Hydrocarbons, C13-18

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to guideline study OECD 411: GLP.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to guideline study OECD 411: GLP.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.

Type of coverage:

semiocclusive

Vehicle:

other: unknown

Details on exposure:

Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing were conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here]. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

Duration of treatment / exposure:

This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks.

Frequency of treatment:

This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks.

Remarks:

Doses / Concentrations:
0, 165, 330 or 495 mg/kg/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

Groups of 12 male and 12 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.
Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing was conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here]. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

The following hematological and clinical chemical parameters were measured.
HEMATOLOGY:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Reticulocyte count
Total leukocyte count
Differential leukocyte count
Morphological examination of erythrocytes and platelets Coagulation determinations (prothrombin time & activated partial thromboplastin time) were also carried out on six animals from each group at week 14 and from the recovery groups at the week 18 necropsy.

CLINICAL CHEMISTRY
Blood urea nitrogen,
Creatinine,
Serum aspartate aminotransferase,
Serum alanine aminotransferase,
Alkaline phosphatase,
Lactate dehydrogenase,
Sorbitol dehydrogenase,
Gamma glutamyl transferase,
Creatinine kinase,
Serum glucose
Total, direct and indirect bilirubin
Total protein
Albumin
Calcium
Phosphorus
Sodium
Potassium
Chloride
A complete necropsy was performed on six rats/sex/group following 13 weeks dosing, and on 6 rats/sex/group of the recovery animals (high dose and controls) at week 18. A limited necropsy was performed on the remaining six animals and their organs were not weighed (see below). Each full necropsy included an examination of the external surface of the body, all orifices, cranial, thoracic, abdominal and pelvic cavities and their contents. Gross observations were recorded and the following organs were weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, spleen, testes, thymus and uterus.

The following tissues were collected, processed and then examined microscopically.
Adrenal glands,
Nose (nasal cavity & turbinates),
Animal identification,
Ovaries,
Bone marrow (from sternum),
Oviducts,
Brain,
Pancreas,
Epididymides,
Parathyroid glands,
Esophagus,
Pituitary gland,
Exorbital lacrimal glands,
Prostate Eyes with optic nerve,
Salivary glands,
Femur (incl. articular surface),
Seminal vesicles,
Gross lesions Skin (application site),
Harderian gland,
Skin (inguinal),
Heart and aorta,
Spinal cord (3 levels),
Intestine (3 levels),
Spleen,
Kidneys,
Stomach,
Larynx and pharynx,
Testes,
Liver,
Thymus,
Lungs with mainstream bronchi,
Thyroid gland,
Lymph nodes (mandibular/mesenteric),
Urinary bladder,
Mammary glands with adjacent skin,
Uterus Muscle (thigh),
Vagina Nerve (sciatic).

The remaining six rats of each group were anesthetized with an intraperitoneal injection of Pentothal ® and transcardially perfused in-situ using 10% neutral-buffered formalin and given a limited necropsy. For these rats, no organs were weighed and the following tissues were collected: Head/skull, Sural nerve, Brain, Tibial nerve, Spinal cord, Gross lesions, Sciatic nerve.

The following tissues were examined microscopically in these animals: Brain (forebrain, cerebrum, midbrain, cerebellum, pons and medulla obligata), Gasserian ganglia, Dorsal root ganglia, Dorsal and ventral root fibers, Sural nerve, Tibial nerve, Spinal cord (cervical and lumbar areas), and Sciatic nerve.

Observations and examinations performed and frequency:

Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.
Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing was conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here].

Sacrifice and pathology:

At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

The following hematological and clinical chemical parameters were measured.
HEMATOLOGY:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Reticulocyte count
Total leukocyte count
Differential leukocyte count
Morphological examination of erythrocytes and platelets Coagulation determinations (prothrombin time & activated partial thromboplastin time) were also carried out on six animals from each group at week 14 and from the recovery groups at the week 18 necropsy.

CLINICAL CHEMISTRY
Blood urea nitrogen,
Creatinine,
Serum aspartate aminotransferase,
Serum alanine aminotransferase,
Alkaline phosphatase,
Lactate dehydrogenase,
Sorbitol dehydrogenase,
Gamma glutamyl transferase,
Creatinine kinase,
Serum glucose
Total, direct and indirect bilirubin
Total protein
Albumin
Calcium
Phosphorus
Sodium
Potassium
Chloride
A complete necropsy was performed on six rats/sex/group following 13 weeks dosing, and on 6 rats/sex/group of the recovery animals (high dose and controls) at week 18. A limited necropsy was performed on the remaining six animals and their organs were not weighed (see below). Each full necropsy included an examination of the external surface of the body, all orifices, cranial, thoracic, abdominal and pelvic cavities and their contents. Gross observations were recorded and the following organs were weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, spleen, testes, thymus and uterus.

