Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422, rat, gavage, NOAEL Systemic, maternal = 30 mg/kg bw/d, NOEAL fertility/reproduction = 300mg/kg bw/d (BASF SE, 2013, 85R0686/01R034)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
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
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
Route of administration:
oral: gavage
Vehicle:
water
Details on 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
Details on mating procedure:
Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 9 days of the mating period. A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Two females (1507 and 4510, control and High dose, respectively were kept with males for 15 days, as no signs of coitus were found, in spite of successful mating). For these females duration of pregnancy was not established.

One male (no. 4008) died during the pre-mating period, therefore its pair female was placed with another male of the same group (no. 4003) on Day 3 of the mating period, as this male has completed mating.

Mating pairs were clearly identified in the data.
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
Details on study schedule:
N/A
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
actual ingested
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
Parental animals: Observations and examinations:
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

OTHER:
Observation of the delivery process, offspring and nursing instinct

Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.

In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-partum, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination (with lung floating test when applicable) in order to identify the cause of death if possible.
All observed abnormalities were recorded.
All pups were terminated on PND4.
Oestrous cyclicity (parental animals):
The Estrous cyclicity was examined
Sperm parameters (parental animals):
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.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
The pups (offspring) were monitored for any behavioural changes.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-partum, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination (with lung floating test when applicable) in order to identify the cause of death if possible.
All observed abnormalities were recorded.
All pups were terminated on PND4.
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
All F1 offspring was terminated on Day 4 post-partum.
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination (with lung floating test when applicable) in order to identify the cause of death if possible.
All observed abnormalities were recorded.
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.
Reproductive indices:
Male Mating Index, Female Mating Index, Male Fertility Index, Female Fertility Index, Gestation Index



Offspring viability indices:
Survival Index, Pre-implantation mortality, Intrauterine mortality, Total mortality, Sex ratio
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

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).

REPRODUCTIVE FUNCTION AND REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Oestrus cycle, reproductive ability assessment and indices:
There was no effect of treatment on the oestrus cycle or reproductive parameters.
There were no differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with test item administration. The mating and fertility indices were 100% in all groups. The gestation index was 100% in control and Low groups while 92% in Mid and High dose animals.
There were two females in Mid and High dose, which were pregnant but not delivered living pups (i.e. 3510 dam from Mid dose had 5 implantations and no pups, while 4507 delivered stillborn pups only). Although the gestation index values are comparable with concurrent control data in Wistar rats, the causative role of the test item cannot be excluded.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 days of pairing (cohabitation).
In one control female (no. 1505) the duration of mating period was 9 days, almost only dioestrus picture characterized the oestrus cycle, however this female successfully paired on Day 9.

Evaluation of the gestation, parturition and post-partal period:
There was mild effect of treatment noted during gestation, parturition or the post-partal period considered related to the maternal toxicity.

The mean duration of pregnancy was similar in the control and test item treated groups and varied from 22.5 days (control), 22.5 days (30 mg/kg, Low dose), 22.7 days (100 mg/kg, Mid dose), to 22.8 days (300 mg/kg, High dose group). However, there were two females in Mid and High dose (3505 and 4504, respectively), which delivered on Day 24. All the parturitions were normal.

Compared to control, the number of corpora lutea was comparable to the control mean in all treated groups.

The number of implantation sites was comparable to the control mean at 30 mg/kg (Low dose group) and slightly lower (by approximately 11-13%) at 100 and 300 mg/kg (Mid and High dose groups). The differences were not statistically significant and were attributable to individual values of single animals (3510 and 4510).

There were no significant differences or effects that could be ascribed to treatment on the pre-implantation mortality values (%).

Compared to control, the intrauterine mortality was increased at 100 and 300 mg/kg bw/day (Mid and High dose groups) and the difference attained statistical significance (p<0.05 and p<0.01). Also the mean postnatal mortality was increased in these groups and was attributed to 100% mortality noted in 3 of 12 females in Mid dose and 4 of 12 High dose females. The differences were not statistically significant and are considered to be possibly related to maternal toxicity.

The total mortality was increased at 100 and 300 mg/kg and the differences attained statistical significance in the High dose group (p<0.01).

Compared to control, the number of born pups was decreased at 100 and 300 mg/kg (Mid and High dose groups) by approximately 17 and 25%, respectively. This mean decrease was mostly attributed to individual values of one Mid dose dam (3510, without pups) and 2 of 12 High dose dams (4506 and 4510, with 5 or 1 pups). The difference attained statistical significance for High dose (p<0.01, litter mean).

The differences seen in foetal/pup mortality are considered to be a consequence of maternal toxicity.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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.
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on fertility, reproductive organs and reproductive performance were observed
Dose descriptor:
NOAEL
Remarks:
systemic and developmental toxicity, local effects
Effect level:
30 mg/kg bw/day
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, intrauterine and foetal/pup mortality due to maternal toxicity
Remarks on result:
other: Generation: P and F1 (migrated information)
VIABILITY AND CLINICAL SIGNS (OFFSPRING)
Compared to control, the number of liveborn pups was decreased at 100 and 300 mg/kg (Mid and High dose groups). Decreased number of live born pups was observed in 4 of 11 Mid dose dams and in 5 of 12 High dose dams. The difference attained statistical significance (p<0.05 and p<0.01, respectively for mean litter values).

Increased mortality of pups was noted on post natal Days 0 and 4 at 100 and 300 mg/kg and was contributed to the litters of the same dams.
The viability indices on postnatal Day 4 were 97% in control, 98% in Low dose, 67% in Mid dose and 65% in the High dose animals.

No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical, external or gross macroscopic examinations of the pups. No efficient or lack of suckling was observed in 30 pups in Mid dose (4 litters) and in 19 pups in High dose (4 litters). Three Mid dose pups were cold and one was cyanotic. Injured or missing tail was observed in 2 Low dose pups.

In single pups at 100 and 300 mg/kg, diffuse dark red discoloration of the lungs was observed at necropsy on PND0.

The differences seen in the Mid and High groups were considered to be a consequence of maternal toxicity, individual observations at the low dose were not considered to be related to treatment.

The sex ratios were similar in the Control and treated groups.

BODY WEIGHT (OFFSPRING)
Compared to control, slightly lower mean body weights were noted at 300 mg/kg on postnatal Days 0 and 4.
On postnatal Day 0, the mean values were approximately 8.7% lower, than controls (litter basis) and the differences attained statistical significance (p<0.05).

The body weight gain of these pups was at the control level between Days 0-4, however, lower mean body weight was still observable On Day 4 (approximately 3.6% on litter basis).

Mean body weights values evaluated for all pups were similar at 30 and 100 mg/kg bw/day treated groups. Slightly lower, than control mean body weight gain was recorded at 100 mg/kg bw/day (p<0.05).

The differences seen in the Mid and High groups were considered to be a consequence of maternal toxicity

GROSS PATHOLOGY (OFFSPRING)
The observations were incidental or a common background.
Dose descriptor:
NOAEL
Remarks:
systemic and developmental toxicity, local effects
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight, clincal chemistry; gross pathology; organ weights, histopathology etc
Remarks on result:
other: Generation: P and F1 (migrated information)
Reproductive effects observed:
not specified

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Additional information

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed to obtain information on the toxicity of the test item following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. The study was conducted according to OECD 422 guideline and GLP (CiToxLab, 2013).

The study also included a reproductive/developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum. 

Male and female Wistar rats (12 animals per sex and dose level) 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. The dose level were 0, 30, 100 and 300 mg/kg bw/day.The control animals were treated with the vehicle only (distilled water).

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, at least weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation and clinical chemistry. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Selected organs were subjected to histopathology examination.

Analysis of test item formulations for concentration and homogeneity was performed three times during the treatment period (during the second, fourth and last weeks of treatment), using validated ICP/AES method. All formulations were found to be in the range of 92 to 99% of nominal concentration and were homogeneous. No test item was detected in the control samples. Based on these results, the test item formulations were considered suitable for the study purposes.

Results

 

One High dose male treated at 300 mg/kg bw/day was found dead on Day 14.

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 tomeningoencephalomyelitis noted microscopically.

 

Clinical signs were limited to slightly decreased activity and soft/liquid faeces noted in 2 of 12 females at 300 mg/kg bw/day and were occasionally observed towards the end of the treatment period. No clinical signs were noted for males, with the exception of clinical signs preceded death of the High dose male (hunched position, piloerection, liquid faeces and red nasal discharge).

 

During neurological assessment, slight differences were recorded for animals treated at 300 mg/kg bw/day. Compared to controls, lower values in the grip strength tests were recorded for both males and females and in landing foot splay tests for females. Taking into account the small magnitude of the effects, these differences were considered to be attributable to general toxicity and not to represent neurotoxicity. No effect was observed in motor activity investigation.

 

Transient slight body weight loss was observed at 300 mg/kg bw/day in males and in some of the females. In spitehigher body weight gain during Week 2, the body weights of males were consistently lower than the controls. The mean terminal body weight was approximately 3% lower,and overall body weight gain was reduced by 21%.

Females hadlower body weight gain during the premating period (by 20%) andentire gestation period (by 26%).This resulted in lower mean body weight on GD 20 (by 10%) and Post Partal Days 0 and 4 (by 15%).

 

Food consumption was decreased at 300 mg/kg bw/day in both males and females.

 

There was no effect of treatment on haematology parameters in males.

In females marked decreases in erythrocyte counts, hemoglobin concentration and haematocrit were recorded for 1/5 High and 1/5 Mid dose animals. The changes were accompanied by increased reticulocyte counts. In addition, both animals had increased relative neutrophil granulocyte and decreased relative lymphocyte counts.

Mild changes in clinical chemistry parameters consisted of slight increase inalanine aminotransferase activityin both sexes at 300 mg/kg bw/day, slightly elevated serum urea concentration in males andlower serum glucose level in femalesin the High and Mid dose. 

 

There was no effect of treatment noted during evaluation of the reproductive parameters,however increasedintrauterine and postnatal mortality values,lower number of born pupsandincreased mortality of pupswere noted at100 and 300 mg/kg bw/day and were attributed to 4/12 Mid dose and 5/12 High Dose dams.

 

Pups born at 300 mg/kg hadslightly lower body weighton postnatal Days 0 and 4, but body weight gain until postnatal Day 4 was comparable with controls.

 

There were no adverse effects on the F1 offspring clinical signs or development.

 

At necropsy,thick mucosa of the non-glandular region part of the stomach was 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 and was related tominimal to moderate parakeratotic hyperkeratosis occasionally associated with influx of mixed cell infiltrate/inflammation, ulceration or erosion predominantly in the High dose group.In one High dose female multifocal, necrotizing ulceration in the cecum and focal capsular fibrosis in the liver was found.

 

In the adrenal glands bilateral minimal to mild diffuse hypertrophy of cortical cells in the zona fasciculata was noted in 3/24 Mid and 6/23 High dose ratsandcorresponded with organ weight changes and with macroscopic observations (enlargement).

 

Minimal to moderate lymphoid atrophy was found in thymus in 1/24 Mid and 7/23 High Dose rats.

 

Conclusion

 

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

 

Treatment at 300 mg/kg bw/day caused transient body weight loss (most pronounced in males) andreduction of body weight gain by 21% 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 corresponding to enlargement of adrenals 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. Increase 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 (30 mg/kg bw/day.

 

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.


Short description of key information:
NOAEL(oral, OECD 422) = 30 mg/kg bw/day (CiTox LAB Hungary, 2013)

Effects on developmental toxicity

Description of key information

OECD 414, rat, gavage, NOAEL maternal = 50 mg/kg bw/d, NOAEL development/teratogenicity = 200 mg/kg bw/d (BASF SE 2018, 30R0686/01R034)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
oct 2017 - july 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: supplied by Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks

- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%.
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours both

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations
were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a calibrated beaker and intensely mixed with a magnetic
stirrer until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0.15g/100 ml, 0,5g/100 ml and 2,0g/100 ml
- Amount of vehicle (if gavage): up to 9,85 ml see above
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany,
under the responsibility of the study director of this test facility (BASF Project No. 02Y0054/08X094).
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day
(GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the
experimental phase) and first administration (GD 6).

The body weight of the pregnant animals on day 0 varied between 143.0 – 192.3 g.
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always
at approximately the same time in the morning.
Frequency of treatment:
daily, GD6-GD19
Duration of test:
till GD 20
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on available study 85R0686/01x043
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs
of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

DETAILED CLINICAL OBSERVATIONS: Yes
see above

BODY WEIGHT: Yes
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION: Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No data


POST-MORTEM EXAMINATIONS: Yes

Pathology
On GD20 all surviving dams were sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross
pathology, special attention being given to the reproductive organs and the gastrointestinal tract.
The following weights were determined in all animals sacrificed on schedule:
1. Adrenal glands
2. Liver
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
3. All gross lesions
4. Adrenal glands
5. Liver
6. Stomach

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Cesarean section
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology
The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI
from uteri from apparently non-pregnant animals and the empty uterus horn in the
case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the
uterus had been opened)
Fetal examinations:
- External examinations: Yes
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia.
Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded.
Thereafter, the fetuses were sacrificed. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.
- Soft tissue examinations: Yes
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR.
- Skeletal examinations: Yes
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL.
- Head examinations: No data
Statistics:
Statistical analyses were performed according to the following:

Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened
uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation
loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

with

Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means


Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

with

Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions


Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

with

Pairwise comparison of each dose group with the control group using the WILCOXONtest (one-sided) for the hypothesis of equal medians


Weight parameters

with

Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians
Indices:
The conception rate (in %), preimplantation loss (in %), postimplantation loss (in %) was calculated
Historical control data:
Historical control data of reproduction toxicology and Historical control data of pathology is given as supplement
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All females of the high-dose group (200 mg/kg bw/d) and five females of the mid-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred
in the respective animals only within the 2-hour examination interval (i.e. 0-2h after treatment) and was initially observed on GD 7 (200 mg/kg bw/d) or GD 19 (50 mg/kg bw/d).
Furthermore, nearly all (18 out of 25) females of the high-dose group (200 mg/kg bw/d) ploughed nose-first into bedding during the treatment period (within 0-2h after treatment). This
finding was initially observed on GD 14. Both findings are considered to be treatment-related, most likely as a local irritation of the upper digestive tract or as a result of the bad taste of the
test substance/vehicle preparation. They are not considered to be a sign of systemic toxicity.
During the 5-hour examination interval (i.e. >2<5h after treatment), no clinical signs or changes
of general behavior were detected in any female of all test groups.
No further clinical signs or changes of general behavior, which may be attributed to the test
substance, were detected in any female at dose levels of 15, 50 or 200 mg/kg bw/d during the
entire study period.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 15, 50 or 200 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (15, 50 and 200 mg/kg bw/d) were generally comparable to the concurrent control group
throughout the entire study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test group 3 (200 mg/kg bw/d) was statistically significantly reduced on GD 6-8, GD 10-13 and GD 19-20 (up to -10 % in comparison to the concurrent control). If calculated for the entire treatment (GD 6-19) period, the high-dose dams consumed about 4% less food than the controls. Although the decrease during GD 6-19 was not statistically significant, three treatment intervals showed a statistically significant decrease in food consumption. Overall, this was assessed as treatment-related and adverse.
The mean food consumption of the dams in test groups 1 and 2 (15 and 50 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Uterus weight
The mean gravid uterus weights of the animals of test groups 1-3 (15, 50 and 200 mg/kg bw/d) were not influenced by the test substance.

Absolute organ weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased for adrenal gland and liver.
The minimally, but significantly increased relative liver weights in test group 3 animals were regarded as treatment-related. Both, absolute (+7%, not significant) and relative (+8%, significant)
liver weights were above the range of historical control values. The significantly decreased weights of adrenal glands in test group 1 animals as well as the significantly increased weights of adrenal glands in test group 3 animals were regarded as incidental, since there was no dose-response-relationship.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The thickened wall of the forestomach (25/25), glandular stomach (9/25) and duodenum (10/25) as well as the focus and the deposition in the glandular stomach (1/25 each) of test
group 3 animals were regarded as treatment-related.

The enlarged livers in 4/25 animals were regarded as treatment-related and correlated to slightly increased liver weights.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No differences of toxicological relevance between the control and the treated groups (15, 50 or 200 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate,
mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No differences of toxicological relevance between the control and the treated groups (15, 50 or 200 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate,
mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No differences of toxicological relevance between the control and the treated groups (15, 50 or 200 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate,
mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
The combination of different individual findings (a reduction in food consumption, decrease in corrected body weight
gain, thickened wall in stomach and duodenum) at 200 mg/kg bw/d was assessed as adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: The combination of different individual findings (a reduction in food consumption, decrease in corrected body weight gain, thickened wall in stomach and duodenum) was assessed as adverse.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (15, 50 and 200 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
No soft tissue malformations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus in test group 3 (200 mg/kg bw/d) had a skeletal malformation (misshapen lumbar vertebra). This isolated finding occurred in one single fetus. No
ontogenetic pattern is recognizable for this individual malformation nor was there any cluster of this individual malformation seen in the other offspring of the test groups. Thus, no association
to the treatment was assumed. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to
several parts of fetal skeletons and appeared in the majority of cases without a relation to dose.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups.
The overall incidences of skeletal variations were comparable to the historical control data.
Details on embryotoxic / teratogenic effects:
External and soft tissue malformations did not occur in any of the fetuses in this study. There was noted one skeletal malformation in test group 3 (200 mg/kg bw/d), i.e. misshapen
lumbar vertebra. This isolated finding occurred in one single fetus (94-08) which had additionally a lower body weight (2.5 g) compared to the group mean value (3.8 g). No ontogenetic
pattern is recognizable for this individual malformation nor was there any cluster of this individual malformation seen in the other offspring of the test groups. It also does neither form
a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. Thus, there is no evidence for
any association of this finding with the treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no embryotoxic / teratogenic effects at highest concentration
Key result
Developmental effects observed:
no
Conclusions:
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose of 50 mg/kg bw/d.
The NOAEL for prenatal developmental toxicity is the highest tested dose of 200 mg/kg bw/d.
Executive summary:

Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts) was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered

as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 15, 50 and 200 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group.The state of health of the animals was checked each day.

The following test substance-related adverse effects/findings were noted: Test group 3 (200 mg/kg bw/d):

Reduced mean food consumption (GD 6-8, GD 10-13 and GD 19-20; up to -10 % in comparison to the concurrent control) together with a decreased corrected body weight

gain (-15% compared to control) in combination with the following findings in pathology: thickened wall of the forestomach, thickened wall of the glandular stomach,

thickened wall of the duodenum, focus in the glandular stomach, deposition in the glandular stomach. No test substance-related adverse effects on gestational parameters or fetuses.

Test group 2 (50 mg/kg bw/d) and Test group 1 (15 mg/kg bw/d): No test substance-related adverse effects on dams, gestational parameters or fetuses

Therefore under the conditions of this prenatal developmental toxicity study, the oral administration of Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts) to

pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 200 mg/kg bw/d caused signs of maternal toxicity. The combination of different individual findings (a reduction in food consumption, decrease in corrected body weight gain, thickened wall in stomach and duodenum) was assessed as adverse. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose of 50 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is the highest tested dose of 200 mg/kg bw/d.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Additional information

Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts) was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered

as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 15, 50 and 200 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group.The state of health of the animals was checked each day.

The following test substance-related adverse effects/findings were noted: Test group 3 (200 mg/kg bw/d):

Reduced mean food consumption (GD 6-8, GD 10-13 and GD 19-20; up to -10 % in comparison to the concurrent control) together with a decreased corrected body weight

gain (-15% compared to control) in combination with the following findings in pathology: thickened wall of the forestomach, thickened wall of the glandular stomach,

thickened wall of the duodenum, focus in the glandular stomach, deposition in the glandular stomach. No test substance-related adverse effects on gestational parameters or fetuses.

Test group 2 (50 mg/kg bw/d) and Test group 1 (15 mg/kg bw/d): No test substance-related adverse effects on dams, gestational parameters or fetuses

Therefore under the conditions of this prenatal developmental toxicity study, the oral administration of Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts) to

pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 200 mg/kg bw/d caused signs of maternal toxicity. The combination of different individual findings (a reduction in food consumption, decrease in corrected body weight gain, thickened wall in stomach and duodenum) was assessed as adverse. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose of 50 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is the highest tested dose of 200 mg/kg bw/d.

Justification for classification or non-classification

Based on the available data, the test substance is not classified with regard to toxicity to reproduction according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP), respectively.