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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 2-[methyloleoylamino]ethane-1-sulphonate
EC Number:
205-285-7
EC Name:
Sodium 2-[methyloleoylamino]ethane-1-sulphonate
Cas Number:
137-20-2
Molecular formula:
C21H41NO4S.Na
IUPAC Name:
sodium 2-[methyl(oleoyl)amino]ethanesulfonate
Test material form:
other: solid
Details on test material:
- Physical state: yellowish pale powder
- Storage condition of test material: room temperature, dry place

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 27-29 days
- Weight at study initiation: 84-89 g
- Acclimation period: approximately 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 16 May 2012 To: 2 July 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The formulation was prepared daily.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed (in RTC Study no. 91450) to confirm that the proposed formulation procedure was acceptable and the stability of formulation was satisfactory. The formulations were found to be stable for 24 hours at room temperature.
Samples of the formulations prepared in weeks 1 and 4 of the study were analysed to check the homogeneity and concentration. The results of these analyses were within the acceptability limits.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study no. 91450). The software used for this activity was the Analyst 1.5.2 (AB Sciex).
Duration of treatment / exposure:
a minimum of 4 consecutive weeks
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 (low and mid dose group)
10 (control and high dose group; five animals of each group were subjected for the 2 week recovery period)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were chosen by the Sponsor as 100, 300 and 1000 mg/kg bw/day, based on information from a preliminary, non GLP compliant study (RTC Study no. 91260EXT).
- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule and cage side observations: Throughout the study, all animals were checked for mortality early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Once before commencement of treatment (data not tabulated, but kept together with the raw data) and once daily during the study, each animal was observed and any clinical signs recorded. During the first 4 days more than one observation was carried out in order to choose the time of the daily observation taking into account the presence, if any, of post-dose reactions. Observations were performed approximately 30 minutes after dosing during the treatment and in the morning during the recovery period. All clinical signs were recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, daily during the first week of treatment, weekly thereafter and just prior to necropsy

FOOD CONSUMPTION: Yes
- The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The interval between allocation and treatment initiation was less than a full week.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of Week 4 of treatment, just prior to necropsy and at the end of Week 2 of the recovery period, from all surviving recovery animals
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: all surviving male and all surviving female animals from each main phase group
- Parameters checked: differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), haematocrit, haemoglobin, heinz bodies, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean red blood cell volume, platelets, prothrombin time, red blood cell count, reticulocyte count, white blood cell count.
Blood samples were collected and analysed in the same order. The blood samples collected were divided into tubes as follows:
EDTA anticoagulant: for haematological investigations
Heparin anticoagulant: for biochemical tests
Citrate anticoagulant: for coagulation tests

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of Week 4 of treatment, just prior to necropsy and at the end of Week 2 of the recovery period, from all surviving recovery animals
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes. Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
- How many animals: all surviving male and all surviving female animals from each main phase group
- Parameters checked: A/G ratio, albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bile acids, calcium, chloride, creatinine, gamma glutamyltransferase, globulin, glucose, phosphorus, potassium, sodium, triglycerides, total bilirubin, total cholesterol, total protein, urea

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of Week 4 of treatment, just prior to necropsy
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- How many animals: all surviving male and all surviving female animals from each main phase group
- Parameters checked: appearance, bilirubin, blood, glucose, ketones, pH, protein, specific gravity, urobilinogen, volume; the sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: crystals, epithelial cells, erythrocytes, leucocytes, spermatozoa and precursors, other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Clinical Observations (Functional Observation Battery tests) - Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena.
- Battery of functions tested: The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
- Grip strength and sensory reactivity to stimuli - Once during Week 4 of treatment and once during Week 2 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed.
- Motor activity assessment (MA) - The motor activity of all animals was measured once during Week 4 of treatment and once during Week 2 of recovery by an automated activity recording. Measurements were performed using a computer generated random order.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
From all animals completing the scheduled test periods, the organs indicated in Annex 1 of study protocol were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

HISTOPATHOLOGY: Yes
Samples of all the tissues listed in Annex 1 of study protocol were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In addition, the testes and the epididymides of control and high dose group animals were dehydrated and embedded in paraffin wax. Sections of the tissues were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS).
In the first instance the examination was restricted as detailed below:
a) Tissues specified from all animals in the control and high dose groups killed after 4 weeks of treatment.
b) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all animals in the control and high dose groups killed after 4 weeks of treatment.
c) All abnormalities in all main phase groups.
The examination was then extended to include, from all other animals killed after 4 weeks of treatment or 2 weeks of recovery, the stomach in which a possible treatment-related change was observed at the high dose level.


Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Results and discussion

Results of examinations

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:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Leucocytosis was recorded in treated males from all treated groups, with a dose-related trend (considered to be secondary effect)
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:
effects observed, treatment-related
Description (incidence and severity):
restricted to forestomach of high dose males
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
restricted to forestomach of high dose animals
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study.
No clinical signs were seen during the study.

BODY WEIGHT AND WEIGHT GAIN
No changes in body weight of toxicological importance were observed during the study in treated animals, compared to controls. No differences in terminal body weight were seen at the end of treatment in both sexes.

FOOD CONSUMPTION
No effect on food consumption was observed.

HAEMATOLOGY
Dosing phase
Leucocytosis was recorded in treated males from all treated groups, with a dose-related trend. Changes were 13% to 61% and involved all white blood cells, with the exception of eosinophils. No changes were recorded at coagulation test.
Recovery phase
Leucocytosis was no longer observed after 2 weeks of recovery.

CLINICAL CHEMISTRY
Dosing phase
Increments of both transaminases (alanine aminotransferase and aspartate aminotransferase) and in bile acids in some males and decreases in bile acids and increases in triglycerides in some females receiving 1000 mg/kg bw/day were seen at the end of treatment. Changes not considered to indicate liver injury.
Recovery phase
Transaminase enzymes, bile acids and triglycerydes values of treated animals were comparable with controls.

URINALYSIS
Dosing phase
No changes were recorded.

NEUROBEHAVIOUR
Neurotoxicity assessement of animals at removal from the cage and in an open arena, and the sensory reactivity to different stimuli, as well as the motor activity performed at the end of treatment period, did not show differences between the groups.

ORGAN WEIGHTS
No differences in organ weights were seen at the end of treatment in both sexes.

GROSS PATHOLOGY
Final sacrifice after 4 weeks of treatment
Treatment-related changes were seen in the forestomach (i.e., nonglandular stomach) of 2 males treated at 1000 mg/kg bw/day. Dark foci were seen in a single animal and in another single animal the mucosa was thickened.
Sacrifice after 2 weeks of recovery period
No treatment-related changes were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
Final sacrifice after 4 weeks of treatment
Treatment-related findings were found in the forestomach (i.e., nonglandular stomach) of animals treated at 1000 mg/kg bw/day. The changes consisted of mostly diffused mucosal hyperplasia (grades mild to moderate), usually associated with hyperkerathosis (grades minimal to mild).
These changes were usually associated with focal to diffused subchronic inflammation located in the submucosa. In two males receiving 1000 mg/kg bw/day focal ulcers were noted. No other treatment related changes observed.
Sacrifice after 2 weeks of recovery period
Treatment related changes were still observed in the forestomach (i.e., nonglandular stomach) of the animals previously treated with 1000 mg/kg bw/day. The changes consisted of diffused mucosal hyperplasia (grade mild), usually associated with hyperkerathosis. This effect was noted in 5/5 males and 2/5 females.

OTHER FINDINGS
No effects were noted on the spermatogenic cycle.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects with regard to systemic toxicity (forestomach effects are considered `first site of contact`and thus local effects which are not relevant for human health hazard / risk assessment)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this study, the high-dose level of 1000 mg/kg bw/day could be considered the NOAEL (No Observed Adverse Effect Level). Effects seen in the forstomach of animals at this dose-level are representing local irritative effects (port-of-entry) and are not considered relevant for human health hazard / risk assessment.
Executive summary: