Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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

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 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
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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)

Results: F1 generation

Details on results (F1)

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.

Effect levels (F1)

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)

Overall reproductive toxicity

Reproductive 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