The following tissues were collected, processed and then examined microscopically.
Adrenal glands,
Nose (nasal cavity & turbinates),
Ovaries,
Bone marrow (from sternum),
Oviducts,
Brain,
Pancreas,
Epididymides,
Parathyroid glands,
Esophagus,
Pituitary gland,
Exorbital lacrimal glands,
Prostate Eyes with optic nerve,
Salivary glands,
Femur (incl. articular surface),
Seminal vesicles,
Gross lesions Skin (application site),
Harderian gland,
Skin (inguinal),
Heart and aorta,
Spinal cord (3 levels),
Intestine (3 levels),
Spleen,
Kidneys,
Stomach,
Larynx and pharynx,
Testes,
Liver,
Thymus,
Lungs with mainstream bronchi,
Thyroid gland,
Lymph nodes (mandibular/mesenteric),
Urinary bladder,
Mammary glands with adjacent skin,
Uterus Muscle (thigh),
Vagina Nerve (sciatic).

The remaining six rats of each group were anesthetized with an intraperitoneal injection of Pentothal ® and transcardially perfused in-situ using 10% neutral-buffered formalin and given a limited necropsy. For these rats, no organs were weighed and the following tissues were collected: Head/skull, Sural nerve, Brain, Tibial nerve, Spinal cord, Gross lesions, Sciatic nerve.

The following tissues were examined microscopically in these animals: Brain (forebrain, cerebrum, midbrain, cerebellum, pons and medulla obligata), Gasserian ganglia, Dorsal root ganglia, Dorsal and ventral root fibers, Sural nerve, Tibial nerve, Spinal cord (cervical and lumbar areas), and Sciatic nerve.

Statistics:

Statistics Normally-distributed in-life data (parametric) were analyzed for test substance effects by analysis of variance and pairwise comparisons made between groups using Dunnett's test. Nonparametric data (non-homogeneous as determined by Bartlett's test) were analyzed using a modified t-test. Statistical significance was reported at the P < 0.05 level. Statistical analyses of neurobehavior data (FOB and motor activity) are described in the results section.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no test substance-related effects on survival, clinical observations (apart from skin irritation), neurobehavioral signs or ophthalmological findings. The only clinical observations during the study were related to skin irritation at the application site. There was a generally dose-related increase in the incidence and severity of erythema, edema, epidermal scaling, scab formation, thickening of the skin and ulceration at the treated site. Males seemed to be more sensitive than females. The FOB screen did not demonstrate any substance-related effects. The areas monitored were: behavioral parameters, including autonomic, muscle tone and equilibrium, sensorimotor responses, central nervous system. In addition the test substance had little effect on motor activity or startle response. Growth rates were unaffected by treatment. At necropsy no substance-related observations were made for males in any group. In the females there was a suggestion of a possible treatment- related effect which occurred in 7 rats across all groups and consisted of skin crusts or ulceration at the site of application of test material. Hematological and serum clinical parameters were unaffected by treatment. The only organ weight effects noted were an increase in spleen/body weight and spleen/brain weight ratios in the high dose group females at the 13 week necropsy and an increase in absolute spleen weight in the same dose group females after the 4 weeks recovery period. Since there were no associated microscopic or clinical chemical findings, these differences were not considered to be of biological relevance. There were no treatment-related microscopic changes in the tissues examined with the exception of the findings in the skin. The skin observations were minimal in nature with a severity score less than 1 on a 1 [low] to 4 [severe] scale. The findings included acanthosis, ulceration, parakeratosis, chronic active inflammation and hyperkeratosis. The males were affected at all doses, however, the effects indicated very little irritation. Recovery group animals revealed complete recovery in the females and minimal hyperkeratosis in the high dose group males. No effects were found in the animals subjected to a detailed neuropathological examination.

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

> 495 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No systemic or neurological effects were noted in the study

Critical effects observed:

not specified

Conclusions:

There were no systemic or neurological effects noted at any of the tested doses. The systemic NOAEL was >495 mg/kg/day.

Executive summary:

Test material was applied at concentrations of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age was administered mineral oil as vehicle controls and an additional high dose group was maintained for a 4-week recovery period following dosing for 13 weeks. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups). There were no systemic or neurological effects noted at any of the tested doses. The systemic NOAEL was >495 mg/kg/day.

