Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 30th October 2012 to 26th July 2013 (Animal study was completed by 8th March 2013)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
EC Number:
294-430-8
EC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Cas Number:
91722-28-0
IUPAC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Test material form:
liquid
Details on test material:
Name of test material (as cited in study report):Internal Olefin Sulfonate (IOS)

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
This strain is widely used in rodent toxicity studies, and there is abundant historical data. Moreover, a sufficient number of animals are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Tsukuba breeding center)
-Number of animals: 58 males and 58 females
- Post-exposure recovery period in satellite groups: 6 males and 6 females each in the control and high dose groups (4 week recovery period)
- Rationale for selecting satellite groups: For observation post-exposure
- Age at study initiation: At arrival: 5 weeks, At start of dosing: 6 weeks
- Weight at study initiation: Males: 192.5 to 220.0 g, Females: 145.0 to 173.4 g (at start of doing)
- Housing: Polycarbonate cages (265W × 426D × 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) with hard woodchip bedding, Two animals of the same sex per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: All animals were acclimatized from arrival to just before the initial dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 to 23.7°C
- Humidity (%): 47.5 to 66.2%
- Air changes (per hr): 6 to 20 times per hour with fresh air
- Photoperiod (hrs dark / hrs light): 12 hours per day (7:00 to 19:00)

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
Oral route is the main exposure route of the test substance for humans, and dietary administration is a commonly used method for assessing the effects of chemicals of which the main route of exposure is ingestion. The period was set for at least 90 days in accordance with the guideline applied.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test substance was uniformly mixed in the basal diet, and the animals were allowed
free access to it.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Stability of the test substance in diet
The test substance was confirmed to be stable in the 250 and 20000 ppm dietary formulations when stored under refrigeration in a dark place up to 42 days, and at room temperature for 8 days in a study entitled “Stability and Homogeneity of Internal Olefin Sulfonate in Dietary Formulation (study No. B120899)” conducted at the test facility using the same lot as that used in this study.

-Confirmation of concentrations and homogeneity of the test substance in diet
Before use of the mixed diet, the concentrations of the test substance, except for the control group, were analyzed by the LC-MS/MS using samples collected from 3 points (top, middle, and bottom layers, n=2 from each layer). The mean concentrations were confirmed to be within ± 15% of the prescribed concentrations in all preparations of the mixed diet (analytical value: 92.3 to 104.3%). Moreover, as for the homogeneity of the test substance in the mixed diet, the coefficient of variation (C.V. value) of the mean concentrations of the top, middle, and bottom layers in all preparations were confirmed to be 15% or below (actual value: 0.8 to 8.2%).
Duration of treatment / exposure:
13 weeks (91 days)
Frequency of treatment:
The test substance was uniformly mixed in the basal diet, and the animals were allowed free access to it.
Doses / concentrationsopen allclose all
Dose / conc.:
2 500 mg/kg diet
Dose / conc.:
5 000 mg/kg diet
Dose / conc.:
10 000 mg/kg diet
No. of animals per sex per dose:
10 animals per sex per dose, and 6 additional recovery animals in control and high-dose (10000 ppm) group.
Control animals:
yes, plain diet
Details on study design:
Administration
- Rationale for dose selection:
The dose levels were selected based on the results of a preliminary study entitled “A 14-Day Feeding Toxicity Study of Internal Olefin Sulfonate in Rats (dietary dose levels: 0, 2500, 5000, 10000, and 20000 ppm, study No. B120896)” conducted at the test facility. At 20000 ppm (male: 1890 mg/kg/day, female: 1740 mg/kg/day), suppressed body weight gain and a transient decrease in food consumption were noted in males. In addition, the following changes were observed at 20000 ppm: increased liver weights in males and females, and decreased total bile acid in males and females, increased ALAT, decreased total protein and albumin, increased A/G ratio, decreased calcium and potassium, and increased chlorine in males in the biochemistry. Decreased reticulocyte count was also observed in males at 20000 ppm in the hematology. At 10000 ppm (male: 938 mg/kg/day, female: 912 mg/kg/day), increased liver weights were noted in males and females. Based on these results and considering the administration period of this study, the high dose for males and females was set at 10000 ppm, at which some toxic changes would occur in males and females, which is the dose level near the dose limit specified in the OECD guideline. The middle and low dose levels were set at 5000 and 2500 ppm, respectively, with a common ratio of 2.

Recovery study
A 4-week recovery period after the end of the dosing period was set for 6 males and 6
females each in the control and high dose groups.

Preparation of the mixed diet with the test substance
- Frequency
The mixed diet was prepared at least once within the period for which their stability was confirmed (within 21 days, 4 times in total; expiration period: 42 days).

- Method
(1) The prescribed amount (calculated with a conversion factor of 2.8409) of the test substance and basal diet was weighed for each dose level using an electronic balance.
*: The sponsor provided information that the component other than the principal ingredient of the test substance is essentially moisture. Therefore, the amount of basal diet was calculated on the assumption that all of the moisture would be vaporized during preparation and storage.
(2) The test substance and an appropriate amount of the weighed basal diet were mixed by shaking in a plastic bag for about 1 minute.
(3) The mixed diet was added with a portion of the weighed basal diet, and it was mixed and ground with a compact mill as the primary mixed diet.
(4) The primary mixed diet was added with a portion of the weighed basal diet to give a total amount of approximately 2 kg, and it was mixed with a tabletop universal mixer for about 10 minutes to make a secondary mixed diet.
(5) The secondary mixed diet and the remaining weighed basal diet were mixed with a V type mixer for about 30 minutes.
(6) The prepared diet was subdivided into portions for about a week in plastic bags.
For the control group, the basal diet packed and sealed in a plastic bag by the supplier was used as is without any preparation.

-Storage conditions
Refrigeration (actual temperature: 4.6 to 6.1°C, permissible range: 1 to 10°C) in a dark
place. The prepared diet was used within 37 days after preparation under refrigerated conditions
and within 8 days at room temperature in the animal room (expiration period: 42 days
under refrigeration in a dark place and 8 days under animal room conditions).
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:

The test substance intake was calculated using the following equation based on the concentrations of the test substance in diet (nominal concentration), body weight, and food consumption of each animal.


All animals were observed once a day

- DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed on all animals once before the start of dosing and once a week in the afternoon during the dosing period. The open field observations were performed for 2 minutes. At the observation, the cage labels were removed and temporary labels were attached to the cages to hide the dose levels and original animal numbers from observers. The temporary animal numbers were assigned to each animal during the examinations. As a result, no treatment-related changes were observed in males or females in any treated group; therefore, detailed clinical observations were not performed during the recovery period.

(1) Hand-held observations
Reactivity on removal from the cage, reactivity to handling, aggression, skin (trauma, color of skin), fur (soiled fur), eyes (exophthalmos, palpebral closure), mucous (color of conjunctiva), secretion, lacrimation, salivation, piloerection, and pupil size

(2) Open field observations
Rearing, arousal, urination, defecation, posture/body position, breathing, co-ordination movement, gait, tremor, clonic convulsion, tonic convulsion, stereotypy, and bizarre behavior


The function test and motor activity measurement were performed on all animals once in the afternoon in the week before the final dosing (week 12; day 79 to 83). The cage labels were removed and temporary labels were attached to the cages to hide the dose levels and original animal numbers from observers during the function tests. The temporary animal numbers were assigned to each animal during the examinations. As a result, no treatment-related changes were observed in males or females in any treated group; therefore, function test and motor activity measurement were not performed during the recovery period.

-Function test
(1) Sensory reactivity to stimuli
Approach response, touch response, auditory response, tail pinch response, and aerial righting reaction were observed in the open field.

(2) Grip strength
After the observation of sensory reactivity to stimuli, grip strength of the forelimb and hindlimb was measured twice using a digital force gauge (DPS-5, Imada Co., Ltd.). The larger value was adopted.

-Motor activity
Animals were acclimated to polycarbonate cages (with bedding, 265W × 426D × 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) without diet and water supply. Animals were transferred to new polycarbonate cages at the start of measurement. Motor activity was recorded in each animal for 1 hour using a motor activity-measuring device (Supermex, Muromachi Kikai Co., Ltd.) without diet and water supply. Data of motor activity were calculated every 10 minutes from the start of the measurement, while a total of 1 hour was calculated. The measurement was carried out in an examination room (No. 2123) for motor activity measurement.


All animals were weighed on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, and 91 during the dosing period and on days 92, 99, 106, 113, and 119 during the recovery period using an electronic balance. The body weight measurement was performed in the morning. On the scheduled necropsy days (days 92 and 120), body weight was measured for calculation of the relative organ weights.


Gross weight of each feeder including diet was weighed on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, and 88 using the balance, and the mean daily food consumption per animal was calculated from the total food consumption over each period, namely, days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 43, 43 to 50, 50 to 57, 57 to 64, 64 to 71, 71 to 78, 78 to 85, and 85 to 88. Gross weight of each feeder including diet was also weighed on days 92, 99, 106, 113, and 116, and the mean daily food consumption of the recovery period was calculated over each period, namely, days 92 to 99, 99 to 106, 106 to 113, and 113 to 116. The mean daily food consumption for each period was expressed as the data of the last day in each period.


The ophthalmoscopic examinations were performed on all animals during the acclimation period and on all animals in the control and 10000 ppm groups in the final week of the dosing period (day 85). The light pupillary reflex was examined with a direct ophthalmoscope (BETA200, Heine Optotechnik Ltd.) under dim light. Then, a mydriatic (Mydrin-P ophthalmic solution, Santen Pharmaceutical Co., Ltd.) was instilled, and the anterior portion and optic media were examined with a slit lamp (SL-15, Kowa Co., Ltd.). The ocular fundus was examined with a binocular indirect ophthalmoscope (OMEGA200, Heine Optotechnik Ltd.).As a result, no treatment-related changes were observed in males or females in the 10000 ppm group; therefore, the examination was not performed in the other groups during the dosing period or in any group during the recovery period.


Urinalysis was performed on 5 males and 5 females (animals with smaller animal numbers) per group in the final week of the dosing period (day 88 to 89). Animals were transferred to the metabolic cages and fresh urine samples were collected within 3 hours without water and diet. A portion of the sample was subjected to the paper test (pH, protein, glucose, ketones, and occult blood) and a portion of the fresh urine sample (about 0.5 mL) was centrifuged (500×g for 5 minutes at room temperature: 20°C) and the urinary sediments were examined microscopically. Urine samples at approximately 20 hours were collected while providing water and diet while being subjected to the other items. During the urine collection, autoclaved polycarbonate bottles were used for water supply. As a result, changes suspected to be treatment-related were noted in urinary electrolyte excretion in males; therefore, urinalysis was performed for all males in the final week of the recovery period (days 116 to 117). The residual urine samples after measurement of Na, K, and Cl were stored in a freezer.

(1) pH by Paper test
(2) Protein by Paper test *
(3) Glucose by Paper test
(4) Ketones by Paper test
(5) Occult blood by Paper test
(6) Urine volume (mL) by Measured with measuring cylinders
(7) Specific gravity by Refractometry
(8) Sodium (Na; mmol) by Ion-selective electrode method
(9) Potassium (K; mmol) by Ion-selective electrode method
(10) Chlorine (Cl; mmol) by Coulometric titration
(11) Sediments Microscopic examination after Sternheimer-Malbin staining [Examination items: cast, crystal, epithelial cell, white blood cell, red blood cell]

< HAEMATOLOGY>
On the days of the scheduled necropsy (days 92 and 120), blood samples (about 3 to 4 mL) were collected from the inferior vena cava of all animals under anesthesia with intraperitoneal injection of sodium thiopental, and the following items were examined. The animals were fasted from the evening (about 16:00) of the day before the examination at the scheduled necropsy for more than 21 hours (specified time: more than 16 hours). A portion of the blood sample (about 0.6 mL) was anticoagulated with 3.2 w/v% trisodium citrate solution and cooled on ice. It was centrifuged (12,000×g for 3 minutes at 4°C), and PT and APTT were examined using the obtained plasma. The other items were examined using the blood samples (about 0.8 mL) anticoagulated with EDTA-2K. The residual blood and plasma samples were discarded just after the examinations.

(1) Red blood cell count (RBC; ×106/μL) by Hydrodynamic focusing DC detection method
(2) Hemoglobin concentration (HGB; g/dL) by SLS-hemoglobin method
(3) Hematocrit (HCT; %) by RBC pulse height detection method
(4) Mean corpuscular volume (MCV; fL) by Calculated from (1) and (3)
(5) Mean corpuscular hemoglobin (MCH; pg) by Calculated from (1) and (2)
(6) Mean corpuscular hemoglobin concentration (MCHC; g/dL) by Calculated from (2) and (3)
(7) Reticulocyte ratio (%) and count (×109/L) by Flow cytometry by using semi-conductor laser
(8) Platelet count (×103/μL) by Hydrodynamic focusing DC detection method
(9) Prothrombin time (PT; sec.) by Scattered light detection method
(10) Activated partial thromboplastin time (APTT; sec.) by Scattered light detection method
(11) White blood cell count (×103/μL) by Flow cytometry by using semi-conductor laser
(12) Differential leukocyte ratio (%) and count (×103/μL) by Flow cytometry by using semi-conductor laser


The remaining blood samples (about 2 to 3 mL) after the hematology test were left to stand at room temperature for 0.5 to 1 hour, and then centrifuged (2,100×g for 10 minutes at 4°C). The following items were examined using the obtained serum. The residual serum samples were stored in a freezer at about –80°C (permissible range: –60°C or lower) and discarded by the completion of the study.

(1) ASAT (GOT; U/L) by UV-rate method (modified JSCC method)
(2) ALAT (GPT; U/L) by UV-rate method (modified JSCC method)
(3) γGT (U/L) by L-γ-Glutamyl-3-carboxy-4-nitroanilide substrate method (modified JSCC method)
(4) ALP (U/L) by p-nitrophenylphosphate substrate method (modified JSCC method)
(5) Total bilirubin (mg/dL) by Enzymatic method (BOD method)
(6) Urea nitrogen (mg/dL) by Enzymatic-UV method (Urease-LEDH method)
(7) Creatinine (mg/dL) by Enzymatic method (Creatininase-POD method)
(8) Glucose (mg/dL) by Enzymatic method (HK-G6PDH method)
(9) Total cholesterol (mg/dL) by Enzymatic method (CO-HMMPS method)
(10) Triglyceride (mg/dL) by Enzymatic method (GPO-HMMPS method, No interference by free glycerin)
(11) Total protein (g/dL) by Biuret method
(12) Albumin (g/dL) by BCG method
(13) A/G ratio Calculated from (11) and (12)
(14) Calcium (mg/dL) by OCPC method
(15) Inorganic phosphorus (mg/dL) by Enzymatic method (PNP-XOD-POD method)
(16) Sodium (Na; mmol/L) by Ion-selective electrode method
(17) Potassium (K; mmol/L) by Ion-selective electrode method
(18) Chlorine (Cl; mmol/L) by Ion-selective electrode method
(19) Total bile acid (μmol/L) by Enzymatic cycling method (3α-HSD method)


-Organs/Tissues
The organs and tissues were removed and subjected to the examinations

- Necropsy
On the days of the scheduled necropsy (days 92 and 120), all animals were euthanized by exsanguination from the abdominal aorta after blood sampling for the hematology and blood chemistry tests, and then subjected to necropsy.

-Organ weights
The organs of the scheduled sacrifice animals were weighed at necropsy using an electronic balance (AW120, Shimadzu Corporation). Bilateral organs were weighed together. The thyroid/parathyroid was weighed after fixation in 10 vol% phosphate-buffered formalin. Relative organ weight was calculated using the body weight on the necropsy day.

-Histopathology
The organs and tissues were removed from all animals, and fixed in 10 vol% phosphate-buffered formalin. The testes were fixed in Bouin’s solution and the eyeballs, optic nerves, and Harderian glands were fixed in Davidson’s solution, and then stored in 10 vol% phosphate-buffered formalin. After fixation, organs and tissues of the scheduled sacrifice animals on day 92 in the control and 10000 ppm groups were processed by the standard method to prepare hematoxylin- and eosin-stained (HE) sections and examined microscopically. Furthermore, the following organs and tissues exhibiting gross abnormalities were examined in the same manner: testis and epididymis in 1 male (#20113) and subcutaneous mass in 1 female (#60112) in the control group sacrificed after the recovery period. The teeth with malocclusion in 1 female (#60306) in the 5000 ppm group sacrificed after the dosing period were not examined according to the SOP of the test facility, because useful information is not obtained by a histopathological examination. As a result, no treatment-related changes were observed in males or females in the 10000 ppm group; therefore, the examination was not performed in the other groups during the dosing period or in any group during the recovery period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table. Pathological examination for Organs/Tissues)
HISTOPATHOLOGY: Yes (see table. Pathological examination for Organs/Tissues)
Statistics:
Safety Study Support System (Provantis™, Instem LSS Ltd.) was used for the statistical analyses.


- Numerical data
[Detailed clinical observation (rearing), function tests (grip strength and motor activity), body weight, food consumption, hematology, blood chemistry, urinalysis (urine volume, specific gravity, Na, K, and Cl), and absolute and relative organ weights]
Above numerical data were analyzed statistically by multiple comparison tests. First, the datawere tested by Bartlett’s test for homogeneity of variance. When the variances were homogeneous, the one-way analysis of variance (ANOVA) was used, and when the variances were heterogeneous, the Kruskal-Wallis test was performed. When there was a significant difference among the groups, the Dunnett (if homogeneous) or Dunnett type (if heterogeneous) multiple comparison test was performed to compare the means between the control group and each test substance-treated group. Numerical data from 2 groups during the recovery period were tested by the F test for homogeneity of variance. When the variances were homogeneous, Student’s t test was used, and when the variances were heterogeneous, the Aspin-Welch test was performed.

- Categorical data
[Urinalysis (pH, protein, glucose, ketones, occult blood, and sediments)]
Above categorical data of urinalysis, the data were tested by the Kruskal-Wallis test. When there was a significant difference among the groups, the Dunnett type multiple comparison test was used. The Wilcoxon rank sum test was performed for categorical data during the recovery period in order to compare the values between the control and 10000 ppm groups. Bartlett’s test, the one-way analysis of variance, and the Kruskal-Wallis test were conducted at the significance level of 5%. The other tests were conducted at the twotailed significance levels of 1% and 5%.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related abnormal findings were observed in males or females in any treated group. Subcutaneous mass in the anogenital region was observed from day 48 in 1 female (#60112) in the control group, and malocclusion from day 41 in 1 female (#60306) in the 5000 ppm group, as incidental findings.

-Detailed clinical observation
No treatment-related abnormalities were observed in males or females in any treated group. All males and females in the control and treated groups showed normal reactions or normal findings in all indices of the hand-held observations and open field observations throughout the observation period. In addition, the rearing counts in both sexes in the treated groups were comparable to those in the control group at all-time points, with no significant differences.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in males or females in any treated group. The body weights of both sexes in the treated groups were comparable to those in the control group throughout the experimental period, including the recovery period, with no significant differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in males or females in any treated group. The food consumption of both sexes in the treated groups was comparable to that in the control group throughout the experimental period, including the recovery period, with no significant differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related abnormalities were observed in males or females in the 10000 ppm group at the examination in the final week of the dosing period. Findings observed in the 10000 ppm group were similar to those observed in the control group, with approximately the same incidences in both sexes.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in males or females in any treated group. Almost all parameters in males and females in the treated groups were comparable to those in the control group at the examination in both dosing and recovery periods, with no significant differences. At the end of the recovery period, MCHC showed a significantly lower value in males in the 10000 ppm group; however, it was judged to be incidental because no similar change was observed at the end of the dosing period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in males or females in any treated group. Almost all parameters in males and females in the treated groups were comparable to those in the control group at the examination in both dosing and recovery periods, with no significant differences. The following statistically significant changes were observed in females at the end of the dosing period; however, they were judged to be incidental or of no toxicological significance, because of the lack of dose-dependency or changes within the historical control range of the test facility, without corresponding changes in any of other examinations, including histopathology: a decrease in glucose in the 2500 and 5000 ppm groups, a decrease in albumin in the 5000 ppm group, and an increase in chlorine in the 5000 and 10000 ppm groups. Moreover, a significantly higher value of A/G ratio observed in males in the 10000 ppm group at the end of the recovery period was judged to be incidental, because no similar change was observed at the end of the dosing period.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At the examination in the dosing period, urinary sodium level in males increased dose dependently in the treated groups, with significant differences in the 5000 and 10000 ppm groups. Meanwhile, no significant changes were observed in urinary electrolytes in females in any treated group. Besides, a significant increase in crystals was observed in males in the 2500 and 5000 ppm groups; however, it was judged to be incidental because no significant increase was detected in the 10000 ppm group. In addition, a significant decrease in epithelial cells observed in females in all treated groups without dose-dependency was judged to be of no toxicological significance. At the examination of males in the recovery period, urinary sodium level in the 10000 ppm group was almost the same as that in the control group, with no significant difference. Moreover, no significant differences were detected in any other parameter between the control and 10000 ppm groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
-Function test
No treatment-related abnormalities were observed in males or females in any treated group. All males and females in the control and treated groups showed normal reactions in all indices of the sensory reactivity to stimuli. In addition, the grip strength of the forelimb and hindlimb in both sexes in the treated groups was comparable to that in the control group, with no significant differences.

