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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1992
Report Date:
1982
Reference Type:
publication
Title:
Unnamed
Year:
1994
Report Date:
1982
Reference Type:
publication
Title:
Unnamed
Year:
1987
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Principles of method if other than guideline:
The subchronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): C-178
- Physical state: liquid
- Analytical purity: 100% active ingredient
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 41 days (age: 28 days at receipt)
- Weight at study initiation: week 0 males: 157-163 g; week 0 females: 133-139 g
- Housing: individually in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
other: not occluded
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: the back of the rats
- % coverage: no data
- Type of wrap if used: not occluded no further data
- Time intervals for shavings or clipplings: all animals were clipped ca. 23 h prior to initial dose. The animals were reclipped when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.077 ml/kg
- Concentration (if solution): 1.0, 3.33 and 10.0 %
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): immiscible with water
- Amount(s) applied (volume or weight with unit): 2.077 ml 7kg of the test substance in corn oil
- Concentration (if solution): 1.0, 3.33 and 10.0 % of the test substance in corn oil

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
the remaining samples of weekly dosing solutions for each dose were returned to the sponsor for analysis, no further data
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 66.66, 200 mg/kg bw
Basis:
other: nominal conc.
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: for mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly including signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting)


OTHER:
- blood was obtained from over night fasted rats via venipuncture of the orbital sinus under light ether anestehsia, the same animals were used that were intended for formalin-fixation (5/sex/dose).
- hematology upon termination: hemoglobin, hematocrit, erythrocytes, clotting time, total and differential, leukocytes, erythrocytes morphology
- clinical chemistry upon termination: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, blood urea nitrogen, fasting glucose, total protein, total bilirubin, sodium, potassium, calcium, inorganic phosphorus
- urinanalysis 6 days before termination: gross appearance, specific gravity, pH, protein, glucose, occult blood
Sacrifice and pathology:
One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed
in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries,
spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused
intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were
performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.
Other examinations:
- neurologic functions were evaluated monthly
- Parameters examined according to a scoring system: posture, gait, muscular tone, reflexes (corneal), righting and toe-pinch
- no further data
Statistics:
Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of
all dose groups were compared to control at each time interval.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled termination of the study; weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control. The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study . Males were generally more susceptible than females to the dermal effects of the test substance
throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.

DERMAL OBSERVATIONS
In the controls dermal effects were absent except for one female rat which had one exfoliation score in Week 8, and one other female rat which
exhibited eschar and exfoliation at various times during the initial 2 months of the study.
In the low dose group, erythema was only noted occasionally during the final 2 months of the study. In the initial 3 weeks, erythema was recorded more frequently (approximately 12-13 % of total observations) than later in the study. Exfoliation was recorded with gradually diminishing frequency in approximately one-haft of the low dose group animals (both sexes) during weeks 2 and 3. During the last 5 weeks of the study, only two female rats and one male rat were observed with this effect. Edema, atonia, and fissuring were not observed in any low dose group animals.
In mid dose group male rats, erythema was noted with a same what higher frequency than was seen for low dose group male rats. Erythema frequency for female mid dose group rats was comparable to low dose group. After week 3 of the study, a somewhat lower frequency of erythema was recorded in both sexes. Exfoliation and eschar were recorded for most animals in the mid dose group by week 3 of the study, with diminishing frequency thereafter. As in the low dose group rats, edema, atonia and fissuring were not observed.
In the high dose group, erythema, exfoliation and eschar were seen in most animals of both sexes beginning in week 1. The highest frequency was
noted in week 2 (both sexes), with diminishing frequency thereafter. Atonia was observed in one male and one female during weeks 3-4 and 5-6,
respectively. Fissures were present in one female rat (on one day during week 2 only). Four male rats showed fissures on week 2 of the study.
A persistant fissuring in one of these rats was observed from week 2 through week 7 of the study. Male rats appeared somewhat more sensitive than females to erythema and eschar formation.

BODY WEIGHT AND WEIGHT GAIN
Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10
and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically
significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.

BIOCHEMISTRY
Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.

HEMATOLOGY
None of the hematologic parameters evaluated differed significantly from control values.

URINALYSIS
Protein (100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose
female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all the singular changes are not considered adverse by the registrant.

TERMINAL ORGAN and BODY WEIGHT AND ORGAN/BODY WEIGHT RATIOS
There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.

NEUROBEHAVIOUR
Month 1 neurological function tests showed two high-dose males with slightly reduced corneal resporse. All other evaluations were normal.
Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreasad corneal reflex was observed in four males and one female. A
moderately decreased toe pinch response (hindtoes only) was also present in one male rat.
Month 3 neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only) . All
neurological observations were normal in both the control and treated female rats at Month 3.

GROSS PATHOLOGY/HISTOPATHOLOGY
Formalin-fixed rats:
No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance. The most common
spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.

NEUROPATHOLOGY
Glutaraldehyde-perfused rats:
Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten rats
(5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar
ones from ten control rats (5/sex). In addition, microscopic examinetion of 50 teased nerve fibers from the tibial nerve of ten high-dose animals
(5/sex) were canparable to those of the controls. When quantitative measurements were taken of myelinated nerve tibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
skin irritation
Effect level:
20 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: skin irritation
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
66.66 mg/kg bw/day
Sex:
male
Basis for effect level:
other: significantly reduced body weights in high dose males
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
200 mg/kg bw/day
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion