Registration Dossier

Administrative data

Description of key information

Oral:
NOEL (28 days, gavage, rat): 1000 mg/kg bw
Oral (read-across using sorbitan stearate as source chemical):
NOEL (28 days, gavage, rat): 1000 mg/kg bw
NOEL (80 weeks, feed, rat): ca. 2600 mg/kg bw
NOAEL (2 years, feed, rat): 5000 mg/kg bw

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-07-31 to 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation: Males: 173 to 198 g (mean 184 g); Females: 124 to 149 g (mean 138 g)
- Housing: in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE: PEG 300

- Concentration in vehicle: 0, 8, 40, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): BCBG8285V
- Source: Aldrich
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using a method described in the analytical report. After experimental start and during week 3, duplicate 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. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days).
Duration of treatment / exposure:
28 days
Frequency of treatment:
once per day
Remarks:
Doses / Concentrations:
0, 40, 200 and 1000 mg/kg/d
Basis:
actual ingested
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a previous non-GLP dose range finding toxicity study in Wistar rats.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once before commencement of administration and once weekly (weeks 1 to 3) thereafter


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration and once weekly (weeks 1 to 3) thereafter

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

FOOD CONSUMPTION):
- The food consumption was recorded once during the acclimatization period and weekly thereafter

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 AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of the treatment
- Anaesthetic used for blood collection: Yes (identity)
- Animals fasted: Yes
- How many animals: All


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of the treatment
- Animals fasted: Yes
- How many animals: All


URINALYSIS: Yes
- Time schedule for collection of urine: during the 18 hours fasting period before blood sampling
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: All
- Battery of functions tested: grip strength and motor activity

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• 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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled treatment or recovery periods. There were no clinical signs of toxicological relevance during the treatment period and no late effects were seen during the recovery period.
From day 25 until the end of the treatment period, slightly soft feces were noted in the males treated with 40 mg/kg/day and in both sexes treated with 200 or 1000 mg/kg/day. This finding considered to be incidental, based upon the late onset and lack of dose-related severity. During the recovery period, one control female and one female previously treated with 1000 mg/kg/day had ocular findings related to the post-treatment blood sampling.

BODY WEIGHT AND WEIGHT GAIN
There were no test item-related effects on mean body weights or mean body weight gain. During the treatment and recovery periods, the mean body weights of the test item-treated male and female rats compared favorably with those of the respective controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no test item-related effects on food consumption.

HAEMATOLOGY
There were no test item-related differences in the mean hematology parameters at any dose level.
At 1000 mg/kg/day, males showed a statistically significant decrease in the mean cell hemoglobin concentration (p<0.05). The mean white blood cell count in these males was also increased (p<0.01) when compared with the controls. The mean absolute differential white cell counts showed significant decreases in the populations of neutrophils, lymphocytes and monocytes (all 0.05), whereas the relative differential white cell counts showed only decreased monocytes (p.0.05) when compared with the controls. The mean methemoglobin level was increased in males (p<0.05) and the activated partial thromboplastin time was prolonged (0.05). In females, the only value that differed significantly from the controls was a significantly increased thromboplastin time (p<0.05).
At 200 mg/kg/day, the mean white cell count was significantly reduced (p<0.05) and the mean absolute differential count showed decreased neutrophil and lymphocyte populations that attained statistical significance.
At 40 mg/kg/day, the mean white cell count was significantly reduced (p<0.05) when compared with the controls, and the mean absolute lymphocyte count was also significantly reduced (p<0.05).
All statistically significant differences noted at the end of the treatment period were either unrelated to dose, within the ranges of the historical control values, or restricted to one sex. During the recovery period, the mean white cell count of males treated previously with 1000 mg/kg/day remained below that of the control males. The mean relative eosinophil count was significantly increased (p<0.05) and the mean absolute lymphocyte count was significantly
increased (p<0.05) in these males. The methemoglobin level was elevated in males and reduced in females (both p<0.05). The mean platelet count was elevated (p<0.05) in previously treated females. All other values compared favorably with the respective control values.
In the case of differences recorded after the recovery period, all were within the ranges of the historical control data and/or either not already evident at the end of treatment period and therefore not considered to represent test item-related effects.

CLINICAL CHEMISTRY
There were no test item-related differences in the mean clinical biochemistry parameters at any dose level.
At 1000 mg/kg/day, males had elevated creatinine levels (p<0.01), reduced calcium levels (p<0.05) and reduced phosphorus levels (p0.05) when compared with the controls. Females at this dose level had significantly reduced total bilirubin levels (p<0.05), reduced lactate dehydrogenase activity (p<0.01) and decreased creatine kinase activity (p<0.05) when compared with the controls. All other values compared favorably with the controls.
At 200 mg/kg/day, glucose levels were increased in males when compared to controls. Total bilirubin levels were significantly reduced in the females (p<0.05) at this dose, which also showed decreased creatine kinase activity (p<0.05) when compared with the controls. All other values were similar to those of the controls.
At 40 mg/kg/day, males had elevated creatinine levels (p<0.01), elevated sodium levels (p<0.01), elevated chloride levels (p<0.05), while females showed decreased total bilirubin )p<0.05), increased sodium (p<0.05).
All statistically significant differences noted at the end of the treatment period or recovery period
were either unrelated to dose, within the ranges of the historical control values, or restricted to one sex.

URINALYSIS
No test item-related effects on the urinalysis parameters were noted.
In males treated with 1000 mg/kg/day, significantly lower urine volume (p<0.05) was noted when compared with the controls. In females, the urine was significantly less acidic (p<0.05) than that of the controls. These differences remained within the range of the historical control data.
All other parameters were unaffected.

NEUROBEHAVIOUR
There were no findings evident during the functional observational battery performed at week 4.
Grip Strength: The mean fore- and hind-limb grip strength values of the test item-treated males and females were similar to those recorded in the respective control animals.
Locomotor Activity: the 10-minute recording intervals. In females treated with 1000 mg/kg/day, the mean locomotor activity was significantly increased (p<0.05) during 0-10 minutes only. The remaining recording intervals were generally similar to those of the control females. This transient difference was considered to be due to biological variation and without toxicological relevance.

ORGAN WEIGHTS
There were no test item-related changes in the mean absolute or relative organ weights.
At 1000 mg/kg/day, the mean absolute and relative liver weights were significantly lower (p<0.05) than that of the controls. The mean adrenal-to-brain weight ratio of the males was also significantly lower (p<0.05) than that of the controls. Females were unaffected.
At 200 mg/kg/day, the mean absolute liver weight and mean absolute spleen weights were significantly elevated (p<0.05) in females. The mean spleen-to-body weight and spleen-to-brain weight ratios were significantly higher (both p<0.01) in females.
At 40 mg/kg/day, the mean absolute liver weights were significantly elevated (p<0.05) in females and the mean kidney-to-brain weight ratio of the males was significantly lower (p<0.05) than that of the control males.
None of these differences coincided with test item-related microscopical changes.
The significantly lower mean liver-to-body weight ratio in males (p<0.05) previously treated with 1000 mg/kg/day was not considered to represent a test item-related effect as there were no commensurate microscopical changes.

GROSS PATHOLOGY
There were no gross lesions that could be attributed to treatment with the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic lesions that could be attributed to treatment with the test item. All
lesions recorded were considered to be within the range of normal background alterations.

OTHER FINDINGS
No
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

no

Conclusions:
Based on the results of this study, 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

In this subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 40, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG300, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

Oral administration of the test item to Wistar rats at doses of 40, 200 and 1000 mg/kg/day, for 28 days resulted in no deaths, no clinical signs of toxicity during daily observations and weekly detailed observations (weeks 1-3), no clinical signs of toxicity during the functional observational battery (week 4), no differences in grip strength or locomotor activity, no differences in mean food consumption or body weight development, no differences in the mean hematology, clinical biochemistry or urinalysis parameters, no effects upon organ weights and no test item-related macroscopical or microscopical findings. Based on the results of this study, 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable and robust; 4 studies/reports are available on target or source chemicals.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

The repeated dose toxicity of sorbitan caprylate was assessed in a 28 -day repeated dose study.

In a subacute toxicity study, oral administration of the test item sorbitan caprylate to Wistar rats at doses of 40, 200 and 1000 mg/kg/day, for 28 days resulted in no deaths, no clinical signs of toxicity during daily observations and weekly detailed observations (weeks 1-3), no clinical signs of toxicity during the functional observational battery (week 4), no differences in grip strength or locomotor activity, no differences in mean food consumption or body weight development, no differences in the mean hematology, clinical biochemistry or urinalysis parameters, no effects upon organ weights and no test item-related macroscopical or microscopical findings. Based on the results of this study, 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL).

The repeated dose toxicity of sorbitan caprylate was also assessed based on the analogue approach using sorbitan stearate as a read-across supporting substance.

The analogue approach using sorbitan stearate as source chemical is justified:

Both chemicals are of comparable structures with minor deviations and can be characterized as an ester of sorbitan and a fatty acid. Compared to the source chemical, the target chemical has a shorter alkyl chains that affect its physicochemical properties. But based on the kinetic / metabolic investigations on both chemicals, the length of the alkyl chain is not considered to have significant impact on the metabolic pathway or toxicological mode of action. Oral gavage studies in rats administered C14 labeled sorbitan stearate in oil solutions have demonstrated that about 90% of the substance was absorbed and hydrolyzed to stearic acid and sorbitan. The metabolic fate of sorbitan caprylate was investigated using a lipase assay. The hydrolysis mediated by porcine pancreas lipase was quantitatively determined. The target chemical sorbitan caprylate is proved to be hydrolyzed and caprylic acid was formed . These findings suggest that metabolism of the sorbitan occur initially via enzymatic hydrolysis, leading to sorbitan and the corresponding natural acids.

Based on the above mentioned information, it is reasonable to consider that these two substances are comparable in their metabolic fate and thereby toxicological profiles. Hence, the source chemical is considered as “suitable with interpretation” analog.

According to the available toxicity studies, the findings are also comparable for target and source chemicals:

·        The findings in acute toxicity studies are comparable. Both chemicals are of no acute toxicity.

·        The findings in subacute dose toxicity studies are comparable. No treatment effects were observed in 28-day repeated toxicity studies in Wistar rats. The same NOEL of 1000 mg/kg bw/d was derived for both chemicals.

·        The findings in genetic toxicity are comparable. Both chemicals did not induce gene mutations in Ames tests, but induced structural chromosomal aberrations in cell lines of Chinese Hamster.

·        The findings in reproduction / developmental toxicity studies are comparable.

In a subacute toxicity study, the test item sorbitan stearate was administered daily by oral gavage to Sprague-Dawley rats of both sexes at dose levels of 40, 200 and 1000 mg/kg body weight/day. A control group was treated similarly with the vehicle only. The study comprised a reproductive/developmental toxicity screening test, intended to provide information on possivle effects on male and female reproductive performance and also on the development of the F1 offspring from conception to day 4 post-partum. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Evaluation of the mating, pregnancy, parturition and post-partal periods, and relevant parameters and/or indices were performed and/or calculated. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues. Based on the results of this study, 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) for repeated toxicity, reproductive and developmental effects.

In a chronic study, sorbitan stearate was given to groups of 48 male and 48 female mice at dose levels of 0 (control), 0.5, 2.0 or 4.0% of the diet for 80 weeks. There was no evidence of carcinogenic activity at any of these dose levels. The treatment had no adverse effects on the death rate or rate of body-weight gain. Both male and female mice receiving 4% test item showed enlargement of the kidneys and a higher incidence of nephrosis compared with controls. Other organ-weight changes appeared unlikely to be directly related to treatment, as did a significant depression in the total leucocyte count in the blood of female but not of male mice given 4% test item. The no-untoward-effect level in this study was considered to be 2.0% of the diet, or approximately 2600 mg/kg body weight/day.

In a chronic study, Wistar rats (25 groups consisting of 12 males and 20 females/dose; F0 generation) were administered with sorbitan monostearate in the diet at 0, 5, 10 or 20% (~ 0, 2500, 5000 or 10,000 mg/kg-bw/day) for two years (spanning four generations). During the course of the study, observations were made of physical appearance, behavior, reproduction, and lactation through three successive generations, and gross and histologic evaluations were made at termination. Growth was normal, except for males in the 20 percent group. These animals had reduced weight gains. Fertility and gestation parameters for the initial generation were similar for control and test groups. Infant deaths for the 10 and 20 percent ester groups were higher than for the control group. The author believed this was due to maternal neglect and reduced milk production. Reproduction and lactation data were also recorded for the F, and F1 generations. The proportions of matings resulting in pregnancy were lower in the 20 percent dietary ester group, as was the proportion of nurselings surviving the lactation period. No deviation from the normal range was found in hemoglobin values, leukocyte counts, blood sugar, or plasma cholesterol values. Urinalysis after 1 and 2 years had sporadic positive tests for the presence of albumin and reducing sugars. No striking differences in number of deaths in any group were found up to 1% years. However, during the last quarter of the study the number of deaths was greater for the 20 percent group. Lungs of both test and control animals were congested. In rats of the 10 and 20 percent ester groups, the livers were enlarged, but the incidence of hepatic necrosis was no greater in these groups than in lower dosed groups or controls. Also at the two higher dietary concentrations, the weights of the kidneys were increased, but no microscopic changes were observed. The stomach, GI tract, heart, spleen, pancreas, adrenals, thyroid, gonads, lymph nodes, bone marrow, and spinal cord had no compound-related lesions. The investigators concluded that chronic consumption of a few tenths of 1 percent of Sorbitan Stearate would pose no hazard to human health. Based on the result, it was concluded: LOAEL ~10,000 mg/kg-bw/day (based on body weight change, liver enlargement); NOAEL ~ 5,000 mg/kg-bw/day.

The same results could be also expected for sorbitan caprylate due to the structural similarities.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study under GLP

Justification for classification or non-classification

Based on the results from the available repeated dose toxicity data, it is concluded that the registered substance is not subject to classification and labelling according to the criteria of the EU Dangerous Substances Directive (67/548/EEC) (DSD) and of the EU Classification, Labelling and Packaging Regulation (1972/2008/EC) (CLP).