-Motor activity
No treatment-related changes were observed in males or females in any treated group.
The motor activity counts in both sexes in the treated groups were comparable to those in the control group in each 10 minute-interval as well as in total at all-time points for 1 hour, with no significant differences.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in males or females in any treated group. Almost all organs of males and females in the treated groups were comparable to those in the control group at the examination in both dosing and recovery periods, with no significant differences in absolute or relative weights. At the end of the dosing period, absolute heart weights of females showed significantly lower values in all treated groups; however, there were no significant differences in their relative weights. In addition, significantly higher values were observed in absolute spleen weights of females in the 2500 and 10000 ppm groups, as were in their relative weights in all treated groups. These changes, however, were judged to be incidental or of no toxicological significance, because they were almost within the historical control range of the test facility, without corresponding changes in any of other examinations, including histopathology.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related abnormal findings were observed in males or females in any treated group. Only the following findings were observed as incidental findings, including those that had already been found in the clinical observation: malocclusion in 1 female in the 5000 ppm group at the end of the dosing period, and unilateral small testis and epididymis in 1 male and mass in the subcutis in 1 female in the control group at the end of the recovery period.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related abnormal findings were observed in males or females in the 10000 ppm group. All the findings observed in the 10000 ppm group were common spontaneous changes found in rats of this age; therefore, they were judged to be incidental. Besides, as for the organs and tissues exhibiting gross abnormalities in the control group, atrophy of the testicular seminiferous tubules and decrease of sperm in the epididymal ducts were observed in the male showing small testis and epididymis, as was malignant mammary gland adenocarcinoma in the female having mass in the subcutis.
Histopathological findings: neoplastic:
no effects observed
Details on results:
-Test substance intake
The overall mean test substance intake throughout the dosing period in the 2500, 5000, and 10000 ppm groups was 150.69, 307.18, and 603.09 mg/kg/day for males, and 169.10, 345.86, and 679.90 mg/kg/day for females, respectively, indicating the exposure increased dose-proportionally among the treated groups, with no gender differences. In addition, the maximum exposure level in the 10000 ppm group (male: 912.19 mg/kg/day, female: 865.06 mg/kg/day) reached near the dose limit specified in the OECD guideline during the first week of dosing.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
603.09 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
NOAEL
Effect level:
679.9 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: overall effects

Applicant's summary and conclusion

Conclusions:
The test substance had no adverse effects in males or females even at the highest dose of 10000 ppm. The only treatment-related finding observed was increased urinary sodium excretion considered to be due to sodium derived from the test substance and of no toxicological significance. Therefore, it was concluded that the noobserved-adverse-effect level (NOAEL) was 10000 ppm for both sexes (male: 603.09 mg/kg/day, female: 679.90 mg/kg/day) under the conditions of this study.
Executive summary:

Internal Olefin Sulfonate was administered via diet at dose levels of 0 (control), 2500, 5000, and 10000 ppm to male and female Crl:CD(SD) rats (10 males and 10 females/group) for 91 days to assess any adverse effects by repeated exposure. Furthermore, 6 males and 6 females were assigned to the control and 10000 ppm groups to assess the reversibility after a 4-week recovery period. The control group was given a basal diet only. The overall mean test substance intake throughout the dosing period in the 2500, 5000, and 10000 ppm groups was 150.69, 307.18, and 603.09 mg/kg/day for males, and 169.10, 345.86, and 679.90 mg/kg/day for females, respectively.

 

No adverse effects attributable to the test substance were found in males of females even in the 10000 ppm group. There were no treatment-related changes in males or females in clinical signs, detailed clinical observations, function tests, motor activity, body weights, or food consumption, nor were there any in ophthalmology, hematology, blood chemistry, organ weights, necropsy, or histopathology. Urinary sodium excretion in males increased dose-dependently in the treated groups and a similar tendency was observed in females. However, no corresponding changes were noted in any other examinations, including serum electrolyte balance or histopathological findings, and the urinary sodium excretion returned to the control level in an examination of males after the recovery period. Therefore, the increased urinary sodium excretion observed in the treated groups was considered to be due to sodium derived from the test substance and of no toxicological significance.

 

In conclusion, the no-observed-adverse-effect level (NOAEL) was judged to be 10000 ppm for both males and females (male: 603.09 mg/kg/day, female: 679.90 mg/kg/day) under the conditions of this study.