Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In Life Phase: 5th November 1990 - 5th December 1990.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to GLP and OECD 410.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium Cocoyl Isethionate
- Molecular formula (if other than submission substance): Na.SO3-CH2-CH2-O-CO-R (Where R=C12-C14)
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: organic
- Physical state: solid
- Analytical purity: 72.4% active SCI
- Impurities (identity and concentrations): Free Fatty Acid: 18.2% (C12-C14 coconut fatty acid); Sodium Isethionate: 5.1%; Sodium Soap: 3.1%; Water: 1.3%
- Composition of test material, percentage of components: see above
- Isomers composition: not applicable
- Purity test date: 1991-03-14
- Lot/batch No.: 1247
- Expiration date of the lot/batch: not applicable
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: SCI was stable in aqueous suspension and the test article was stable during the study
- Storage condition of test material: refrigerated (4.4 - 7.2 degrees C), kept for 8 days then analysed

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
The rat was selected as the test subject since it is a well-recognized model in repeat dermal application studies. Fifty-two male and fifty-two female
young adult Charles River COBS CDR rats were purchased from Charles River Breeding Laboratories, Portage, MI. Males and females were purchased in a weight range of from 176 to 200 grams (approximately 5 to 7 weeks of age), and forty males and forty females were randomly selected for the study using the PSRNWT program of the URUS Subacute Toxicology Computer System which randomizes by body weight (±2 standard deviations) per sex in order to obtain similar averaged initial body weights for males and females per group.

Upon arrival, the animals were individually identified with consecutively numbered metallic ear tags, and housed individually in single wire-bottom cages. Animal husbandry and housing conformed to the standards recommended in the "Guide for Care and Use of Laboratory Animals." ) The test subjects were received at least two weeks prior to study initiation to allow for acclimation to laboratory conditions. All animals were observed on a daily basis during the acclimation period for signs of ill heath. An extensive microbiological profile, including viral profile was obtained for representative animals from the same animal colony and was included in the study file for this investigation. Environmental conditions were maintained within the following ranges, temperature 65°F to 78°F, relative humidity between 40% and 70%, and a light cycle of 12 hours on and 12 hours off. The animals were provided with Purina ground stock certified diet no. 5002 and water ad libitum via an automatic watering system upon arrival, during the acclimation period and throughout the investigation.

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Dorsal surface (neck to pelvis), 32cm2.
- % coverage: 32cm2 = 10% (350g rat). As rats gained weight to 400g, area increased to 40cm2 to maintain relative constant dose per area.
- Type of wrap if used: Porous gauze wrapped several times around test subject, secured with elastic tape.
- Time intervals for shavings or clipplings: Once per week.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): Held under warm tap water, rinsed and dried with paper towels.
- Time after start of exposure: 6 hours.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 ml
- Concentration (if solution): 1%, 14% and 36%
- Constant volume or concentration used: yes, constant volume of 1 ml.
- For solids, paste formed: yes - 1% concentration was an aqueous suspension, 14% concentration was a foaming suspension and 36% concentration was a cream


VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Amount(s) applied (volume or weight with unit): See above.
- Concentration (if solution): See above
- Lot/batch no. (if required): No data.
- Purity: No data.


USE OF RESTRAINERS FOR PREVENTING INGESTION: No.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to and during this investigation, test article characterization, stability and verification analyses were conducted by the Unilever Analytical Chemistry Section. The results of these analyses showed that 1) the test article was adequately characterized as SCI, 2) SCI was stable in aqueous suspension, 3) the test article aqueous suspensions were accurately prepared, and 4) the test article was stable during the 28 day study.
During the investigation, 1.0%, 14.0% and 36.0% (w/w) test article aqueous suspensions were prepared fresh on a weekly basis. After preparation an aliquot of each batch was obtained for analytical verification of the concentration, and the remaining batch was divided into seven aliquots for each day of treatment and stored in a refrigerator (40°-45°F). In order to demonstrate the stability of SCI under these storage condi tions, the remaining test article suspension after each day of treatment during the first week of the study was retained and submitted for analysis eight days after preparation.
A variation of ± 10% is considered to be acceptable.
Analysis using the Lever Standard Method has shown the test material to be 72.45% active (SCI). Using this level of activity, the dosages of SCI to be administered in the current investigation were revised as 0g/kg, 0.08g/kg, 0.91g/kg, and 2.07g/kg.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
once per day for 28 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 g/kg
Basis:
analytical per unit body weight
Remarks:
Doses / Concentrations:
0.08 g/kg
Basis:
analytical per unit body weight
Remarks:
Doses / Concentrations:
0.91 g/kg
Basis:
analytical per unit body weight
Remarks:
Doses / Concentrations:
2.07 g/kg
Basis:
analytical per unit body weight
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 10-day range-finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: 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 EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: Yes
- Time schedule for examinations: no data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes (identity) Carbon dioxide
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: all

URINALYSIS: No
- Time schedule for collection of urine: not applicable
- Metabolism cages used for collection of urine: not applicable
- Animals fasted: not applicable

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable
- Battery of functions tested: not applicable
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Not applicable
Statistics:
All raw and transformed data for body weight, food consumption, hematologic and clinical biochemistry tests and organ weights will be statistically analyzed by the URUS Computer and Applied Mathematics Group using SAS Version 6.06 statistical procedures and user written programs.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Gross examination of the test subjects during the twenty-eight day test failed to reveal any signs of systemic toxicity related to treatment with test item. One male test subject in the mid-dose test item-treated group died during the test... (see below)
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Daily observation of the treated sites prior to each treatment revealed only very slight erythema for four of the ten males in the high dose test item group during the third and fourth weeks of the test. Erythema at the treated sites was..(see below)
Mortality:
mortality observed, treatment-related
Description (incidence):
Gross examination of the test subjects during the twenty-eight day test failed to reveal any signs of systemic toxicity related to treatment with test item. One male test subject in the mid-dose test item-treated group died during the test... (see below)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although body weight gain for males in the high dose group was statistically lower than the vehicle control group during the second and third weeks of the test, examination of the individual data revealed that lower weight gains and weight...(see below)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was normal in all groups
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Averaged hematologic test parameters showed a statistically lower hemoglobin level in male test subjects in the high dose group, and a significantly lower averaged percent hematocrit for males in the mid-dose group but did not indicate...(see below)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Evaluation of the clinical biochemical test data revealed a slight decrease in serum glucose concentration for males in the high dose test item-treated group. However, examination of all other biochemical test data, especially the parameters...(see below)
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Evaluation of the organ weight data revealed a slight increase in averaged heart weight relative to final body weight for males, and an increase in averaged adrenal weight relative to final body weight for females in the high dose test item-treated group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathology at necropsy did not reveal any abnormal morphologic conditions considered to be related to treatment with test item.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic examination ofselected tissues and vital organs from all test subjects in the vehicle control and high dose groups did not reveal any abnormal pathology attributable to treatment with test item. One vehicle control female and one...(see be
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: Gross examination of the test subjects during the twenty-eight day test failed to reveal any signs of systemic toxicity related to treatment with test item. One male test subject in the mid-dose test item-treated group succumbed during the test due to mechanical trauma.

BODY WEIGHT AND WEIGHT GAIN: Although body weight gain for males in the high dose group was statistically lower than the vehicle control group during the second and third weeks of the test, examination of the individual data revealed that lower weight gains and weight loss occurred sporadically in each of the control and test item-treated test groups.

FOOD CONSUMPTION: food consumption was normal in all groups

FOOD EFFICIENCY: no data

WATER CONSUMPTION: no data

OPHTHALMOSCOPIC EXAMINATION: no data

HAEMATOLOGY: Averaged hematologic test parameters showed a statistically lower hemoglobin level in male test subjects in the high dose group, and a significantly lower averaged percent hematocrit for males in the mid-dose group but did not indicate a dosage-related response and the averaged data in both instances was within the historical control ranges for the Charles River CD rat. In addition, none of the other hematologic test data provided any evidence of an effect due to treatment with test item. Analysis of the hematologic test parameters for females failed to reveal any indication of a treatment-related response.

CLINICAL CHEMISTRY: Evaluation of the clinical biochemical test data revealed a slight decrease in serum glucose concentration for males in the high dose test item-treated group. However, examination of all other biochemical test data, especially the parameters for liver and kidney function failed to reveal any indication of an adverse effect due to treatment which could explain the slight decrease in serum glucose concentration observed for males in the high dose group.

URINALYSIS: no data

NEUROBEHAVIOUR: no data

ORGAN WEIGHTS: Evaluation of the organ weight data revealed a slight increase in averaged heart weight relative to final body weight for males, and an increase in averaged adrenal weight relative to final body weight for females in the high dose test item-treated group. Statistical analysis of the absolute organ weight data and organ weight relative to brain weight for heart and adrenal glands failed to reveal any significant differences among groups. In addition, organ weight relative to final body weight data for the heart and adrenal glands observed for high dose treated males and females, respectively were within the historical control ranges for Charles River CD rats of the same age, and histopathologic examination of the tissues failed to reveal any adverse morphology.

