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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Test Item: Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts)
- BASF Test Item No.: 01/0686-2
- Batch Number: 12000229U0
- Purity: 100% (UVCB, for details see analytical report No. 12L00196)
- Expiration Date: February 21, 2013
- Physical state, appearance: Liquid, yellowish
- Storage conditions: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: Young adult rats, approximately 12 weeks old at starting and 14 weeks at mating
- Weight at study initiation: Males: 375 - 445 g; Females: 211 - 272 g
- Housing: Rodents were group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 – 23.5°C
- Humidity (%): 30 - 61 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd., in the following way:
The required quantity of the test item was weighed into calibrated mixing vessels and was warmed up to 40ºC on a heated stirrer plate. Dosing solutions were made by adding the required amount of warm (40ºC) distilled water to achieve the desired concentrations (3, 10 and 30 mg/mL) of the test item for each dose level (30, 100 and 300 mg/kg bw/day, respectively) with continuous stirring using a magnetic stirrer for approximately 20 minutes. After 20 minutes the heating was switched off and the formulation will be allowed to reach room temperature on the plate with continuous stirring. Prior to and during administration to the animals, the dose formulation(s) were stirred on a magnetic stirrer at room temperature.
Formulations were prepared fresh prior to administration to animals. No stability of the test item in the vehicle was assessed.

VEHICLE
- Justification for use and choice of vehicle: water was a suitable vehicle
- Concentration in vehicle: 0, 3, 10, 30 mg/mL
- 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 of test item formulations for concentration and homogeneity was performed at Test Site using a validated ICP/AES method to determine the sulfur content. Top, middle and bottom triplicate samples were taken from test item formulations on 3 occasions, at the beginning, approximately mid and end of the treatment period, one duplicate set to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in triplicate from the Group 1 (control) solution for concentration measurements.

Two sets of samples were transferred from CiToxLAB Hungary Ltd. to the Test Site for analysis of concentration (Groups 1-4 solutions) and homogeneity (Groups 2-4, conducted by comparative analysis of top, mid, bottom samples of the same solution). The samples were dispatched at room temperature, by courier to the following address:
To the attention of Stefan Bauer, B.Sc., Chemical Analytics
Seibersdorf Labor GmbH, 2444 Seibersdorf, Austria

In addition from the first occasion two middle samples were taken from all three levels and sent to the test site for method validation. All samples were digested and filled up the same way like in the main study. From the resulting solutions at least five different dilutions were prepared giving similar end concentrations for all concentration steps. All resulting solutions were measured against an external standard. This procedure covered the whole analysis process and proved the independence of dilution. For correctness of the data a standard addition experiment was applied, two different concentrations, carried out as triplicates each.
(Additional middle samples were sent at the second occasion for method validation, but were not used.)

Description of the analytical method:
An aliquot of the sample was digested with appropriate acid, filled up to a definite volume and after proper dilution measured with ICP/AES.

Acceptance criteria of the concentration analysis set according to the analytical method validation were 100 ± 15% and all samples results were within this range and varied between 89.4 and 100.4%.

Any samples not employed in the primary analysis (back-up samples) are stored frozen (approximately - 20 ºC) at CiToxLAB Hungary Ltd., until it is determined by the analyst and Study Director that are not required for confirmatory analysis, prior to finalization of the study report. These samples will then be discarded and their disposition will be recorded in the raw data.
Duration of treatment / exposure:
Male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 29 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:

No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.

- Rationale for animal assignment (if not random): random
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon (pm) as practical during the working day, as no peak period of effects was noted after dosing during the first days of treatment.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.

On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then weekly, in the morning (am) or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.

BODY WEIGHT: Yes
All adult animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination.

Parent females were weighed on gestation Days GD0, 7, 10, 14, 17 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before termination).


FOOD CONSUMPTION:
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly.

CLINICAL PATHOLOGY
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter has been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

For terminal blood sampling all selected animals (subgroup B, 5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (1.4 mL blood in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.

HAEMATOLOGY: Yes
Haematology and blood clotting times
The following parameters were evaluated in selected animals:
Red Blood Cell (erythrocyte) count, White Blood Cell (leukocyte) count, Haemoglobin concentration, Haematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration, Red Cell (erythrocyte) volume, Platelet (thrombocyte) count, Mean Platelet Thrombocyte volume, Reticulocyte count, Neutrophil, Lymphocyte, Monocyte, Basophil, Eosinophil, Large Unstained Cells, Activated Partial Thromboplastin Time, Prothrombin Time
Blood smears were prepared (fixed, then stained) for all subgroup B animals. The smears were not examined, as no adverse effects were observed at the standard haematology evaluation.

CLINICAL CHEMISTRY: Yes
The following parameters were evaluated in selected animals:
Blood sugar concentration, Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration, Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ration, Aspartate Aminotransferase activity, Alanine Aminotransferase activity, Alkaline. Phosphatase – activity, Gamma Glutamyltransferase -activity, Bile acids
Additional sera samples for potential measurement of TSH, T3 and T4 were stored (approximately - 20ºC) at CiToxLAB Hungary Ltd. Since there were no treatment-related effects on the thyroid, these examinations were not necessary.

NEUROBEHAVIOURAL EXAMINATION: Yes
Randomly selected animals, 5 males and 5 females/group, “subgroup A”:

Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 25 am, females, on PND 3 am).

Selected animals were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay, grip strength and motor activity.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots of the hind paws was measured.

Fore/hind grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs are allowed to grip the support bar and gently pulled back until they release the bar; the device measures the maximum grip strength. This was performed at least 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support.

Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. At the first instance the data from the high dose and control groups was evaluated for distance travelled in 5 minute segments. No treatment related changes were observed in the high dose groups, therefore the evaluation was not extended to the lower groups. The results are tabulated for information with individual data for each parameter (distance travelled, average speed, % of time spent immobile, number of rearings and average duration of rearing).

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.

Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
Sacrifice and pathology:
GROSS PATHOLOGY/HISTOPATHOLOGY: Yes

PATHOLOGY
Terminal procedures and macroscopic evaluation

Gross necropsy was performed on each animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under anaesthesia (details are presented in "Details of Other Materials") by exsanguination.

After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

The number of implantation sites and of corpora lutea was recorded in the females as applicable.


Organ weight measurements
At the time of termination, body weight and weight of the following organs of all adult animals was determined:

- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids

Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised.

Tissue preservation and microscopic evaluation


On completion of the macroscopic examination the following tissues and organs were retained from all animals.

Gross findings
Adrenal glands
Animal identification 1
*Aorta (thoracic and abdominal)
Brain 2
Clitoral gland / Preputial gland
Epididymides
Eyes with the optic nerves 7
*Oesophagus
Femur with marrow incl. joint
Heart 3
Kidneys
Large intestine 4
*External lachrymal glands
Harderian glands
Liver
Lungs with bronchi 5
Lymph nodes 6
*Larynx, *Nasopharynx
Ovaries with oviduct
*Pancreas
Pituitary
Prostate
*Salivary glands 10
Sciatic nerve
Seminal vesicles with coagulating glands
Skeletal muscle (quadriceps)
*Skin, subcutis and mammary gland (inguinal)
Small intestine 8
Spinal cord (cervical, lumbar, and thoracic levels)
Spleen
Sternum with marrow
Stomach
Testes
Thymus
Thyroid with parathyroids 7
*Tongue
Trachea
Urinary bladder
Uterus 9
Vagina

1. Fixation and preservation only.
2. Cerebral cortex, midbrain, cerebellum and medulla.
3. Section including both ventricles and atria, septum with papillary muscle.
4. Caecum, colon and rectum.
5. Lungs of euthanized animals were infused with formalin.
6. Mandibular and mesenteric.
7. Parathyroids and optic nerves were examined histologically only if present in routine sections.
8. Duodenum, ileum and jejunum with Peyer’s patches.
9. Horns, body and cervix.
10. Salivary glands (including mandibular, sublingual and parotid glands)
*Organs and tissues marked by asterisk were not examined as no macroscopic changes indicative of a compound-related effect were observed.

The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, and all other organs in 10% buffered formalin solution.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• found dead male no.4008
• all macroscopic findings (abnormalities), except of minor order from all animals
• reproductive organs of all animals of the control and high dose group and all females (uterus, cervix, clitoral gland, ovary and vagina) that failed to deliver healthy pups (no. 3501, 3505, 3508 and 3510, Mid dose).

From all the males of the Control and High dose group additional slides of the testes were prepared to examine staging of spermatogenesis.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Examination of tissues from Low and Mid groups 2 and 3 were not performed, except treatment-related gross findings.

Pups euthanized at PND 4 were carefully examined externally for gross abnormalities.
Statistics:
Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.7, as appropriate. Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations. The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
One male (no. 4008) at 300 mg/kg bw/day (High dose) was found dead on Day 14 of the study. Hunched position, piloerection, liquid faeces and red nasal discharge preceded the death of this animal. In addition a marked body weight loss (17%) was observed during the week before the death. No cause of death was established for this animal, although the causative role of the test item cannot be excluded as characteristic test item related changes were noted in the stomach and adrenals, in addition to meningoencephalomyelitis noted microscopically.

In one male (no. 4008) at 300 mg/kg bw/day (High dose) hunched position, piloerection, liquid faeces and red nasal discharge were observed between Days 12-13, before death of this animal on Day 14 of the study.

In females at 300 mg/kg bw/day, slightly decreased activity was noted in 2 of 12 animals (no. 4506 and 4507) on one or three occasions, on Day 36 or between Days 42-44, respectively). In addition, in both females soft/liquid faeces was observed.

Transient, slight to moderate salivation was recorded immediately following dosing at 300 mg/kg bw/day (in 4 of 12 males and 8 of 12 females). This sign was observed in males up to 4 occasions from Day 12 up to Day 15 and in females from 1 up to 11 occasions, between Days 12-15 and in 1-2 animals on few occasions from Day 21 up to day 41. Slight salivation was observed on one occasion in one male at 100 mg/kg bw/day. This observation was considered to be procedure related.
Thin fur was observed in 1 of 12 females at 300 mg/kg bw/day from Day 22 up to 42. This observation was regarded as incidental finding. However, all animals were clinically normal.

BODY WEIGHT AND WEIGHT GAIN
Following treatment at 300 mg/kg bw/day, transient slight body weight loss (approximately 4%) was observed in males during the first week of treatment. Mean body weight value was approximately 8% lower, than control on Day 7 (p<0.01). In spite of higher body weight gain during Week 2 (p<0.01), the body weights were consistently lower than the controls throughout the treatment period (by approximately 4%) and the differences attained statistical significance on Day 14 (p<0.05). Compared to the controls, mean value at the termination was approximately 3% lower, but the differences were not statistically significant.

In females at 300 mg/kg bw/day, transient body weight loss was observed in 4 of 12 animals, during the first week of treatment. The differences attained statistical significance for mean body weight gain on Week 1 (p<0.01) and on Week 2 (p<0.05, better gain).

Compared to controls, lower body weight gain values were recorded for entire gestation period GD0-20 and between GD0-7 and GD14-20 (p<0.01). This resulted in lower mean body weight on GD 20 (by 10%) and Post Partal Days 0 and 4 (by 15%). The differences attained statistical significance (p<0.01).

Body weight and body weight gain values of males and females at 30 and 100 mg/kg bw/day were comparable to the control throughout the treatment period.

FOOD CONSUMPTION
Compared to control, significantly lower food consumption was noted for males at 300 mg/kg bw/day during Week 1 (p<0.01) and slightly lower during Week 2 and following the mating period to Day 21 (p<0.05).

For the High dose females, lower than control food consumption was measured on Week 1, from GD7 up to PP0 (p<0.01) and PP Days 0-4 (p<0.05).

Food consumption of males and females in the at 30 and 100 mg/kg Low and Mid dose groups were comparable with the controls, with exception of slightly lower food consumption noted for females at 100 mg/kg (Mid dose) during GG20 and PP0 (p<0.01).

HAEMATOLOGY
In males, there were no test item related adverse effects at any dose level, all parameters falling with the normal physiological ranges for the age, strain and sex of rats on test.
Although slightly higher erythrocyte distribution width values were measured at 300 mg/kg (p<0.05), which can be a consequence of slightly higher reticulocyte counts, the changes were minimal and not regarded as adverse.

In females significantly lower values for erythrocyte counts, hemoglobin concentration and haematocrit were recorded for single animals in High and Mid dose groups (no. 4506 and 3508). Compared to control mean, the erythrocyte counts were lower by approximately 40-35% and hemoglobin concentration by 46-28% for the High and Mid dose female, respectively. The changes were accompanied by marked increase in reticulocyte counts. In addition, both animals had increased relative neutrophil granulocyte and decreased relative lymphocyte counts. This finding may be related to the potential stomach ulceration detected at histopathology.

Minor changes, on occasion attaining statistical significance were noted in the haematology parameters in males (i.e. higher mean platelet counts at 100 and 300 mg/kg) or in females (i.e. higher mean corpuscular haemoglobin at 100 mg/kg). As the mean and individual values remained within the normal ranges, the differences were of low magnitude, therefore the finding was not considered toxicologically significant or to reflect an adverse effect of the test item.

Blood coagulation

There was no adverse effect of the test item on blood clotting parameters Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PTT).

CLINICAL CHEMISTRY
Alanine aminotransferase (ALT) activity was slightly higher in both males and females at 300 mg/kg bw/day (High dose). The values were within the physiological range. The differences attained statistical significance for males.
For High and Mid dose males, slightly elevated serum urea concentration was measured, and the differences were statistically significant.
Lower values of creatinine and calcium concentration were measured in all treated males.
In females, slightly lower serum glucose level was measured at 300 and 100 mg/kg bw/day.
In one female at 300 mg/kg bw/day (no. 4506) markedly decreased albumin concentration (by approximately 30%, compared to the control mean), elevated alkaline phosphatase and gamma GT activity and low glucose and cholesterol concentration were found. In this female multifocal, necrotizing ulceration in the cecum and focal capsular fibrosis in the liver was found.
In addition, minor changes were noted in cholesterol concentration in females at 100 mg/kg, (higher values, related to relatively low individual values of control). The difference attained statistical significance (p<0.05), however were related to individual animal variation and not to treatment with the test item.

NEUROBEHAVIOUR
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.
Increased vocalization was observed on occasion throughout all groups of males and in control, Low and High dose groups of females at the modified Irwin test (functional observation battery). However, there were no treatment-related differences to the Control, and the incidence was similarly low in all groups including control, and these variations were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations.

At evaluation of the landing foot splay test, slightly lower values were recorded for females at 300 mg/kg bw/day (High dose), when compared to control means. The differences attained statistical significance (p<0.05). When evaluating individual data, the values of 4 of 5 females were within the control values and only performance of one female (4504) was lower.
It should be noted, that control values were relatively high.
In males, the landing foot splay values were comparable with the control mean in all dose groups.

At evaluation of grip strength results of the fore and hind limbs, slightly lower mean value for fore limbs was recorded for males at 300 mg/kg bw/day (High dose). The difference attained statistical significance (p<0.05). The values of hind limbs were at control level.

In females, slightly lower values for hind limbs were recorded at 100 and 300 mg/kg bw/day, when compared to control means (p<0.05 and p<0.01, respectively). The control mean was relatively high and high individual variation was observed for these values. At 300 mg/kg bw/day, the mean value of fore limb was also slightly lower, than control, but did not attain statistical significance. Significantly lower performance of the test for both limbs was noted in 1 of 5 females (4507).

Taking into account the relative low magnitude of the differences on one hand and the general toxicity observed in this dose level on the other, these effects were no considered indicative for a neurotoxic action of the test compound but rather attributable to general toxicity.

During evaluation of motor activity, the total travelled distance was slightly lower in females at 300 mg/kg bw/day (High dose). The mean values were lower than the controls by approximately 30%, when evaluated for the 60 minute duration. The difference was not statistically significant. When evaluated as individual values, the control values for overall duration were in the range of 6001 and 8519 cm, while values of the high dose females were in the range of 4128 and 8486 cm. In the control group there was one female (1501) with markedly high individual value (16813 cm). The individual values recorded during 5-minute segments were comparable with these of the control. The patterns of activity were similar in all experimental groups including controls.

No significant differences to control were noted at this test for the High dose males.

ORGAN WEIGHTS
At 300 mg/kg bw/day (High dose) test item related differences were found in adrenals weights in both sexes and thymus weights in females.

Compared to control means, the weight of adrenals in males and females at 300 mg/kg bw/day was higher, by approximately 22 and 20% for the absolute values in males and females respectively. The differences attained statistical significance for both absolute and relative values. Slightly higher adrenal gland weight was found in females at 100 mg/kg bw/day. The differences were in the range of 9-15% (absolute and relative values) and attained statistical significance for relative values only.

Lower absolute and relative weights of thymus were noted in females at 300 mg/kg bw/day. The differences were in the range of 23-35% and attained statistical significance.

Females at 300 mg/kg bw/day had lower absolute weights of heart, kidneys, liver and uterus weight.

Compared to the controls, higher spleen weights were recorded for females at 100 mg/kg bw/day.

GROSS PATHOLOGY
Macroscopic findings considered related to the test item were observed in the stomach and in adrenal glands in both males and females at 300 and 100 mg/kg bw/day (High and Mid dose). The stomach changes consisted of thick mucosa with raised areas in the non-glandular region part of the stomach and were noted in all males and in 10 of 12 females at 300 mg/kg bw/day and in 8 of 12 males and in 3 of 12 females at 100 mg/kg bw/day. The change was diffuse mostly in the high dose animals, while focal in mid dose animals.

Enlargement of adrenal glands was noted in 3 of 11 males and 2 of 12 females at 300 mg/kg bw/day and in 3 of 12 males at 100 mg/kg bw/day. Bilateral, diffuse pale discoloration was noted in 1 of 12 females in Mid and High dose.

Spleen enlargement was observed in females only, in 3 of 12 females at 100 mg/kg bw/day and 1 of 12 females at 300 mg/kg bw/day.

In one High dose female (4506) multifocal ulceration was observed in cecum, confirmed by histopathology.

In the animal found dead the following observation was made:
adrenals were enlarged, mucosa of the non-glandular region of the stomach was thick and greyish, the lungs were dark red.

Incidental gross observations

Other observations were regarded as incidental or common findings.


HISTOPATHOLOGY
Test item related microscopic effects were observed in the adrenal glands, stomach, thymus and spleen at dose levels of 100 and 300 mg/kg bw, with clear dose relationship.

Bilateral minimal to mild diffuse hypertrophy of cortical cells in the zona fasciculata was noted in 3/24 Mid and 6/23 High Dose rats. This change was characterized by enlarged cytoplasm and/or nuclei of cortical cells. In the stomach non-glandular mucosa, minimal to moderate parakeratotic hyperkeratosis was observed in 11/24 (mainly minimal/mild intensity) Mid and 23/23 (mainly mild/moderate severity) High Dose animals. This lesion was occasionally associated with influx of mixed cell infiltrate/inflammation, ulceration or erosion predominantly in the High Dose group rats.

In one female at 300 mg/kg bw/day multifocal, necrotizing ulceration in the cecum and focal capsular fibrosis in the liver was found.

Minimal to moderate lymphoid atrophy of thymus in 1/24 Mid and 7/23 High Dose rats was accompanied with decreasing number of lymphocytes leading to decreased cell density, decreased compartment size (more pronounced in cortex), which corresponded with organ weight changes related brain weight.

In the spleen, there was increasing of degree in extramedullary hematopoiesis from minimal/mild seen in Control females to minimal to moderate (predominantly mild/moderate) in High Dose females.

No test item-related microscopic changes were noted in the reproductive organs, brains or pituitaries at a dose level of 300 mg/kg bw. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.

In the found dead animal (4008) the following observations were made:
Mild bilateral hypertrophy of cortical cell in the zona fasciculata of the adrenal glands, mild diffuse parakeratotic hyperkeratosis of the stomach non-glandular mucosa and mild lymphoid atrophy of thymus observed by light microscopy, were considered to be test item administration-related. Agonal congestion of the lungs corresponded with gross pulmonary changes. Additionally, mild subacute meningoencephalomyelitis of the brain/spinal cord noted microscopically could have attribution to the death of this male.

Other observations were incidental or a common background.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic and local effects
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; clinical chemistry; gross pathology; organ weights; histopathology; local effects in the stomach

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Administration of the test itemsulfonium compounds, C11-14-alkylbis (hydroxyethyl), 2-hydroxyethyl sulfates (salts) at dose levels of 30, 100 and 300 mg/kg body weight/day to Wistar rats for at least 29 consecutive days was associated withthe following relevant findings:

 

Treatment at 300 mg/kg bw/day caused transient body weight loss (most pronounced in males) andreduction of body weight gain by21% inmales and in females by approximately 20-26% during premating and gestation periods,mild changes in clinical chemistry parameters (slight increase inalanine aminotransferase activityin both sexesslightly elevated serum urea concentration in males andlower serum glucose level in females)minimal to moderate parakeratotic hyperkeratosis occasionally associated with influx of mixed cell infiltrate/inflammation, ulceration or erosion in stomach, hypertrophy of cortical cells in the zona fasciculate of adrenal glands corresponded with enlargement of adrenal glands and lymphoid atrophy in the thymus.An increasedintrauterine and postnatal mortality,lower number of born pups,increased mortality and lower body weight of pupswere considered to be probably secondary to maternal toxicity.

 

Treatment at 100 mg/kg bw/day caused parakeratotic hyperkeratosis changes in mucosa of stomach, hypertrophy of cortical cells in the zona fasciculate of adrenal glands and thymus atrophy. Increased intrauterine and postnatal mortality,lower number of born pups andincreased mortality of pupswere considered to be probably secondary to maternal toxicity.

 

No adverse effects or test item related findings were observed at the low dose level.

 

In conclusion, for systemic and developmental toxicity as well as local effects, the NOAEL of sulfonium compounds, C11-14-alkylbis (hydroxyethyl), 2-hydroxyethyl sulfates (salts) administered by the oral route to Wistar rats for at least 29 consecutive days is considered to be the low-dose level of 30 mg/kg bw/day.

 

The NOAEL for reproductive performance and fertility was 300 mg/kg bw/d in male and female Wistar rats.

Applicant's summary and conclusion