Registration Dossier

Administrative data

Description of key information

There are Klimisch 1 28 day oral (dietary) and dermal studies, and there is a Klimisch 1 90 oral gavage study on sodium 2-hydroxyethanesulfonate  (sodium isethionate) the potentially toxic metabolite of Coco fatty acids 2-sulfoethyl ester, sodium salt, which can be used as a basis for read across to a 90 NOAEL for Coco fatty acids 2-sulfoethyl ester.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2008-08-14 till 2009-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Füllinsdorf / Switzerland
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation:
Males: 183 g to 202 g (mean 194 g)
Females: 138 g to 156 g (mean 146 g)
- Fasting period before study: for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat / mouse maintenance diet, ad libitum
- Water (e.g. ad libitum): Community tap-water from Itingen was available, ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%).
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Purity: bidistilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After experimental start, samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration and stability. Samples of about 2 g of each concentration were taken during week 3, 8 and 13 after commencement of dosing to confirm homogeneity and concentration. The test item was used as analytical standard.
The test item concentrations were determined by IC coupled to a conductivity detector. The content was determined by external standard quantitation referring to the area under the test item peak.
Duration of treatment / exposure:
91/92 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Group 1: 15 males and 15 females (control group, where 10 animals were satellite A and the other 5 animals formed satellite B)
Group 2: 10 males and 10 females
Group 3: 10 males and 10 females
Group 4: 15 males and 15 females ( 10 animals were satellite A and the other 5 animals formed satellite B)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (Harlan Laboratories Study B96974).
- Rationale for selecting satellite groups: the animals were allowed to a 28-day treatment-free recovery period
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once before commencement of administration as well as twice daily on days 1 to 3, once daily from on day 4 to the end of the treatment period, and once daily during days 1 to 28 (recovery period).
- Cage side observations checked: appearance (piloerection, salivation, hunched posture), motor (ataxia, tremor, prostration), behaviour (hyperactivity, somnolence), respiration (dyspnea, tachypnea, bradypnea)


BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment and recovery periods and before necropsy


FOOD CONSUMPTION:
once during the acclimatization period and weekly thereafter


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once in all animals during acclimatization period; once in animals of the control and high dose groups, as well as in animals of the middle dose groups if test item-related changes were seen in the high dose (treatment period); once in the remaining animals of the control and high dose groups during the recovery period.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the dosing period, and after 17 weeks (satellite B animals)
- Anaesthetic used for blood collection: Yes (under light isoflurane anesthesia)
- Animals fasted: Yes (for approximately 18 hours before blood sampling but allowed access to water ad libitum)
- How many animals: all group animals at week 13, while 6 males and 6 females/group after 17 weeks, only groups 0 and 4
- Parameters checked in table No 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the dosing period, and after 17 weeks (satellite B animals)
- Animals fasted: Yes (for approximately 18 hours before blood sampling but allowed access to water ad libitum)
- How many animals: all group animals at week 13, while 6 males and 6 females/group after 17 weeks, only groups 0 and 4
- Parameters checked in table No. 2 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: during the 18 hours fasting period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No. 3 were examined.


NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- functional observational battery (week 13)
- grip strength (week 13)
- locomotor activity (week 13)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 4)
HISTOPATHOLOGY: Yes (see table No. 4)

Statistics:
The following statistical methods were used to analyze body weight, locomotor activity, fore- and hindlimb grip strength, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
- The Dunnett-test
- The Steel-test
- Fisher's exact-test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats survived until scheduled necropsy.
No test item-related clinical signs of toxicological relevance were noted during daily observations or weekly observations (weeks 1 - 12) at any dose level.

BODY WEIGHT AND WEIGHT GAIN
A trend to slightly lower mean absolute and relative body weights values were ascertained in males treated with 1000 mg/kg/day when compared with controls. This was considered to be a mild test item-related change. All other animals were unaffected.

FOOD CONSUMPTION
No differences of toxicological relevance were noted in the mean daily food consumption.


OPHTHALMOSCOPIC EXAMINATION
None of the ophthalmoscopic findings noted after 13 weeks’ treatment in rats treated with 1000 mg/kg/day were considered to be related to the test item.

HAEMATOLOGY
A number of test item-related differences were noted in the hematology parameters of the rats treated with 1000 mg/kg/day. These changes were manifested by reduced mean corpuscular hemoglobin concentration values, elevated mean absolute and relative reticulocyte counts, and a ‘left-shift’ in the reticulocyte maturity indices indicative of increased reticulocyte turnover. Females treated with 1000 mg/kg/day also had reduced hemoglobin distribution width. All remained within the upper limits of the historical control data. Both sexes showed slightly elevated methemoglobin. In the absence of differences which exceeded the historical control values and/or Heinz body formation, these were considered to be of no toxicological relevance The remaining changes noted in animals at 1000 mg/kg/day (elevated mean absolute lymphocytes and monocytes in females) did not exceed the ranges of the historical control values or were not accompanied by concomitant changes in related parameters and were therefore considered to be incidental. All changes noted at 200 mg/kg/day or at 50 mg/kg/day were considered to be incidental. No test item-related findings remained nor were late effects in the animals previously treated with 1000 mg/kg/day noted after 4 weeks of recovery.

CLINICAL CHEMISTRY
Test item-related changes in the parameters of the clinical biochemistry were noted, and included reduced glucose levels in males and females at 1000 mg/kg/day. Although some outlying values were found, the overall mean lactate dehydrogenase activity was generally higher in males and females at 1000 mg/kg/day. The mean total bilirubin level was elevated in males and females at 1000 mg/kg/day when compared with the respective contr ls, possibly indicating some degree of cell friability. Elevated mean cholesterol levels and phospholipid levels were noted in males, whereas these
parameters were only marginally elevated in females at this dose level. In rats treated previously with 1000 mg/kg/day, the mean cholesterol level remained elevated after the recovery period in males when compared with the controls. Aspartate aminotransferase activity was elevated in females treated with 1000 mg/kg/day, whereas alanine aminotransferase activity was elevated in males of this dose level when compared with their respective controls. Differences in electrolytes included elevated sodium levels in males at 200 and 1000 mg/kg/day and in females at 50 mg/kg/day and 1000 mg/kg/day, reduced potassium levels in males at 1000 mg/kg/day and females at 200 mg/kg/day and 50 mg/kg/day, increased calcium levels at 1000 mg/kg/day, increased phosphorus in females at 1000 mg/kg/day, and increased chloride levels in males at 200 mg/kg/day were noted. The mean total protein levels were elevated in males and females; both values exceeded the upper limit of the historical control values.

URINALYSIS
Changes in the urine parameters of males treated with 1000 mg/kg/day included elevated mean urine volume and reduced mean relative density. Despite two marked outliers, the differences noted in both parameters of the remaining males at 1000 mg/kg/day also diverged from the values recorded in the control males and were considered to be test item related. The mean urinary pH values of the males treated with 1000 mg/kg/day and 200 mg/kg/day were lower than the control males and this was also considered to be a test item-related change. The protein level and ketones were reduced in males and females treated with 1000 mg/kg/day when compared with the controls. These changes were not considered to be associated with any toxicological relevance. The mean relative density and the mean protein level were also significantly lower in females at 200 mg/kg/day (p<0.05), but considered to be toxicologically irrelevant. All other parameters showed either minor differences which were unrelated to dose or were unaffected. No test item-related findings remained nor were late effects in the animals previously treated with 1000 mg/kg/day noted after 4 weeks of recovery.


ORGAN WEIGHTS
Reduced thymus weights were noted in test item treated males at all dose levels. A similar but less marked trend for lower thymus weights were noted in the test item-treated females. The mean spleen-to-body weight ratio of the females treated with 1000 mg/kg/day was elevated when compared with controls. The mean absolute spleen weight and the mean spleen-to-brain weight ratio were slightly elevated. The changes in spleen weights reflected the microscopical changes (increased hemopoiesis) seen in this organ.

GROSS PATHOLOGY
Macroscopically, males and females treated with 1000 mg/kg/day showed elevated incidence of liver foci were noted when compared with the respective controls. This finding was considered to be test item related. This finding was also noted in one female at 200 mg/kg/day after 13 weeks of treatment, but was not seen in any males at this dose level. It was not evident in either sex at 50 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically, the livers of animals treated with 1000 mg/kg/day showed degeneration, necrosis (focal or of single hepatocytes), bile duct hyperplasia, focal hepatocytic hyperplasia (very likely regenerative), peribiliary fibrosis and an increased incidence and severity of mixed inflammatory cells infiltration in the parenchyma. Increased hemopoiesis was seen in the spleen of animals at 1000 mg/kg/day. After 28 days of recovery, there was the total reversibility of the findings: the livers and spleens of animals treated previously with 1000 mg/kg/day reverted to normal.


HISTORICAL CONTROL DATA (if applicable)
Used


OTHER FINDINGS:
Functional Observational Battery: No clinical signs were evident at any dose level during the functional observational battery performed at week 13 of treatment.

Grip Strength: At 1000 mg/kg/day, the mean hindlimb grip strength value of males was lower that that of the control males and considered to be related to the slight difference in mean body weight. The mean forelimb grip strength of these males compared favorably with those of the control males.
All other animals were unaffected.

Locomotor Activity: There were no test item-related effects on the mean locomotor activity of either gender.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology
Dose descriptor:
NOAEL
Effect level:
426 mg/kg bw/day (actual dose received)
Based on:
other: Calculated as equimolar amount Coco Isethionate
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Recalculated as the equimolar amount of Coco Isethionate. Molweight sodium isethionate = 148 g/mol. Molweight Coco Isethionate = 315 g/mol. NOAEL = (200 mg/kg bw/day) *(315 g/mol)/ (148 g/mol) = 426 mg/kg bw/day
Critical effects observed:
not specified

Tabelle 5: Mean severity of Findings

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/g/day)

0

50

200

1000

0

50

200

1000

Number of animals examined

12

12

12

12

12

12

12

12

Liver

Mixed Cell Infiltration

7/1.0

8/1.0

9/1.0

11/1.8

5/1.2

5/1.0

7/1.0

12/1.5

Degeneration

-

-

-

3/1.3

-

-

-

4/2.0

Focal Necrosis

-

-

-

6/1.3

-

-

-

3/1.3

Single Cell Necrosis

-

-

-

7/1.1

-

-

-

4/1.5

Focal Hepatic Hyperplasia

-

-

-

3/1.7

-

-

-

3/1.7

Bile Duct Hyperplasia

-

-

-

½.0

-

-

-

6/1.5

Peribiliary fibrosis

-

-

-

½.0

-

-

-

5/1.4

Spleen

Incr. Hemopoiesis

-

-

½.0

4/1.8

2/2.0

-

-

8/2.3

Conclusions:
Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Sodium 2-hydroxyethanesulphonate, was considered to be 200 mg/kg body weight/day.
Executive summary:

Oral administration of Sodium 2-hydroxyethanesulphonate to Wistar rats at doses of 50, 200 and 1000 mg/kg/day for 91/92 days resulted in no deaths, no clinical signs during daily or weekly observations, no clinical signs during the functional observational battery, no test item-related differences in the mean fore-or hindlimb grip strength or mean locomotor activity, no toxicologically relevant ophthalmoscopic changes, no differences in the mean food consumption, no changes in hematology parameters at 200 mg/kg/day or at 50 mg/kg/day, and no changes in urinalysis parameters at 50 mg/kg/day.

Although statistically significant differences were noted in the mean hindlimb grip strength values of males treated with 1000 mg/kg/day, these were considered to be secondary effects to the lower body weights.

Test item-related findings were generally restricted to slightly lower mean absolute and relative body weights in males treated with 1000 mg/kg/day, changes in the hematology parameters of the rats treated with 1000 mg/kg/day (reduced mean corpuscular hemoglobin concentration values, elevated mean absolute and relative reticulocyte counts, and a ‘left-shift’ in the reticulocyte maturity indices indicative of increased reticulocyte turnover, reduced hemoglobin distribution width in females only), the clinical biochemistry parameters at 1000 mg/kg/day (reduced glucose levels, elevated total bilirubin levels, elevated cholesterol and phospholipid levels, elevated aspartate or alanine aminotransferase activities at 1000 mg/kg/day) including electrolytes (elevated sodium levels at 50, 200 or 1000 mg/kg/day, reduced potassium levels at 50, 200 or 1000 mg/kg/day, increased calcium levels at 1000 mg/kg/day, increased phosphorus in females at 1000 mg/kg/day, increased chloride levels in males at 200 mg/kg/day), increased spleen weights in rats at 1000 mg/kg/day, macroscopical changes in the liver (a higher incidence of tan foci reported in the liver of males and females treated with 1000 mg/kg/day) after the treatment period only, microscopical changes in the liver (presence of degeneration, necrosis (focal or of single hepatocytes), bile ducts hyperplasia, focal hepatocytic hyperplasia, peribiliary fibrosis and an increased incidence and severity of mixed inflammatory cells infiltration in the parenchyma) and spleen (increased hemopoiesis) with complete post-recovery reversibility.

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Sodium 2-hydroxyethanesulphonate, was considered to be 200 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
426 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
We have a Klimisch 1 28 day dietary study on Coco fatty acids 2-sulfoethyl ester, sodium salt and a Klimisch 1 90 study on sodium 2-hydroxyethanesulfonate , the more toxic metabolite. The adjusted NOAEL for this study is lower than that from the 28 day study, so it is considered a more appropriate study to use as the basis for calculating DNELs.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The requirement for a study is waived due to the availability of Klimisch 1 studies by the oral and dermal routes.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The requirement for a study is waived due to the availability of Klimisch 1 studies by the oral and dermal routes.

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:
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.
Reason / purpose:
reference to other study
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
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.
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
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
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.
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.
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.
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 070 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study Unilever 28 Day Dermal Repeat Dose 7782, was conducted according to OECD guideline 410 to GLP and the test substance is clearly identified and is Klimisch 1.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The study Unilever 28 Day Dermal Repeat Dose 7782, was conducted according to OECD guideline 410 to GLP and the test substance is clearly identified and is Klimisch 1, the DNEL form this for systemic effects is expected to also prevent local effect on the skin.

Additional information

There are Klimisch 1 28 day oral (dietary) and dermal studies on Coco fatty acids 2-sulfoethyl ester, sodium salts howing no adverse effects at high limit doses. There is a Klimisch 1 90 oral gavage study onsodium 2-hydroxyethanesulfonate (sodium isethionate)the more toxic metabolite of Coco fatty acids 2-sulfoethyl ester, sodium salt , which provide a conservative NOAEL from which DNELs can be calculated for the oral and inhalation routes. The Dermal 28 day study provides a good basis for the calculation of dermal DNELs. There are no significant adverse organ effects that would indicate a need for classification for Specifc Target Organ Toxicity (STOT).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

There is a 28 day oral study in the rat Unilever 28 Day Oral Diet study, where the highest level of 1% in the diet corresponded to approximately 1000mg/kg bodyweight/day.  The NOAEL was >1% in the diet (ca. >1000mg/kg bodyweight/day).  Studies carried out on a related substance to Coco fatty acids 2-sulfoethyl ester, sodium salt, Sodium [14C]lauryl Isethionate   (Fatty acids, C12-18 and C18-unstaurated, 2-sulfoethyl esters, sodium salt  CAS No 85408-62-4), details of which can be found in the section on Toxicokinetics, metabolism and distribution, showed it  to be expected to be quickly hydrolysed in the gastrointestinal tract and metabolized in the liver.  This would result in the fatty acid chain being removed to leave as a metabolite sodium  isethionate.  There is a 90 day is a Klimisch 1 rated GLP study available on sodium 2-hydroxyethanesulfonate CAS No 1562-00-1 (sodium isethionate), KEY 408 2209 Harlan Braun.  As the fatty acid portion of the molecule in Coco fatty acids 2-sulfoethyl ester, sodium salt can be expected be rapidly metabolized in the body and would not be expected to exhibit any toxic effect, it can be assumed that any toxic effects seen would be due to the remaining sodium 2-hydroxyethanesulfonate  (sodium isethionate).  This is confirmed by the NOAEL in the 90 day study which was 200mg/kg bodyweight/day, based primarily on adverse effects in the liver and on reduced bodyweights in males seen at the highest dose level of ca. 1000mg/kg bodyweight/day compared to the ca. 1000mg/kg bodyweight /day seen in the 28 day dietary study on the Coco fatty acids 2-sulfoethyl ester, sodium salt.  The NOAEL based on sodium isethionate was recalculated as the equimolar amount of Coco Isethionate.  Molecular weight sodium isethionate = 148, g/mol. Molecular weight of Coco Isethionate = 315 g/mol.  Therefore the NOAEL = (200 mg/kg bw/day) *(315 g/mol)/ (148 g/mol) = 426 mg/kg bw/day giving a revised NOAEL of 426mg/kg bodyweight/day.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

We have oral and dermal repeat dose studies; inhalation is not expected to be a significant route of exposure, dermal exposure being the most likely route in industrial manufacture and use of the Coco fatty acids 2-sulfoethyl ester, sodium salt.  The oral route has shown more absorption than the dermal route based on the difference in DNELs, therefore following the REACH guidance a DNEL for systemic effect from inhalation can be calculated for the oral NOAEL.  Therefore it is not scientifically justified to carry out an inhalation study; this avoids the use of additional animals.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

While Coco fatty acids 2-sulfoethyl ester, sodium salt as an eye irritant, inhalation might be expected to cause some local irritant effects in the upper respiratory tract, inhalation is not expected to occur to any significant degree.  Based on the low toxicity of Coco fatty acids 2-sulfoethyl ester, sodium salt, low potential for significant inhalation and the fact that local effect in the respiratory tract would be expected to limited to irritation rather than tissue damage, it is not scientifically justified to perform additional animal tests to establish a NOAEL for local effects in the respiratory tract.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

There is a Klimisch 1 28 day dermal study available for Coco fatty acids 2-sulfoethyl ester, sodium salt CAS No 61789-32-0.  This study Unilever 28 Day Dermal Repeat Dose 7782, was conducted according to OECD guideline 410 to GLP and the test substance is clearly identified.  The study defines a clear NOAEL.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

There is a Klimisch 1 28 day dermal study available for Coco fatty acids 2-sulfoethyl ester, sodium salt CAS No 61789-32-0.  This study showed some initial local irritant effects at the highest dose level but these subsided and were not visible at the end of the study.  As the highest dose level in this study was clearly on the borderline for local irritant effects it can be expected that the DNEL for systemic effects via the dermal route will also protect against local irritant effect in the skin.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Classification for Specific Target Organ Toxicity STOT, by the EU CLP(GHS) guideline requires serious adverse effects on organs at dose levels of less than 100mg/kg bodyweight/day in a 90 day study or less than 300mg/kg bodyweight/day in a 28 day study. The lack of any serious adverse effects on the organs of the rats in any of the test for repeat dose toxicity and certainly no adverse effect within the range where classification could be considered clearly results in no classification for STOT.