control. For each dose solution, preparation was performed by weighing a specified amount of test article into a clean, tared 500 ml volumetric flask. The exact weight of the test article was recorded. Then reverse osmosis (RO) water was added to the flask to q.s. to the 500 ml volume, and a stir bar was added. The flask was then stoppered, inverted and placed on a magnetic stirrer as necessary to completely mix the components into solution. Solutions, with stir bars, were then transferred to screw cap containers for sampling, storage, and dosing. For the vehicle control dose solution, approximately 500 ml of RO water was added to a clean, capped container. Containers were capped and stored at room temperature when not in use. The test article dose solutions and the vehicle control dose solution (water alone) were delivered daily at a constant dose volume of 2 ml/kg body weight.

study groups design----see table 1 in the field for other informations on method
Rationale for Dosage Level Selection
The dose levels selected by the Sponsor Representative and utilized during the study were
0, 350, 700 and 1000 mg/kg/day. This dose regimen was designed to demonstrate a gradient of limited toxic effects. The high dose was not expected to cause lethality, but to possibly produce signs of toxicity such as decreased body weight. The intermediate dose level was selected for the purposes of evaluating any potential toxicological effects. The low dose was not expected to produce signs of toxicity. The data from this study will be used for human risk assessment.

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Body weight, food consumption, and routine detailed clinical observations were recorded weekly. Routine detailed clinical observations, performed in conjunction with body weights, consisted of a detailed examination, including, but not limited to changes in: skin and fur, eyes and mucous membranes, morbidity, posture, respiration, salivation, and behavior Expanded clinical observations were performed in random order weekly, at approximately the same time of day, on the first ten animals/sex/dose group. Expanded clinical observations were performed in a blind fashion and consisted of hand-held and open field observations. During study week 13 expanded clinical observations also included elicited behaviors observations and locomotor activity testing. Ophthalmology examinations were performed prior to study initiation and during study week 13. Estrous cycle evaluations were performed on all female animals for 21 consecutive days, beginning in study week 11 and inclusive through study week 13. After 91-93 days of treatment, the animals were fasted overnight and urine was collected over a period of 16-18 hours overnight prior to necropsy.
In addition, the treated skins of animals in the mid and low dose treatment groups were also processed and evaluated by light microscopy.

Sacrifice and pathology:

Just prior to necropsy, blood samples (for hematology, clinical chemistry, and coagulation evaluation) were collected via the vena cava after rats were anesthetized with carbon dioxide. At necropsy, a portion of the midsection of the right epididymis was excised from each male rat to collect samples for sperm analysis. Also at necropsy, all macroscopic observations were recorded, and terminal body weights, organ weights, and tissues specified in the protocol were collected for all animals. Histopathology was performed on specified organs from the vehicle control and high dose animals.
Unscheduled Deaths and Euthanasia
On study day 14, one male control animal (G1M2634) was euthanized for humane reasons and necropsied according to the procedure described below under Anatomic Pathology. A final body weight was collected immediately prior to euthanasia of the animal. No terminal body weight, organ weights, or clinical pathology blood samples were collected for this animal. All tissues were collected, processed, and evaluated by light microscopy.
Scheduled Euthanasia
All surviving animals were necropsied according to the procedure described below under Anatomic Pathology. Just prior to necropsy, animals were weighed, anesthetized by carbon dioxide inhalation, and euthanized by exsanguination. The date of death and terminal body weights were recorded,
Pre-Necropsy Sample Collection
On each afternoon prior to a scheduled necropsy (study days 91, 92 and 93), animals were placed into metabolism cages in random order for the scheduled necropsy and fasted overnight (approximately 16-24 hours).
Anatomic Pathology
All animals submitted for necropsy received a macroscopic evaluation made under the supervision of a board-certified veterinary pathologist. The evaluation included a comprehensive necropsy, defined as the macroscopic examination of the external surfaces of the body and orifices, and the cranial, thoracic, abdominal, and pelvic cavities and their contents.

Other examinations:

1, Organs and tissues were dissected free and placed in tissue collection trays. Tissues were collected in 10% neutral buffered formalin.
2, The weight of the selected organs listed befow were recorded at necropsy. Organ weight data was collected at all scheduled necropsies.
3, Male Reproductive Assessment
(1) Sperm Mobility Sample Collection and Evaluation
(2) Total Sperm Count Sample Collection and Determination

Statistics:

The observed values for body weight, food consumption, clinical pathology parameters, absolute organ weights, grip strength, and Digiscan data were analyzed separately for each sex using either normal distribution or distribution-free techniques. Organ-to-body weight and organ-to-brain weight ratios were calculated and analyzed. If LeveneTs test for homogeneity of variance was not significant (p > 0.05)( comparisons with the control group were made using Dunnett，s multiple comparison procedure. If Levene's test was significant (p £ 0.05), these comparisons were made using a nonparametric analog of Dunnett's procedure. The Dunnett’s procedure and its nonparametric analog fixed the two-sided risk level at a 5% probability that one or more of the comparisons to the control were significant, when in fact a treatment effect does not exist Significance at the 1% and 0.1% levels, and p-values, were also be reported, where appropriate.
Tail flick data was evaluated statistically using a survival analysis method, and the Kaplan-Meier method was used for estimating the 25 ( 50th, and 75th quartile for each group response. This is a protocol deviation, but does not affect the integrity of the study. Pair-wise comparisons between control and treated groups were made using the Wilcoxon test.
The sperm motility, total count, and sperm morphology data were compared across groups using the Kruskal-Wallis nonparametric ANOVA test. If a significant result was obtained at the 5% probability level, the Wilcoxon (Mann Whitney U) test served as the post-hoc group comparison test (at the 5% one-tailed probability level).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

one control male that was euthanized for humane reasons on study day 14. Treatment-related clinical findings included rough coat and fur staining.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

By study week 4, all animals in the 1000 mg/kg/day treatment group were exhibiting dermal findings.

Mortality:

mortality observed, treatment-related

Description (incidence):

one control male that was euthanized for humane reasons on study day 14. Treatment-related clinical findings included rough coat and fur staining.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The study animals in the 1000 mg/kg/day treatment group exhibited statistically significant decreases in body weight gains per day and in cumulative absolute body weight gains.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

when compared to controls, male animals in the 1000 mg/kg/day treatment groups exhibited a statistically significant (p<0.05) increase in mean feed consumed per body weight per day at the measurement interval on study day 77.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Corneal mineralization was observed in animals from all treatment groups, but with a higher incidence in animals from the 350 and 1000 mg/kg/day treatment groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology findings were restricted to statistically significant changes in the female animals in this study.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The mild increase in urine protein in females at 1000 mg/kg/day suggests a test article-related effect at this dose level.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treated female animals exhibited increases in heart-to-body weight ratio, kidney-to-body weight ratio, and kidney-to-brain weight ratio when compared to controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

macroscopic observations were limited to the treated skin and included slight focal erythema and/or slight dermal thickening and eschar formation in 1/15 and 2/15 males in the 700 and 1000 mg/kg/day treatment groups, respectively.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluations of tissues from surviving animals indicated that treatment-related changes were restricted to epidermal hyperplasia and hyperkeratosis in treated skin.

Histopathological findings: neoplastic:

not examined

Details on results:

Mortality
One male animal from the control treatment group was euthanized for humane reasons on study day 14. The remaining animals survived to the scheduled euthanasia.
Dermal Observations
During the study, dermal findings exhibited by treated animals were restricted to very slight (barely perceptible) erythema, desquamation, and slight (10% of the test site) eschar. After the first week of dosing, compared with control animals, the incidence of these findings was slightly increased in the 350 mg/kg/day treatment group with 6/30 animals affected, and significantly increased in the 700 and 1000 mg/kg/day treatment groups with 26/30 and 28/30 animals affected, respectively. In these animals affected in study week 1, <50% exhibited the very slight erythema (i.e., the great majority of the findings were desquamation and eschar). In study weeks 2 and 3, the total dermal findings in each dose group persisted at approximately the same rate of incidence observed in study week 1. However, in study week 2, approximately 50% of the animals in the 700 and 1000 mg/kg/day treatment groups were exhibiting very slight erythema, with one animal in the 700 mg/kg/day treatment group exhibiting well-defined erythema.
By study week 4, all animals in the 1000 mg/kg/day treatment group were exhibiting dermal findings, and this trend continued throughout the remainder of the 13 week study with the exception of one animal in study week 13. During this time, the very slight erythema response peaked in these high dose animals at week 5 with approximately 66% of the animals affected. For animals in the 700 mg/kg/day treatment group, the majority (25-30) of the animals were exhibiting dermal findings from study week 4 continuing through the remainder of the study. During this time, the very slight erythema response peaked in these mid dose animals at week 8 with approximately 33% of the animals affected. In study week 4. the incidence of dermal findings in animals treated with 350 mg/kg/day of the test article was 8/30, and this incidence continually decreased to 0/30 animals affected by the end of the 13 week study. During this time, the very slight erythema response peaked in these low dose animals at week 5 with approximately 17% of the animals affected.
Routine Clinical Observations
Rough coat was the primary clinical finding considered to be treatment-related. Study records indicate that the rough coat was oftentimes accompanied by stiff fur. Rough coat was observed in 0/15 females and 0/15 males in the control group, 4/15 females in the 350 mg/kg/day treatment group, 10/15 females and 4/15 males in the 700 mg/kg/day treatment group, and 12/15 females and 7/15 males in the 1000 mg/kg/day treatment group. Fur staining also appeared treatment-related, and it was observed in 0/15 females and 0/15 males in the control group, 1/15 females and 1/15 males in the 350 mg/kg/day treatment group, in 4/15 females and 1/15 males in the 700 mg/kg/day treatment group, and in 5/15 females and 1/15 males in the 1000 mg/kg/day treatment group. Other clinical signs observed included alopecia, scab, open sore/ulcer, and dark dry material excretion. These findings were distributed approximately evenly across all treatment groups, including controls, for both sexes. In addition, one control male animal sustained a broken upper incisor tooth noted on study day 14. Based on the recommendation of the facility veterinarian, this animal was humanely euthanized as a result of this injury.
Expanded Clinical Observations
In male animals, the following week/parameter were found to demonstrate a dose-related trend at a statistically significant level; week 1 - muscle tone (p<0.030), week 2 - muscle tone (p<0.006), week 3 - muscle tone (p<0.004), week 4 - reactivity to handling (p<0.035), week 4 - locomotor activity (p<0.007)f week 4 - presence of urine (p<0.040), week 12 - appearance of fur-staining (p<0.004)t week 12 - muscle tone (p<0.025), and week 13 - appearance of fur-staining (p<0.010). In female animals, the following week/parameter were found to demonstrate a dose-related trend at a statistically significant level; week 12 - appearance of fur-staining (p<0.010) and week 13 - appearance of fur-staining (p<0.000). For these parameters demonstrating statistically significant trend test results, pair-wise comparisons were performed between treated and control animals. The response for the following parameters for treated male animals were found to be statistically different from control male animals for the same week: appearance of fur- staining in 1000 mg/kg/day group in week 12 (p<0,030), appearance of fur-staining in 700 mg/kg/day group in week 13 (p<0,030), muscle tone in the 1000 mg/kg/day group in weeks (p<0-011), 3 (p<0.002), and 12 (p<0.011)_ and muscle tone in the 350 mg/kg/day group in week 3 (p<0.003). The response for the following parameters for treated female animals were found to be statistically different from control female animals for the same week: appearance of fur-staining in 1000 mg/kg/day group in weeks 12 (p<0.020) and 13 (p<0.000), appearance of fur-staining in 700 mg/kg/day group in weeks 12 (p>0,001) and 13 (p<0,000), and appearance of fur-staining in 350 mg/kg/day group in weeks 12 (p<0.001) and 13 (p<0.011). There were no statistically significant changes in any other hand-held or open field parameters in male or female animals from any treatment group when compared to controls.
Bodv Weight
Statistically significant decreases were observed in mean body weight gains per day for both male and female animals in the 1000 mg/kg/day treatment group on study day 7 (p<0.05), and for male animals in the 1000 mg/kg/day grcnjp on study day 35 (p<0,01). Similarly, statistically significant (p<0.05) decreases were observed in mean cumulative body weight gains for both male and female animals in the 1000 mg/kg/day treatment group on study day 7, and for male animals in the 1000 mg/kg/day group on study days 35 and 42.
There were no statistically significant differences in mean absolute body weights for either sex in any treatment groups when compared to controls. However, male animals in the 700 and 1000 mg/kg/day treatment groups showed slight but consistent decreased mean absolute body weights relative to controls throughout the study.
All individual male animals exhibited positive weight gain through study day 42. Beginning on study day 49, some males in all treatment groups exhibited weight loss over certain measurement intervals. Throughout the study, some female animals in all treatment groups exhibited weight loss over certain measurement intervals, with more individuals in all treatment groups exhibiting weight loss toward the end of the study. By study day 91, weight loss occurred in more individual female animals from the 1000 mg/kg/day treatment group than in any other treatment group
Food Consumption
Individual animal feed consumed per day from interval to interval was positive for all animals on study. There were no statistically significant differences in mean feed consumed per day for either sex in any treatment groups when compared to controls.
Individual animal feed consumed per body weight per day from interval to interval was positive for all animals on study. However, when compared to controls, male animals in the 1000 mg/kg/day treatment groups exhibited a statistically significant (p<0.05) increase in mean feed consumed per body weight per day at the measurement interval on study day 77. Female animals in the 1000 mg/kg/day treatment group also exhibited statistically significant increases in mean feed consumed per body weight per day, relative to controls, for measurement intervals on study days 14 (p<0.05), 42 (p<0.05), 63 (p<0.001)t 84 (p<0.05), and 91 (p<0.001). Additionally, for the measurement interval on study day 63, female animals in both the 350 and 700 mg/kg/day treatment groups exhibited statistically significant increases, p<0.05 and p<0.01 respectively, in mean feed consumed per body weight per day when compared to controls.
Ophthalmic Examinations
Animals exhibiting corneal mineralization and scars were placed in the 350 and 1000 mg/kg/day treatment groups at randomization.
During pre-necropsy ophthalmic examinations, a full data set (i.e., data for both eyes) was not recorded for animal G2M2670. As a result, this animal was not considered In the data summary for group 2 males. The major findings observed at the pre-necropsy ophthalmic examination were corneal mineralization and scars, cataract, and retinal degeneration. Corneal mineralization was observed in animals from all treatment groups, but with a higher incidence in animals from the 350 and 1000 mg/kg/day treatment groups. Of the cataracts observed, 14 occurred in animals that did not exhibit cataracts pre-study, with 2, 2,6, and 4 in treatment groups 0t 350, 700, and 1000 mg/kg/day, respectively. Retinal degeneration occurred in one male animal in the 1000 mg/kg/day treatment group.
Vaginal Cytology
Vaginal smears were performed on all female animals during study weeks 11 through 13 to evaluate the phases of the estrus cycle.
see table 1 and 2 in the field for additional information of results.
Estrous cycle data for the treated female animals were comparable to that of the vehicle control female animals. Based on these data, repeated dermal administration of the test article at levels of up to 1000 mg/kg/day for 91 days to female rats does not appear to affect the estrous cycle.
Male Reproductive Assessment
There were no statistically significant differences in mean percent sperm motility, mean total sperm count, or mean percent abnormal sperm morphology in any treatment groups when compared to controls. Group mean values for percent sperm motility were comparable across treatment groups and ranged from 93% to 97%. The number of sperm/gram of caudal epididymis was comparable across treatment groups and ranged from 699.2 to 867.1 million sperm/gram. A low incidence of head abnormalities was observed across treatment groups and ranged from 0.1 to 0.2 percent abnormal sperm.
Clinical Pathology
Hematology findings were restricted to statistically significant changes in the female animals in this study. Females in the 1000 mg/kg/day treatment group experienced statistically significant increases in total leukocyte count (WBC) and monocytes (MONO A) (p<0.01), and in segmented neutrophils (SEGS A), lymphocytes (LYMPH A), and large unstained cells (LUC A) (p<0.05)f when compared with controls. There were no statistically significant changes in hematology parameters in female rats from any other treatment group or in male rats from any treatment group when compared to controls
Clinical chemistry findings were restricted to a minor but statistically significant decrease (p<0.05) in chloride (CL) in female animals from the 350 mg/kg/day treatment group and to a minor but statistically significant increase (p<0,05) in total bile acids (TBA) in female animals from the 700 mg/kg/day treatment group. There were no statistically significant changes in clinical chemistry parameters in female animals in the 1000 mg/kg/day treatment group, or in male animals from any treatment group when compared to controls.
There were no statistically significant changes in coagulation parameters or protein electrophoresis parameters in male or female animals from any treatment group when compared to controls.
When compared with controls, urine pH (UR PH) was decreased in a dose dependent manner in both male and female animals in the study, but the decrease was only statistically significant in the males and females (p<0.05) from the 1000 mg/kg/day treatment group. Urine specific gravity (UR SPGR) was increased in a dose-related manner in all treated female animals when compared to controls, and the Increase was statistically significant in females from the 700 mg/kg/day (p<0.01) and 1000 mg/kg/day (p<0.001) treatment groups. This increase in UR SPGR in treated females correlated with a decrease in urine volume (UR VOL) in treated females, but the decrease was only statistically significant (p<0.05) in the 1000 mg/kg/day females. Treated males also exhibited a non-significant decrease in UR VOL Statistically significant increases were observed in urine potassium (UR K) and urine chloride (UR CL) in female animals from the 700 mg/kg/day treatment group (p<0.05) and the 1000 mg/kg/day treatment group (p<0.01)( when compared to controls. Urine sodium (UR NA) concentration was also increased in females in ail dose groups, but these changes ware not statistically significant* Relative to controls, excretion rates of urine chloride (URCL) were decreased in treated males and increased in treated females, but the changes were not statistically significant. Excretion rates of urine potassium (URK) were significantly increased in female animals from the 350 mg/kg/day (p<0.05), 700 mg/kg/day (p<0.01), and 1000 mg/kg/day (p<0.05) treatment groups when compared to controls. Other urinalysis parameters (macroscopic and microscopic evaluations and test strip analysis) were not analyzed statistically, but were evaluated. In female animals, small concentrations of urine Ketones and protein were noted to be present in more treated rats than in control rats. All other macroscopic and microscopic evaluations and test strip analysis parameters were found to be within normal limits in female animals. All macroscopic and microscopic evaluations and test strip analysis parameters were found to be within normal limits in male animals.
Anatomic Pathology
For surviving animals, macroscopic observations were limited to the treated skin and included slight focal erythema and/or slight dermal thickening and eschar formation in 1/15 and 2/15 males in the 700 and 1000 mg/kg/day treatment groups, respectively. No macroscopic observations were noted in the treated skin of any other surviving animals.

Microscopic evaluations of tissues from surviving animals indicated that treatment-related changes were restricted to epidermal hyperplasia and hyperkeratosis in treated skin. These findings were observed in 6/15 males and 10/15 females in the 1000 mg/kg/day treatment group and in 4/15 males in the 700 mg/kg/day treatment group. However, there was no evidence of inflammation or other changes that would be expected to progress to ulceration or chronic skin damage. In addition, minimal hyperkeratosis was also noted in 4/14 control male animals. Microscopic findings in all other tissues were consistent with common, spontaneous alterations in laboratory rats.
Absolute terminal body and organ weights did not differ significantly for either male or female animals in any treatment group relative to controls. However, all treated female animals exhibited significantly increased heart-to-body weight ratios. In addition, kidney-to- body weight ratios were also increased in all treated females, but the increase was only statistically significant in the 350 and 1000 mg/kg/day treatment groups. Kidney-to-brain weight ratios were also increased all treated females, but the increase was only statistically significant in the 1000 mg/kg/day treatment group.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table1. Total Days in Each Phase of the Estrous Cycle (Mean Number of Days土SD)

Dose Level (mg/kg/day)	Number of Animals	Phase of Cycle
		Proestrus	Estrus	Metestrus	Diestrus	Combined Estrus and Proestrus	Combined Metestrus and Diestrus |
0	15	2.9±1,2	5.1±1.1	6.3±2.2	6.6±1.7	8.1±1.5	12.9±1.5
350	15	2.7± 2.2	5.3±1,2	6.5±2-2	6.5±1,9	8.1±1.7	12.9±1.7
700	15	2.5± 1,8	5.3±1.2	7.0±2.1	6.1±1.2	7.9±1.6	13.1±1.6
1000	15	2.1± 1.6	6.1±1.3	7.6±2.0	5.2±1.7	8.2±1.9	12.8±1.9

Table 2. Estrous Cycle Length (Mean Number of Days土SD}

Dose Level (mg/kg/day)	Number of Animals	Duration of Estrous Cycle
0	15	3.9±0.7
350	15	4.2±0.6
700	15	4.1±0.6
1000	15	4.0± 0.3

Applicant's summary and conclusion

Conclusions:

Under the condition of this study, the NOAEL for systemic toxicity of test article based on dermal exposure is 700 mg/kg/day, and the highest dose level of 1000 mg/kg/day is considered to be a minimal LOAEL.

Executive summary:

The study was conducted following OECD guideline and GLP principles to assess the toxic effects of test article when administered dermally once per day for 91 -93 consecutive days to SD rats, and this report can provide the information on the potential hazards to confirm exposure levels associated with no-observed-adverse-effect-level. Fifteen animals/sex/dose group received a daily dermal dose of test article at dose levels of 0, 350, 700, or 1000 mg/kg .

Based on the experimental data, daily dermal administration of test article to Sprague-Dawley rats for 91-93 days was associated with treatment-related rough coat, fur staining, and slight dermal irritation. In 1000mg/kg/day females, mild increases in total leukocyte counts and urine protein suggest a test article related effect at this dose level. Alterations at 700 mg/kg/day or less were not considered to be of toxicological concern. Therefore, under the conditions of this study, the NOAEL for systemic toxicity of test article based on dermal exposure is 700 mg/kg/day, and the highest dose level of 1000 mg/kg/day is considered to be a minimal LOAEL.