Data source

Reference

Reference 1

Reference Type:

other: HPV Summary

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydrodesulfurized kerosene

IUPAC Name:

Hydrodesulfurized kerosene

Constituent 2

Reference substance name:

8006-20-6

IUPAC Name:

8006-20-6

Details on test material:

Hydrodesulfurized kerosene

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: unknown

Details on exposure:

Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing were conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here]. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

Duration of treatment / exposure:

This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks.

Frequency of treatment:

This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 165, 330 or 495 mg/kg/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

Groups of 12 male and 12 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.
Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing was conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here]. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

The following hematological and clinical chemical parameters were measured.
HEMATOLOGY:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Reticulocyte count
Total leukocyte count
Differential leukocyte count
Morphological examination of erythrocytes and platelets Coagulation determinations (prothrombin time & activated partial thromboplastin time) were also carried out on six animals from each group at week 14 and from the recovery groups at the week 18 necropsy.

CLINICAL CHEMISTRY
Blood urea nitrogen,
Creatinine,
Serum aspartate aminotransferase,
Serum alanine aminotransferase,
Alkaline phosphatase,
Lactate dehydrogenase,
Sorbitol dehydrogenase,
Gamma glutamyl transferase,
Creatinine kinase,
Serum glucose
Total, direct and indirect bilirubin
Total protein
Albumin
Calcium
Phosphorus
Sodium
Potassium
Chloride
A complete necropsy was performed on six rats/sex/group following 13 weeks dosing, and on 6 rats/sex/group of the recovery animals (high dose and controls) at week 18. A limited necropsy was performed on the remaining six animals and their organs were not weighed (see below). Each full necropsy included an examination of the external surface of the body, all orifices, cranial, thoracic, abdominal and pelvic cavities and their contents. Gross observations were recorded and the following organs were weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, spleen, testes, thymus and uterus.

The following tissues were collected, processed and then examined microscopically.
Adrenal glands,
Nose (nasal cavity & turbinates),
Animal identification,
Ovaries,
Bone marrow (from sternum),
Oviducts,
Brain,
Pancreas,
Epididymides,
Parathyroid glands,
Esophagus,
Pituitary gland,
Exorbital lacrimal glands,
Prostate Eyes with optic nerve,
Salivary glands,
Femur (incl. articular surface),
Seminal vesicles,
Gross lesions Skin (application site),
Harderian gland,
Skin (inguinal),
Heart and aorta,
Spinal cord (3 levels),
Intestine (3 levels),
Spleen,
Kidneys,
Stomach,
Larynx and pharynx,
Testes,
Liver,
Thymus,
Lungs with mainstream bronchi,
Thyroid gland,
Lymph nodes (mandibular/mesenteric),
Urinary bladder,
Mammary glands with adjacent skin,
Uterus Muscle (thigh),
Vagina Nerve (sciatic).

The remaining six rats of each group were anesthetized with an intraperitoneal injection of Pentothal ® and transcardially perfused in-situ using 10% neutral-buffered formalin and given a limited necropsy. For these rats, no organs were weighed and the following tissues were collected: Head/skull, Sural nerve, Brain, Tibial nerve, Spinal cord, Gross lesions, Sciatic nerve.

The following tissues were examined microscopically in these animals: Brain (forebrain, cerebrum, midbrain, cerebellum, pons and medulla obligata), Gasserian ganglia, Dorsal root ganglia, Dorsal and ventral root fibers, Sural nerve, Tibial nerve, Spinal cord (cervical and lumbar areas), and Sciatic nerve.

Examinations

Observations and examinations performed and frequency:

Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.
Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing was conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here].

Sacrifice and pathology:

At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

The following hematological and clinical chemical parameters were measured.
HEMATOLOGY:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Reticulocyte count
Total leukocyte count
Differential leukocyte count
Morphological examination of erythrocytes and platelets Coagulation determinations (prothrombin time & activated partial thromboplastin time) were also carried out on six animals from each group at week 14 and from the recovery groups at the week 18 necropsy.

CLINICAL CHEMISTRY
Blood urea nitrogen,
Creatinine,
Serum aspartate aminotransferase,
Serum alanine aminotransferase,
Alkaline phosphatase,
Lactate dehydrogenase,
Sorbitol dehydrogenase,
Gamma glutamyl transferase,
Creatinine kinase,
Serum glucose
Total, direct and indirect bilirubin
Total protein
Albumin
Calcium
Phosphorus
Sodium
Potassium
Chloride
A complete necropsy was performed on six rats/sex/group following 13 weeks dosing, and on 6 rats/sex/group of the recovery animals (high dose and controls) at week 18. A limited necropsy was performed on the remaining six animals and their organs were not weighed (see below). Each full necropsy included an examination of the external surface of the body, all orifices, cranial, thoracic, abdominal and pelvic cavities and their contents. Gross observations were recorded and the following organs were weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, spleen, testes, thymus and uterus.

The following tissues were collected, processed and then examined microscopically.
Adrenal glands,
Nose (nasal cavity & turbinates),
Ovaries,
Bone marrow (from sternum),
Oviducts,
Brain,
Pancreas,
Epididymides,
Parathyroid glands,
Esophagus,
Pituitary gland,
Exorbital lacrimal glands,
Prostate Eyes with optic nerve,
Salivary glands,
Femur (incl. articular surface),
Seminal vesicles,
Gross lesions Skin (application site),
Harderian gland,
Skin (inguinal),
Heart and aorta,
Spinal cord (3 levels),
Intestine (3 levels),
Spleen,
Kidneys,
Stomach,
Larynx and pharynx,
Testes,
Liver,
Thymus,
Lungs with mainstream bronchi,
Thyroid gland,
Lymph nodes (mandibular/mesenteric),
Urinary bladder,
Mammary glands with adjacent skin,
Uterus Muscle (thigh),
Vagina Nerve (sciatic).

The remaining six rats of each group were anesthetized with an intraperitoneal injection of Pentothal ® and transcardially perfused in-situ using 10% neutral-buffered formalin and given a limited necropsy. For these rats, no organs were weighed and the following tissues were collected: Head/skull, Sural nerve, Brain, Tibial nerve, Spinal cord, Gross lesions, Sciatic nerve.

The following tissues were examined microscopically in these animals: Brain (forebrain, cerebrum, midbrain, cerebellum, pons and medulla obligata), Gasserian ganglia, Dorsal root ganglia, Dorsal and ventral root fibers, Sural nerve, Tibial nerve, Spinal cord (cervical and lumbar areas), and Sciatic nerve.

Statistics:

Statistics Normally-distributed in-life data (parametric) were analyzed for test substance effects by analysis of variance and pairwise comparisons made between groups using Dunnett's test. Nonparametric data (non-homogeneous as determined by Bartlett's test) were analyzed using a modified t-test. Statistical significance was reported at the P < 0.05 level. Statistical analyses of neurobehavior data (FOB and motor activity) are described in the results section.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no test substance-related effects on survival, clinical observations (apart from skin irritation), neurobehavioral signs or ophthalmological findings. The only clinical observations during the study were related to skin irritation at the application site. There was a generally dose-related increase in the incidence and severity of erythema, edema, epidermal scaling, scab formation, thickening of the skin and ulceration at the treated site. Males seemed to be more sensitive than females. The FOB screen did not demonstrate any substance-related effects. The areas monitored were: behavioral parameters, including autonomic, muscle tone and equilibrium, sensorimotor responses, central nervous system. In addition the test substance had little effect on motor activity or startle response. Growth rates were unaffected by treatment. At necropsy no substance-related observations were made for males in any group. In the females there was a suggestion of a possible treatment- related effect which occurred in 7 rats across all groups and consisted of skin crusts or ulceration at the site of application of test material. Hematological and serum clinical parameters were unaffected by treatment. The only organ weight effects noted were an increase in spleen/body weight and spleen/brain weight ratios in the high dose group females at the 13 week necropsy and an increase in absolute spleen weight in the same dose group females after the 4 weeks recovery period. Since there were no associated microscopic or clinical chemical findings, these differences were not considered to be of biological relevance. There were no treatment-related microscopic changes in the tissues examined with the exception of the findings in the skin. The skin observations were minimal in nature with a severity score less than 1 on a 1 [low] to 4 [severe] scale. The findings included acanthosis, ulceration, parakeratosis, chronic active inflammation and hyperkeratosis. The males were affected at all doses, however, the effects indicated very little irritation. Recovery group animals revealed complete recovery in the females and minimal hyperkeratosis in the high dose group males. No effects were found in the animals subjected to a detailed neuropathological examination.

Effect levels

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

> 495 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No systemic or neurological effects were noted in the study

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

There were no systemic or neurological effects noted at any of the tested doses. The systemic NOAEL was >495 mg/kg/day.

Executive summary:

Test material was applied at concentrations of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age was administered mineral oil as vehicle controls and an additional high dose group was maintained for a 4-week recovery period following dosing for 13 weeks. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups). There were no systemic or neurological effects noted at any of the tested doses. The systemic NOAEL was >495 mg/kg/day.