Registration Dossier

Administrative data

Description of key information

Dermal administration of the test item on rats produced no significant observations of systemic toxicity. A no effect level for dermal irritation was not established in this study and thus is considered to be below 0.03 mL/kg/day (ca. 26.7 mg/kg bw/day).

Key value for chemical safety assessment

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Nov 5, 1984 - Dec 3 to 4, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Remarks:
US EPA GLP, 1984
Limit test:
no
Specific details on test material used for the study:
- Identification: HLD (2,4-dicyclohexyl-2-methyl pentane)
- clear liquid
- Lot No.: G-15842
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan
- Age at study initiation: 7 wks old
- Weight at study initiation: males: 233 to 257 g; females: 155 to 169 g
- Fasting period before study: Overnight prior to start of study
- Housing: individually in wire mesh cages
- Diet (e.g. ad libitum): Certified Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 °C ± 0.6 °C
- Humidity (%): 49% ± 2.1%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: near the shoulder blades
- % coverage: 4 sq. in area (6-8%)
- Type of wrap if used: None
- Time intervals for shavings or clippings: Fur was shaved prior to initial dosing. Thereafter, shaving was performed as necessary to maintain the treatment area. Care was taken not to damage the treated skin area.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No washing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03, 0.15, 0.75 mL/kg/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: No (no attempts were made by the use of collars or other similar restraining procedures to prevent the rats from ingesting the test item)
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
A 5 g sample of the test article was collected after study termination and shipped under ambient conditions to the Sponsor (Monsanto) for analysis.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Administered dermally once daily, 5 days a week
Dose / conc.:
27 mg/kg bw/day
Remarks:
Calculated from dosage level of 0.03 mL/kg/day; based on specific gravity of test item of 0.886 g/cm3 at 25 deg C (from SDS).
Dose / conc.:
133 mg/kg bw/day
Remarks:
Calculated from dosage level of 0.15 mL/kg/day; based on specific gravity of test item of 0.886 g/cm3 at 25 deg C (from SDS).
Dose / conc.:
665 mg/kg bw/day
Remarks:
Calculated from dosage level of 0.75 mL/kg/day; based on specific gravity of test item of 0.886 g/cm3 at 25 deg C (from SDS).
No. of animals per sex per dose:
10 males and 10 females per dose group (40 males and 40 females in total)
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for mortality, moribundity, and gross signs of toxicologic and pharmacologic effects
- Detailed observations of appearance and condition, behavior and activity, excretory functions, respiration, orifices, eyes and palpation for masses were conducted at least once each week.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD CONSUMPTION:
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: On all rats pre-test and at week 4
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On all rats at 4 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters: Hematocrit, hemoglobin concentration, erythrocyte count, MCH, MCV and MCHC (calculated), total and differential leukocyte count, platelet count, reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On all rats at 4 weeks
- Animals fasted: No data
- How many animals: All
- Parameters: (Serum used) asparate aminotransferase, alanine aminotransferase, alkaline phosphatase, glucose, urea nitrogen, total bilirubin, total cholesterol, albumin, globulin (calculated), total protein, creatinine, sodium, potassium, chloride, calcium, phosphorus, creatine phosphokinase.

URINALYSIS: Yes
- Time schedule for collection of urine: On all rats at 4 weeks
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters: Volume, color and appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrites, bilirubin, occult blood, microscopy of spun deposit.
Sacrifice and pathology:
ANATOMIC PATHOLOGY
- macroscopic pathology: conducted on all rats. Tissues fixed in formalin (except eye, fixed in glutaraldehyde)
- organ weights: collected from all rats. Absolute and relative (to brain and to body weights) weights of adrenal (2), brain, heart, kidney (2), liver, testis (2)
- microscopic pathology: conducted on all rats. Full tissue examined for rats in 0and 0.75 mL/kg/day dosage levels. Liver, kidney (2), skin (treated and untreated) and lung (2) examined for rats from 0.03 and 0.15 mL/kg/day dosage levels.
Statistics:
Body weights, food consumption, absolute and relative organ weights and clinical laboratory values analyzed using Bartlett's test and analysis of variance. Treatment groups compared to the control group, by sex, using the appropriate t-statistic (equal or unequal variance) and Dunnett's multiple comparison tables. Nonparametric analysis of total bilirubin, chloride, urine specific gravity and volume by using rank transformations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations which were attributed to test item administration included red areas, scabbed areas, thickened skin and abraded areas. All these signs were evident on the test site of the rats. Red areas were most predominant and were first noted on study day 3. Abraded areas, which were quite large at times, and thickened skin were evident only at the 0.15 and 0.75 mL/kg/day dosage levels.

Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic lesions were observed at the dermal application sites in all treated groups. Scab formation was observed in all treated groups. These lesions tended to increase in both incidence and severity as dosage increased. Crust formation was observed in the 0.15 and 0.75 mL/kg/day groups. The incidence and severity of this lesion was greater in the 0.75 mL/kg/day group. Thickening of the skin at the application site was also observed in the 0.15 and 0.75 mL/kg/lay groups. The incidence and severity of this lesion was somewhat greater in the 0.75 mL/kg/day group. All of the above lesions (scab formation, crust formation and thickening of the skin) were considered to be direct effects of the test article. In addition, a dose response was evident.

Abrasions of the skin at the application site were observed in all treated groups. These changes tended to increase in both incidence and severity as dosage increased. Abrasions are mechanical injuries of the skin. In this case they most likely represent self-trauma; and, as such, were regarded as indirect effects of the test article. Some differences in the incidence and severity of macroscopic dermal lesions between males and females in all treated groups were observed. These differences were not considered meaningful.

Enlargement of the axillary and/or prescapular lymph nodes was observed in four males and nine females of the 0.75 mL/kg/day group. The difference in incidence of this change between males and females in this group was not considered meaningful. The changes in these lymph nodes were regarded as secondary to the development of lesions at the dermal application sites. As such, they were regarded as indirect effects of the test article.

All other macroscopic changes observed were regarded as incidental or spontaneous in nature. They were not considered to be related to the test article.

The principal lesions observed microscopically at the dermal application sites in all treated groups were epidermal exudates, dermatitis and acanthosis. Minor differences in incidence and severity between males and females were not considered meaningful.

The development of exudative epidermitis and underlying dermatitis was considered to be a direct effect of the test article. 'The incidence and severity of these lesions indicate a dose effect. The development of a bacterial dermal infection was regarded as a secondary complication of epidermal injury, as such an occurrence is not expected in otherwise normal skin. Acanthosis was also considered to be direct effect of the test article. The incidence and severity of this lesion, however, do not
indicate a dose effect.

Some degree of hyperkeratosis was seen in nearly all animals in all treated groups. In addition, this change was seen in nine males and two females in the 0 mL/kg/day group. Hyperkeratosis was also seen infrequently in untreated skin. There was no meaningful difference in severity. Hyperkeratosis is a nonspecific reaction of the epidermis to irritation. Its greater incidence in males in the 0 mL/kg/day group and in untreated skin probably reflects normal physiologic variation and anatomic location. The occurrence of hyperkeratosis in the 0 ml/kg/day group is most likely a result of mechanical factors, i.e., sham application to the skin. At least part of the hyperkeratosis in the treated groups, therefore, may be due to the same mechanical factors. The rats were not prevented from grooming the application site and thus probably some test article was ingested subsequent to this grooming activity.
Mortality:
no mortality observed
Description (incidence):
None of the animals in the study died or were sacrificed in extremis during the course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weights were slightly decreased in the 0.75 mL/kg/day male group over most of the study and in the female rats at this dose level during the last week of study. Body weights in the 0.03 and the 0.15 mL/kg/day dosage groups were considered comparable to those of the controls. Statistical significance was noted at weeks 2, 3, and 4 in the 0.75 mL/kg/day dose group.

Body weights of the 0.75 mL/kg/day male dosage group were depressed throughout the study and were 8% less than control values at the study termination. Meanwhile high-dose female animals had body weights 7% less than their respective controls at the end of the study.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Based on g of diet consumed/kg of body weight/day, food consumption appeared to be increased slightly in the 0.75 mL/kg/day male group and to a greater extent in the female rats at this dose level. Based on g of diet/animal/day, food consumption appeared generally comparable in all groups. Slight increases in food consumption were also seen in the 0.15 mL/kg groups.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test article related abnormalities were detected in the eye examinations. The observations that were noted were representative of pathology that would be expected for this group of animals considering age, sex and strain.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slight decreases in the erythrocyte count, hemoglobin and hematocrit were noted for the 0.75 mL/kg/day males and for hematocrit for the female rats at this dose level. Changes in total leukocyte counts and segmented neutrophil counts were consistent with a secondary bacterial infection at the high dose level.

No changes were noted in the calculated erythrocyte indices including MCV, MCH and MCHC, and reticulocytes were considered to be within normal limits and similar to control values.

Leukocytes were elevated in both the male and female high-dose animals, and significant increases were noted in segmented neutrophils from animals in those groups. These changes were likely related to the bacterial infection observed at the treatment sites. Similar findings were not reported for hematological parameters in the other treated groups. The changes reported in hematological values were not considered to be related to the test article administration.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry and urinalysis values were considered unremarkable.

Several serum biochemistry values were significantly different from controls at the high dosage. In the males these included an increase in alanine aminotransferase and a decrease in albumin and glucose. Female high-dose animals showed increases in globulin, aspartate aminotransferase, alanine aminotransferase and urea nitrogen with a decrease in glucose; although these changes were considered to be in the range of normal biological variability. The low-dose males also had significantly lower potassium values. The statistically significant values reported for serum biochemistry were not considered to be treatment-related since they were within the normal limits for this species/strain and did not show a dose-effect. Also, no histopathological correlates were observed in any organ that may influence changes in these clinical chemistries.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Clinical chemistry and urinalysis values were considered unremarkable.
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Enlargement of the lymph nodes in some animals in the high dose group was considered a response to the secondary bacterial infection. Initial irritation of the skin resulted in self-inflicted wounds and other changes favoring secondary bacterial infection.

Leukocytes were elevated in both the male and female high-dose animals, and significant increases were noted in segmented neutrophils from animals in those groups. These changes were likely related to the bacterial infection observed at the treatment sites.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Occasional values for absolute organ weights or organ weight relative to body weight and/or brain weight were noted to be statistically significantly different from those of the control group.

The increase in heart/body weight ratio only in the 0.75 mL/kg/day male group was considered related to the decrease in body weight in this group compared with the controls.

Changes in organ weight indices for the heart in female rats lacked a clear dose response relationship. Similarly, increases in the 0.75 mL/kg/day female group kidney/body weight ratios and all liver and all liver organ weight parameters were considered the result of normal biological variation in the absence of microscopic changes in kidney and liver to account for the increases. These changes were associated with decreased body weights, were isolated, and were not dose-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological observations were seen that could be attributed to systemic toxicity of the test item. No toxicologic significance was attributed to increases in testes ratios in the 0.15 and 0.75 mL/kg/day groups in the absence of, changes in the absolute weight of testes, and of microscopic changes.

No microscopic changes in the heart of any significance were observed for female rats.No microscopic changes observed in kidney and liver.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic lesions associated with systemic toxicity were noted. Observations of the livers, hearts, kidneys, and testes indicated no lesions were present which would correlate with the sporadic changes in organ weights and ratios, or with the elevated serum biochemical values. Spleen and bone marrow tissues were within normal limits. Microscopic examination of the treated skin sites demonstrated lesions which correlated well with the incidence and severity of the macroscopic observations at these sites. The histopathological observations included: dermatitis, acanthosis, and an epidermal exudate composed of epidermal cells, bacteria, and red blood cells. No indication of skin corrosion was seen at any dosage.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOEL
Effect level:
ca. 665 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Remarks on result:
other: Based on dosage of 0.75 mL/kg/day
Dose descriptor:
NOEL
Effect level:
< 26.7 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Remarks on result:
other: Based on dosage of 0.03 mL/kg/day

By the third day, inflammation and irritation occurred at the application site in a dose related manner. There was no indication of corrosion at any dose level in this study. The 0.75 mL/kg/day level was considered an irritant to the intact skin. The 0.03 mL/kg/day level was considered a minimally irritant dose level. There was no indication of systemic toxicity at any dose level used in this study.

Conclusions:
Dermal administration of the test item on rats produced no significant observations of systemic toxicity. Treatment-site observations of scabs, abrasions, crusted material and thickening, and corresponding histological observations of dermatitis, acanthosis and epidermal exudate were noted with incidence and severity increasing in a dose-dependent manner. No evidence of corrosivity to skin was seen at any treatment level. Based on the results of this study, a no effect level for systemic toxicity is considered to be at least 0.75 mL/kg/day (ca. 665 mg/kg bw/day). A no effect level for dermal irritation was not established in this study and thus is considered to be below 0.03 mL/kg/day (ca. 26.7 mg/kg bw/day).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Nov 5, 1984 - Dec 3 to 4, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Remarks:
US EPA GLP, 1984
Limit test:
no
Specific details on test material used for the study:
- Identification: HLD (2,4-dicyclohexyl-2-methyl pentane)
- clear liquid
- Lot No.: G-15842
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan
- Age at study initiation: 7 wks old
- Weight at study initiation: males: 233 to 257 g; females: 155 to 169 g
- Fasting period before study: Overnight prior to start of study
- Housing: individually in wire mesh cages
- Diet (e.g. ad libitum): Certified Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 °C ± 0.6 °C
- Humidity (%): 49% ± 2.1%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: near the shoulder blades
- % coverage: 4 sq. in area (6-8%)
- Type of wrap if used: None
- Time intervals for shavings or clippings: Fur was shaved prior to initial dosing. Thereafter, shaving was performed as necessary to maintain the treatment area. Care was taken not to damage the treated skin area.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No washing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03, 0.15, 0.75 mL/kg/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: No (no attempts were made by the use of collars or other similar restraining procedures to prevent the rats from ingesting the test item)
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
A 5 g sample of the test article was collected after study termination and shipped under ambient conditions to the Sponsor (Monsanto) for analysis.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Administered dermally once daily, 5 days a week
Dose / conc.:
27 mg/kg bw/day
Remarks:
Calculated from dosage level of 0.03 mL/kg/day; based on specific gravity of test item of 0.886 g/cm3 at 25 deg C (from SDS).
Dose / conc.:
133 mg/kg bw/day
Remarks:
Calculated from dosage level of 0.15 mL/kg/day; based on specific gravity of test item of 0.886 g/cm3 at 25 deg C (from SDS).
Dose / conc.:
665 mg/kg bw/day
Remarks:
Calculated from dosage level of 0.75 mL/kg/day; based on specific gravity of test item of 0.886 g/cm3 at 25 deg C (from SDS).
No. of animals per sex per dose:
10 males and 10 females per dose group (40 males and 40 females in total)
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for mortality, moribundity, and gross signs of toxicologic and pharmacologic effects
- Detailed observations of appearance and condition, behavior and activity, excretory functions, respiration, orifices, eyes and palpation for masses were conducted at least once each week.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD CONSUMPTION:
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: On all rats pre-test and at week 4
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On all rats at 4 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters: Hematocrit, hemoglobin concentration, erythrocyte count, MCH, MCV and MCHC (calculated), total and differential leukocyte count, platelet count, reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On all rats at 4 weeks
- Animals fasted: No data
- How many animals: All
- Parameters: (Serum used) asparate aminotransferase, alanine aminotransferase, alkaline phosphatase, glucose, urea nitrogen, total bilirubin, total cholesterol, albumin, globulin (calculated), total protein, creatinine, sodium, potassium, chloride, calcium, phosphorus, creatine phosphokinase.

URINALYSIS: Yes
- Time schedule for collection of urine: On all rats at 4 weeks
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters: Volume, color and appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrites, bilirubin, occult blood, microscopy of spun deposit.
Sacrifice and pathology:
ANATOMIC PATHOLOGY
- macroscopic pathology: conducted on all rats. Tissues fixed in formalin (except eye, fixed in glutaraldehyde)
- organ weights: collected from all rats. Absolute and relative (to brain and to body weights) weights of adrenal (2), brain, heart, kidney (2), liver, testis (2)
- microscopic pathology: conducted on all rats. Full tissue examined for rats in 0and 0.75 mL/kg/day dosage levels. Liver, kidney (2), skin (treated and untreated) and lung (2) examined for rats from 0.03 and 0.15 mL/kg/day dosage levels.
Statistics:
Body weights, food consumption, absolute and relative organ weights and clinical laboratory values analyzed using Bartlett's test and analysis of variance. Treatment groups compared to the control group, by sex, using the appropriate t-statistic (equal or unequal variance) and Dunnett's multiple comparison tables. Nonparametric analysis of total bilirubin, chloride, urine specific gravity and volume by using rank transformations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations which were attributed to test item administration included red areas, scabbed areas, thickened skin and abraded areas. All these signs were evident on the test site of the rats. Red areas were most predominant and were first noted on study day 3. Abraded areas, which were quite large at times, and thickened skin were evident only at the 0.15 and 0.75 mL/kg/day dosage levels.

Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic lesions were observed at the dermal application sites in all treated groups. Scab formation was observed in all treated groups. These lesions tended to increase in both incidence and severity as dosage increased. Crust formation was observed in the 0.15 and 0.75 mL/kg/day groups. The incidence and severity of this lesion was greater in the 0.75 mL/kg/day group. Thickening of the skin at the application site was also observed in the 0.15 and 0.75 mL/kg/lay groups. The incidence and severity of this lesion was somewhat greater in the 0.75 mL/kg/day group. All of the above lesions (scab formation, crust formation and thickening of the skin) were considered to be direct effects of the test article. In addition, a dose response was evident.

Abrasions of the skin at the application site were observed in all treated groups. These changes tended to increase in both incidence and severity as dosage increased. Abrasions are mechanical injuries of the skin. In this case they most likely represent self-trauma; and, as such, were regarded as indirect effects of the test article. Some differences in the incidence and severity of macroscopic dermal lesions between males and females in all treated groups were observed. These differences were not considered meaningful.

Enlargement of the axillary and/or prescapular lymph nodes was observed in four males and nine females of the 0.75 mL/kg/day group. The difference in incidence of this change between males and females in this group was not considered meaningful. The changes in these lymph nodes were regarded as secondary to the development of lesions at the dermal application sites. As such, they were regarded as indirect effects of the test article.

All other macroscopic changes observed were regarded as incidental or spontaneous in nature. They were not considered to be related to the test article.

The principal lesions observed microscopically at the dermal application sites in all treated groups were epidermal exudates, dermatitis and acanthosis. Minor differences in incidence and severity between males and females were not considered meaningful.

The development of exudative epidermitis and underlying dermatitis was considered to be a direct effect of the test article. 'The incidence and severity of these lesions indicate a dose effect. The development of a bacterial dermal infection was regarded as a secondary complication of epidermal injury, as such an occurrence is not expected in otherwise normal skin. Acanthosis was also considered to be direct effect of the test article. The incidence and severity of this lesion, however, do not
indicate a dose effect.

Some degree of hyperkeratosis was seen in nearly all animals in all treated groups. In addition, this change was seen in nine males and two females in the 0 mL/kg/day group. Hyperkeratosis was also seen infrequently in untreated skin. There was no meaningful difference in severity. Hyperkeratosis is a nonspecific reaction of the epidermis to irritation. Its greater incidence in males in the 0 mL/kg/day group and in untreated skin probably reflects normal physiologic variation and anatomic location. The occurrence of hyperkeratosis in the 0 ml/kg/day group is most likely a result of mechanical factors, i.e., sham application to the skin. At least part of the hyperkeratosis in the treated groups, therefore, may be due to the same mechanical factors. The rats were not prevented from grooming the application site and thus probably some test article was ingested subsequent to this grooming activity.
Mortality:
no mortality observed
Description (incidence):
None of the animals in the study died or were sacrificed in extremis during the course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weights were slightly decreased in the 0.75 mL/kg/day male group over most of the study and in the female rats at this dose level during the last week of study. Body weights in the 0.03 and the 0.15 mL/kg/day dosage groups were considered comparable to those of the controls. Statistical significance was noted at weeks 2, 3, and 4 in the 0.75 mL/kg/day dose group.

Body weights of the 0.75 mL/kg/day male dosage group were depressed throughout the study and were 8% less than control values at the study termination. Meanwhile high-dose female animals had body weights 7% less than their respective controls at the end of the study.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Based on g of diet consumed/kg of body weight/day, food consumption appeared to be increased slightly in the 0.75 mL/kg/day male group and to a greater extent in the female rats at this dose level. Based on g of diet/animal/day, food consumption appeared generally comparable in all groups. Slight increases in food consumption were also seen in the 0.15 mL/kg groups.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test article related abnormalities were detected in the eye examinations. The observations that were noted were representative of pathology that would be expected for this group of animals considering age, sex and strain.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slight decreases in the erythrocyte count, hemoglobin and hematocrit were noted for the 0.75 mL/kg/day males and for hematocrit for the female rats at this dose level. Changes in total leukocyte counts and segmented neutrophil counts were consistent with a secondary bacterial infection at the high dose level.

No changes were noted in the calculated erythrocyte indices including MCV, MCH and MCHC, and reticulocytes were considered to be within normal limits and similar to control values.

Leukocytes were elevated in both the male and female high-dose animals, and significant increases were noted in segmented neutrophils from animals in those groups. These changes were likely related to the bacterial infection observed at the treatment sites. Similar findings were not reported for hematological parameters in the other treated groups. The changes reported in hematological values were not considered to be related to the test article administration.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry and urinalysis values were considered unremarkable.

Several serum biochemistry values were significantly different from controls at the high dosage. In the males these included an increase in alanine aminotransferase and a decrease in albumin and glucose. Female high-dose animals showed increases in globulin, aspartate aminotransferase, alanine aminotransferase and urea nitrogen with a decrease in glucose; although these changes were considered to be in the range of normal biological variability. The low-dose males also had significantly lower potassium values. The statistically significant values reported for serum biochemistry were not considered to be treatment-related since they were within the normal limits for this species/strain and did not show a dose-effect. Also, no histopathological correlates were observed in any organ that may influence changes in these clinical chemistries.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Clinical chemistry and urinalysis values were considered unremarkable.
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Enlargement of the lymph nodes in some animals in the high dose group was considered a response to the secondary bacterial infection. Initial irritation of the skin resulted in self-inflicted wounds and other changes favoring secondary bacterial infection.

Leukocytes were elevated in both the male and female high-dose animals, and significant increases were noted in segmented neutrophils from animals in those groups. These changes were likely related to the bacterial infection observed at the treatment sites.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Occasional values for absolute organ weights or organ weight relative to body weight and/or brain weight were noted to be statistically significantly different from those of the control group.

The increase in heart/body weight ratio only in the 0.75 mL/kg/day male group was considered related to the decrease in body weight in this group compared with the controls.

Changes in organ weight indices for the heart in female rats lacked a clear dose response relationship. Similarly, increases in the 0.75 mL/kg/day female group kidney/body weight ratios and all liver and all liver organ weight parameters were considered the result of normal biological variation in the absence of microscopic changes in kidney and liver to account for the increases. These changes were associated with decreased body weights, were isolated, and were not dose-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological observations were seen that could be attributed to systemic toxicity of the test item. No toxicologic significance was attributed to increases in testes ratios in the 0.15 and 0.75 mL/kg/day groups in the absence of, changes in the absolute weight of testes, and of microscopic changes.

No microscopic changes in the heart of any significance were observed for female rats.No microscopic changes observed in kidney and liver.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic lesions associated with systemic toxicity were noted. Observations of the livers, hearts, kidneys, and testes indicated no lesions were present which would correlate with the sporadic changes in organ weights and ratios, or with the elevated serum biochemical values. Spleen and bone marrow tissues were within normal limits. Microscopic examination of the treated skin sites demonstrated lesions which correlated well with the incidence and severity of the macroscopic observations at these sites. The histopathological observations included: dermatitis, acanthosis, and an epidermal exudate composed of epidermal cells, bacteria, and red blood cells. No indication of skin corrosion was seen at any dosage.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOEL
Effect level:
ca. 665 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Remarks on result:
other: Based on dosage of 0.75 mL/kg/day
Dose descriptor:
NOEL
Effect level:
< 26.7 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Remarks on result:
other: Based on dosage of 0.03 mL/kg/day

By the third day, inflammation and irritation occurred at the application site in a dose related manner. There was no indication of corrosion at any dose level in this study. The 0.75 mL/kg/day level was considered an irritant to the intact skin. The 0.03 mL/kg/day level was considered a minimally irritant dose level. There was no indication of systemic toxicity at any dose level used in this study.

Conclusions:
Dermal administration of the test item on rats produced no significant observations of systemic toxicity. Treatment-site observations of scabs, abrasions, crusted material and thickening, and corresponding histological observations of dermatitis, acanthosis and epidermal exudate were noted with incidence and severity increasing in a dose-dependent manner. No evidence of corrosivity to skin was seen at any treatment level. Based on the results of this study, a no effect level for systemic toxicity is considered to be at least 0.75 mL/kg/day (ca. 665 mg/kg bw/day). A no effect level for dermal irritation was not established in this study and thus is considered to be below 0.03 mL/kg/day (ca. 26.7 mg/kg bw/day).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Justification for classification or non-classification