Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1976
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Does not meet important criteria of today standard methods
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1976

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female New Zealand White rabbits were used in the study. 6 animals/sex were used in each group. The hair at the site of application on the back and sides of each animal was clipped short throughout the study. The thoracic-lumbar area (one on each side of the midline) was re alternated to minimize skin irritation. The application sites on three animals of each sex in each group were abraded on the first treatment day of each week.
Animals were observed for clinical signs, dermal irritation and body weight changes during the study period. Urine and blood samples were collected at 0, 3, 7, and 13 weeks of the study to determine the various parameters. Animals were sacrificed after 13 wk and examined for gross abnormalities. Tissue samples were collected to perform histology. Organ-body weight ratios were determined for liver, kidneys, adrenals, heart, thyroid, spleen, and brain.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: Two formulations containing 0.5% (P22) and 1% (P21) of N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate
- Substance type: Other
- Physical state: Not reported
No other information on details of formulations was provided in the study report.

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: No other information on test animals was provided in the study report.
- Housing: Animals were housed in cages.

ENVIRONMENTAL CONDITIONS: No details on environmental conditions were provided in the study report.

Administration / exposure

Type of coverage:
open
Vehicle:
water
Details on exposure:
TEST SITE: The thoracic-lumbar area (one on each side of the midline) was alternated to minimize skin irritation.
- Area of exposure: Not reported
- % coverage: Not reported
- Type of wrap if used: Not reported
- Time intervals for shavings or clipplings: The hair at the site of application on the back and sides of each animal was clipped short throughout the study.
- The application sites on three animals of each sex in each group were abraded on the first treatment day of each week.

REMOVAL OF TEST SUBSTANCE
- Washing: Animals were shampooed, rinsed, dried, and returned to their cages.
- Time after start of exposure: 1 h

TEST MATERIAL
- Amount applied: 1 mL/Kg bw, this was the maximum dose that could be applied on the side of the rabbits without runoff.
- Concentration: Formulation 1 (P21) and 2 (P22) contained 1% and 0.5% of test substance, respectively. Formulation 1 containing test substance (1%) was mixed with an equal volume of 6% hydrogen peroxide just before use.
- Constant volume or concentration used: Yes

VEHICLE: Water

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, the animals were restrained in holding stocks for 1 h following each application.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Twice/week
Doses / concentrationsopen allclose all
Remarks:
Prior to treatment, formulation 1 was mixed with equal volume of 6% hydrogen peroxide.
No. of animals per sex per dose:
6 animals/sex/group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose selected was the maximum dose that could be applied on the side of the rabbits without runoff.
- Rationale for animal assignment: Not reported

TREATMENT: The treatment schedule was as follows:
- Control 1, 2 and 3: Animals were shaved, not treated.
- Group 1: Formulation 1 (P21) containing 1% of test substance was prepared. Prior to treatment, the formulation was mixed with equal volume of 6% hydrogen peroxide.
- Group 2: Formulation 2 (P22) contained 0.5% of test substance.

Examinations

Observations and examinations performed and frequency:
MORTALITY: Yes
- Time schedule: Time schedule was not specified in the study report.

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: Details on time schedule on observation of dermal irritation was not provided in the study report.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 0, 3, 7, and 13 week of the study, the blood was collected by cardiac puncture
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Complete blood count (HCT, Hb, RBC, WBC and Meb-Hb)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 0, 3, 7, and 13 week of the study. The clinical chemistry determinations were performed on blood obtained by cardiac puncture by SMA-12 analysis using the Technicon auto analyzer.
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Methemoglobin, fasting blood sugar, blood urea nitrogen, alkaline phosphatase and serum glutamic oxaloacetic transaminase

URINALYSIS: Yes
- Time schedule for collection of urine: At 0, 3, 7, and 13 week of the study.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Color, pH, albumin, glucose, occult blood and microscopic elements

NEUROBEHAVIOURAL EXAMINATION: No

ORGAN TO WEIGHT RATIO: Organ-body weight ratios were determined for liver, kidneys, adrenals, heart, thyroid, spleen, and brain.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all survivors were sacrificed after 13 wk and examined for gross abnormalities.

HISTOPATHOLOGY: Yes
- Tissues collected: Twenty-five tissues {skin from treated and untreated areas, lymph nodes (mesenteric, axillary, and cervical), spleen, stomach, duodenum, colon, liver, gall bladder, adrenals, nerve with adjacent muscle, eyes, pancreas, kidneys, urinary bladder, ovaries, testes, bone, bone marrow, heart, lung, thyroid, brain, and skeletal muscle under the site of application and elsewhere (thigh) from each animal were stained with hematoxylin and eosin and examined microscopically.
Statistics:
- Statistical analysis of the data on body weight gains, hematology, clinical chemistries, and absolute and relative organ weights was performed using the analysis of variance F test and Student's t test.
- When variances differed significantly, Student's t test was modified (t') and Cochran's approximation was utilized (Snedecor and Cochran, 1967).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
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:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
- Five control and five test animals died during the study due to complications resulting from cardiac puncture while collecting blood.

CLINICAL SIGNS:
- No evidence of test substance induced toxicity was seen. The treated skin showed slight thickening in some groups, particularly Group 1. This was not unexpected, due to the frequency of dye application.

BODY WEIGHT AND WEIGHT GAIN
- Body weight gain of all test groups was at least equal to that of the controls.

HAEMATOLOGY
- There were scattered statistically significant differences in the hematologic values between test and control groups at the various sampling intervals. However, these differences were not considered to be of toxicologically significant because of either the direction or continuity of the differences or the fact that they fell within the range of historical control values.

CLINICAL CHEMISTRY
- There were scattered statistically significant differences in the clinical chemistry values between test and control groups at the various sampling intervals. However, these differences were not considered to be of toxicologically significant because of either the direction or continuity of the differences or the fact that they fell within the range of historical control values. These symptoms were not accompanied with histomorphologic changes.

URINALYSIS
- The results of the urinalyses were unremarkable. No dye discoloration of the urine was seen at any time during the test.

GROSS PATHOLOGY
- No gross abnormalities were seen at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
- No microscopic lesions were seen that were judged to be due to the administration of the formulations.

ORGAN TO BODY WEIGHT RATIO:
- There were a few instances when there were statistically significant differences in relative organ weights between a test group and the combined controls where the differences were not significant when the group was compared with each control group separately.

OTHER
- The incidence and severity of disease processes was common to laboratory rabbits and it was not affected by the experimental treatments.

Effect levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Dermal application of P21 formulation containing 1% N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate mixed with equal volume of hydrogen peroxide and dermal applicaton of P22 formulation containing 0.5% of test substance to hair clipped skin of rabbits for 13 weeks (twice/week) did not induce any evidence of systemic toxicity.
Executive summary:

The study was performed to determine the toxicity of N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate after repeated dermal application by following methods similar to the OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study).

Male and female New Zealand White rabbits were used in the study. 6 animals/sex were used in each group. The hair at the site of application on the back and sides of each animal was clipped short throughout the study. The thoracic-lumbar area (one on each side of the midline) was re alternated to minimize skin irritation. The application sites on three animals of each sex in each group were abraded on the first treatment day of each week.


1 mL/Kg bw of test substance formulation was applied. Animals were treated for 13 weeks (twice/week). The treatment schedule was as follows:

Control 1, 2 and 3: Animals were shaved, not treated.

Group 1: Formulation 1 (P21) containing 1% of test substance was prepared. Prior to treatment, the formulation was mixed with equal volume of 6% hydrogen peroxide.

Group 2: Formulation 2 (P22) contained 0.5% of test substance

After 1 h of treatment, animals were shampooed, rinsed, dried, and returned to their cages.

Animals were observed for clinical signs, dermal irritation and body weight changes during the study period. Urine and blood samples were collected at 0, 3, 7, and 13 weeks of the study to determine the various parameters. Animals were sacrificed after 13 wk and examined for gross abnormalities. Tissue samples were collected to perform histology. Organ-body weight ratios were determined for liver, kidneys, adrenals, heart, thyroid, spleen, and brain.

Five control and five test animals died during the study due to complications resulting from cardiac puncture while collecting blood. Treated skin showed slight thickening in some groups, particularly Group 1. This was not unexpected, due to the frequency of dye application. No evidence of test substance induced toxicity was observed. Body weight gain of all test groups was at least equal to that of the controls.

There were scattered statistically significant differences in the hematologic and clinical chemistry values between test and control groups at the various sampling intervals. However, these differences were not considered to be of toxicological significance because of either the trend or persistence of the differences or the fact that they fell within the range of historical control values. The results of the urinalyses were unremarkable.

No gross abnormalities were seen at necropsy. No microscopic lesions were seen that were judged to be due to the administration of the formulations.

Based on above, dermal application of P21 formulation containing 1% N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate mixed with equal volume of hydrogen peroxide and dermal application of P22 formulation containing 0.5% of test substance to hair clipped skin of rabbits for 13 weeks (twice/week) did not induce any evidence of systemic toxicity.