GROSS PATHOLOGY: Gross pathology at necropsy did not reveal any abnormal morphologic conditions considered to be related to treatment with test item.

HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathologic examination of skin from treated and untreated sites for all test subjects in the vehicle control and each of the low, mid- and high dose test item-treated groups revealed a slight thickening of the epithelium accompanied by a slight increase in cornification and mitotic rate in the basal cell layer at the treated sites when compared to untreated skin. These morphologic changes were similar among the control and treated groups indicating that the effects observed were due to the distilled water vehicle and the gauze wrap and not due to the test article. Microscopic examination of skin sections also showed that there was no indication of dermal inflammation due to topical application of test item. Based on these findings it was concluded that topical application of test item did not result in any adverse morphologic effects on skin or internal tissues in male or female rats.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): no effects

HISTORICAL CONTROL DATA (if applicable): not applicable

OTHER FINDINGS: Dermal irritancy: Daily observation of the treated sites prior to each treatment revealed only very slight erythema for four of the ten males in the high dose test item group during the third and fourth weeks of the test. Erythema at the treated sites was observed for females in each of the test item-treated groups and was significantly different from the vehicle control group on several days during the first week of the test. Very slight edema was also observed for females in the high dose group only during the first week. However, both the incidence of the test subjects responding and the severity of the responses decreased during the remainder of the investigation.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 2 070 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Under the conditions of the twenty-eight day dermal application study, daily topical dosages of test item as high as 2070 mg/kg was without significant adverse effect in the rat.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the twenty-eight day dermal application study, the "no observed adverse effect level" is 2070 mg/kg test item in the rat.
Executive summary:

The effects of the test item were investigated in a 28 day repeat dose dermal study in the rat.

Gross observation of the test subjects during the investigation failed to reveal any signs of systemic toxicity which could be attributed to treatment with test item. One male test subject in the mid-dose group succumbed during the test but the death was unrelated to the test article.

Dermal irritancy at the treated sites was observed for males and females predominantly in the high dose test item-treated group. Local irritancy was statistically significant (p < 0.05) for females only during the first week of the test. Thereafter, both the incidence and severity of the responses, in most instances considered to be very slight, decreased during the remainder of the test.

Statistical analysis of the body weight and food consumption data revealed a significant decrease (p < 0.01) in body weight gain for the high dose test item-treated-males during the second week of the study and again during the third week (p < 0.05) but not at the conclusion of the test. EvaluatIon of total body weight gain during the study did not reveal any statistical differences between the vehicle control and each of the test groups. Analysis of the body weight gain data for females failed to reveal any significant differences among group means. Averaged food consumption data for male and female test subjects did not indicate any adverse effect due to treatment with test item.

Evaluation of the hematologic test parameters revealed a decrease (p < 0.05) in averaged hemoglobin level for males in the-high dose group, although none of the other erythrocyte parameters for this group were affected, and the averaged hemoglobin data was within the historical control range for male Charles River CD rats. None of the other hematologic data for males or for female test subjects revealed any indication of a treatment-related effect.

Analysis of the clinical biochemical test data revealed a significant decrease (p < 0.05) in serum glucose for males in the high dose group. Analysis of all other biochemical test data, and histopathology failed to reveal any indication of an effect due to treatment with test item. Statistical analysis of the biochemical test data for females did not show any statistical differences among groups.

Evaluation of the absolute and relative organ weight data revealed a statistical increase (p < 0.05) in heart weight relative to final body weight for-males and adrenal weight relative to final body weight for females in the high dose group. Examination of these data

showed that the organ weights relative to final body weight were within the historical control ranges for this strain of rat, and histopathologic evaluation of these organs failed to reveal any morphologic alterations. In addition, statistical analysis of the absolute and relative organ weight to brain weight data for males and females failed to reveal any significant differences among groups.

Gross and microscopic pathology of selected tissues and vital organs excised from all test subjects in the vehicle control and high dose groups did not reveal any abnormal morphology attributable to treatment with test item. Histopathologic examination of skin sections obtained from the treated sites of all test subjects in the control and test article-treated groups showed a slight increase in the thickness and cornification of the epithelium accompanied by a slight increase in the mitotic rate of the basal cell layer. This finding was

similar among the control and test groups and unrelated to treatment with the test article.

Under the conditions of the twenty-eight day dermal application study, the "no observed adverse effect level" was determined to be 2070 mg/kg test item in the rat.