trisodium 2-(2-sulfoethoxy)ethane-1-sulfonate ethenesulfonate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Procured from Charles River, USA and bred at IIBAT animal house facility.
- Age at study initiation: Young adult rats, between 12 and 14 weeks old. Females were virgin.
- Weight at study initiation: Males: 326-407g; Females: 241-287g. The weight variation in animals involved in the study were not exceeded ± 20 % of the mean weight of each sex.
- Fasting period before study: no data
- Housing: Females were housed in groups in cages, each cage containing five animals in pre mating period. Males were housed individually during pre mating. One male and one female kept together in a cage until the confirmation of mating occurs. After confirmation of mating females were caged individually.
- Diet (e.g. ad libitum): Animals received ad libitum standard gamma irradiated pelleted food supplied by M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India.
- Water (e.g. ad libitum): Reverse osmosis water was provided to animals ad libitum.
- Acclimation period: Five days prior to experiment in the test room.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 19.6 – 22.0°C
- Humidity (%): relative humidity between 50 – 59%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h light and 12h dark

IN-LIFE DATES: From: To:78

Route of administration:

oral: gavage

Vehicle:

other: Distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

VEHICLE
distilled water

Details on mating procedure:

1:1 (one male to one female) The females were housed with the same males until pregnancy occurs. Each morning the females were examined for the presence of sperm and/or vaginal plug. Day 0 of pregnancy is defined as the day on which vaginal plug or sperm is observed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quantification was done by HPLC-UV method. Stability study showed that 87 % recovery was observed for all concentrations tested, i.e. 500; 1000; 2000 mg/kg bw

Duration of treatment / exposure:

28 days

Frequency of treatment:

The test substance was administered daily. Dosing of both the sexes began 2 weeks prior to mating, after acclimatization. Dosing was continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum dosing period of 28 days has been completed and then sacrificed.
Dosing of mating confirmed females was continued throughout gestation and including day 3 post partum.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Eighty animals were randomized into four groups, each consisting of 10/sex.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of range finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no data
- Section schedule rationale (if not random):

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: no data
- Time schedule: Males were observed for 28 days and females were observed during premating (14 days), mating (1-6 days), and gestation (22-23 days), parturition and till day 4 post partum.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Once before the first exposure and once a week thereafter, detailed clinical observations will be made in all animals. These observations will be made in the home cage, handling observations and in a standard arena (open field), preferably at the same time, each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of individual male and female were recorded prior to the administration of the test substance (day 0) and weekly thereafter. During pregnancy, females were weighed on days 0, 7, 14 and 20 of pregnancy and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed consumption was recorded daily during pre-mating (cage wise), pregnancy and lactation in females. The Feed consumption was not recorded during mating period. In males, feed consumption was recorded daily only during pre-mating.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood:Blood was collected from orbital sinus in heparinised vials (for biochemistry) as well as in vials containing EDTA (for hematology). In males, it was done at the end of the pre-mating period and just prior to, the procedure for killing the animals. In females, it was done at the end of the pre-mating period and just prior to, the procedure for killing the animals.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: females and males but not specified how many
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology


URINALYSIS: No data


NEUROBEHAVIOURAL EXAMINATION: No data


OTHER:

Sperm parameters (parental animals):

parameters examined in parent generations: testes weight, an depididymus weigt

Litter observations:

the duration of gestation was recorded and is calculated from day 0 of pregnancy to the day of parturition. each litter was examined as earliest after delivery to establish the numbers and sex of pups,still births,live births,runts and the presence of gross abnormalities. live pups were counted and sexed,litters were weighed within 24 hours of parturition ( day 0post postpartum) and day 4 post partum.sex ratio (m/f) was calculated

Postmortem examinations (parental animals):

All surviving animals were sacrificed by CO2.
Parent animals were examined microscopically for any abnormality or pathological change special attention was paid to the organs of the reproductive system.
All gross pathological changes were recorded individually for each animal.
the number of implantation sites were recorded,and the counting of corpora lutea was done

Postmortem examinations (offspring):

the pups will be euthanized by injection of 0.12 ml Thiopenal sodium solution on day 4 post partum, and were carefully examined externally for gross abnormalities.
Histopathology/organ weight: not conducted.

Statistics:

Body weight, food consumption, detail signs of toxicity, FOB, hematology, biochemistry and organ weight, corpora lutea, implantations, litter data of rats belonging to the experimental groups assured for homogeneity. When the data is homogenies then it was analysed using ANOVA. (Student’s Newman – Keul’s Test was employed for post - hoc comparison). When the data is not homogeneous it was analysed with Kruskal-Wallis One-Way ANOVA on Rank basis.

Reproductive indices:

reproductive indices: Sex ratio's were calculated
Reproductive parameters were mean gestation lenght , mean litter size,number of implantation losses,mean corpura lutea

Offspring viability indices:

mean live litter size, mean litter weight

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

No morbidity/mortality was observed in any of the animals during the entire observation period

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistical , significant changes were observed in body weight of males and females of treated groups when compared to control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Test substance related statistically changes were not observed in fed consumption of males and females of treated groups when compared with control group.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

Statistically significant changes were not observed in hematology parameters of all dose groups when compared with control, except a slight decrease in MCV of G4 male at day 28, which is well within normal limit and considered of no biological significant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Statistically significant changes were not observed in biochemistry parameters of all dose groups when compared with control.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance related effects were observed in functional observation battery parameters in treated groups of males and in the control group of males.However in G2 females auditory function (acoustic startle) parameter was increased when compared to control group female animals

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

detailed histological examination was performed on all animals of the control and the HD group on the ovaries, no test substance related effects were observed

Reproductive function: sperm measures:

not specified

Description (incidence and severity):

detailed histological examination was performed on all animals of the control and the HD group on the testes and epididymis, no test substance related effects were observed

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance related effect were observed on mating/mating period of females treated groups , when compared to the control
There was also no test substance related effect on gestation length of dams in any of the treated groups , when, compared to the control.
no test substance related effect was observed on mean implantation in any of the treated groups of animals when compared to the control.
no test substance related effect was observed on mean litter size in any of the treated groups of animals when compared to the control at day 0 and day 4 post partum.
no test substance related effect was observed on mean litter weight in any of the treated groups of animals when compared to the control.
no test substance related effect was observed on the number of delivered with live pups in any of the treated groups of animals when compared to the control.
no test substance related effect was observed on loss offspring (pre implantation,post implantation, and post natal) of in any of the treated groups of animals when compared to the control.
no test substance related effect was observed on sex ratio of the pups in any of the treated groups of animals when compared to the control.

no effects observed in any of the examined parameters

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Gross external examination of live pups sacrificed on day 4 post-partum did not reveal any abnormality that could be attributed to the treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No test substance related effect was observed on loss of offspring (pre implantation,post implantation, and post natal) in any of the treated groups when compared with the control group of anaimals

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross external examination of live pups sacrificed on day 4 post partum did not reveal any abnormality that could be attributed to the treatment

Histopathological findings:

no effects observed

No test substance related external effect of the pups was observed.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Based on the above findings it can be concluded that the dose 2000 mg/kg b.w. of Sodium Vinyl Sulphonate Solution (Provichem 2202) is non-toxic to Wistar rats in respect to combined repeated dose and Reproduction/Developmental toxicity, therefore the NOAEL of the test substance is regarded as >=2000 mg/kg body weight.

Executive summary:

In a combined Repeated  Dose Toxicity  study  with the reproductive/developmental  toxicity screening Test (OECD 422), performed to GLP potential toxicity of the test item was evaluated in the rat.

In a preliminary 7 days range finding study the test item was administrated orally to three  dose groups with three males and  three  females. 500, 1000 and 2000 mg/kg Bw/day of the test item was administrated dally for 7  days, and observed for mortality and sings of toxicity daily. Control animals were  treated similar but with distilled water. No test item related mortality , signs of toxicity or macroscopic observations were observed. Based on the results of the  DRF study the same three  doses ie 500, 1000, an 2000 mg/kg Bw/day were elected for the main study.

The  test item was administrated gavage to the rats of both sexes two weeks prior to mating, after acclimatization. Dosing was continued in both sexes during mating period. Males were further dosed after the mating period until the minimum dosing period of  28 days has been  completed and then     sacrificed. Dosing of mating confirmed females was continued throughout gestation and including day 3 post-partum. All  dams  were allowed to litter naturally and the size, weight of litter and sex of litter-mates were recorded at parturition( day 0) and at day 4 post-partum.

Females  which failed to deliver were sacrificed 26 days after day of mating period.

No animals died (table 13) in any test article group ,and no test article  related changes  were observed in clinical signs (Table 6,13 and  14), body weight( Table 1,2,3,15,16 and 17), feed  consumption (Table 4,5,19 and 20) ,Heamatology (Table 7, and 22)  ,Biochemistry( table 8 and 23), Functional observation  battery (FOB)  (Table 12 and 27), bone marrow cytology and reproduction performance like fertility, mating period (Table 11and  24), gestation length (table 11and 25), mean corpora lutea( table 26), mean implantation losses (table 10 and 26), mean litter size( table 10 and 26), mean litter weight ( table 10,11,26) and sex ratio of offspring.(table 11,26)

Absolute and relative  weight changes of testis and epididymis and other organs of treated groups were not statistically different when compared  to control. Gross and histopathological observations of parent animals did not reveal any test substance related lesion in any of treated group. No test substance related external observations of pups were observed.

From the above observations  it can be concluded  that 500, 1000, and 2000 mg/kg Bw/day of sodium vinyl sulphonate is non toxic to the  parent and the pup born to them and therefore ,the NOAEL of the the test  substance  is arrived at 2000 mg/kg Bw

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

In an OECD Guideline 414 (Prenatal Developmental Toxicity Study) the potential of Reaction mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate to induce developmental toxicity was determined after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via drinking water to time-mated female New Zealand White Rabbits from Days 7 to 29 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 1671,5031 and 16711 ppm (0, 99, 326,1256 mg/kg bw/day),

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chantillon sur Chalaronne france )
- Age at study initiation: 17-19 weeks
- Weight at study initiation: 3000 to 4300 g
- Fasting period before study: no
- Housing: Caging:
Cages with perforated floors (Ebeco, Germany, dimensions
67 x 62 x 55 cm) equipped with water bottles.
Animals will be individually housed.
These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records.
Cage Identification: Color coded cage card indicating at least Test Facility Study No., group, animal identification number.

- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):at libitum ( exposure via drinking water)
- Acclimation period:5 days prior to commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21 °C
- Humidity (%): 40-70 %
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light):12-hours light and 12-hours dark

IN-LIFE DATES: From: To:Day7-to day 29 post coitium

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

The amount of test material incorporated in the drinking water will be kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test material intake will be estimated based on the body weight and water consumption values.
Drinking water will be refreshed daily. Batch codes will be entered via Provantis as additional check to verify the dosing procedure according to Standard Operating procedures.

Group nr 1: vehicle (control) dose level 0 ppm, Corrected dose ppm Anticipated Dose level 0mg/kg/day.
Group nr 2: test item(*), 1671 ppm, Corrected dose 4965 ppm Anticipated Dose level 100 mg/kg/day.
Group nr 3: test item(*), 5013 ppm, Corrected dose 14895 ppm Anticipated Dose level 300 mg/kg/day.
Group nr 4: test item(*), 16711 ppm, Corrected dose 49650 ppm Anticipated Dose level 1000 mg/kg/day.

(*) test item: The test material has a dry residue percentage of 33.65%, therefore dose calculations were corrected for purity using a correction factor of 2.971.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was based on the analytical procedure validated for the test material in Test Facility Study No. 20343523.
Accuracy and homogeneity were determined for drinking water preparation samples prepared for use in Week 1.
For determination of accuracy of Group 1, duplicate samples were taken at random position from the municipal tap water, tapped on 23 May 2022.
For determination of accuracy of Group 2, 3 and 4, duplicate samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the drinking water preparation samples.
Calibration solutions were injected in duplicate. Study samples and QC samples were analyzed by single injection.

Chemical analyses of drinking water formulations were conducted once during the study and confirmed that drinking water formulations of test material were prepared accurately and homogenously.

The mean recoveries of the QC samples were within the criterion range of 90 -110%. It demonstrated that the analytical method was adequate for the determination of the test material in the study samples.

Accuracy
In the Group 1 drinking water preparation samples, no test material was detected.
The concentrations analyzed in the drinking water preparation samples of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90 -110% of target concentration).

Homogeneity
The drinking water preparation samples of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalarone , France). The females arrived on Day 1 post-coitum ( Dag 0 post coitium is defined as the day old successful mating) . they were 17-19 weeks old and weighed between 3000-4300 g at initiation of administration.

Duration of treatment / exposure:

The test item and control were administrated to the appropriate animals by inclusion in the drinking water at libitum from day 7to day 29 post coitum, inclusive.

Frequency of treatment:

At libitum.

Duration of test:

Animals were euthanized on day 29 post-coitum.

Dose / conc.:

0 ppm

Dose / conc.:

1 671 ppm

Remarks:

Corresponding to 99 mg/kg bw/day

Dose / conc.:

5 031 ppm

Remarks:

Corresponding to 326 mg/Kgbw/day

Dose / conc.:

1 671 ppm

Remarks:

Corresponding to 1256 mg/kg Bw/day

No. of animals per sex per dose:

Number of females =22 per group/dose

Control animals:

yes, concurrent no treatment

Details on study design:

Justification for Test System and Number of Animals
The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The Study Plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. and approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Maternal examinations:

Mortality/Moribunditry: FO -generation .At least twice daily beginning upon arrival through termination/release.
Except on days of receipt and necropsy where frequency will be at least once daily. Animals will be observed within their cage unless necessary for identification or confirmation of possible findings

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily starting on Day 7 post-coitum up to the day prior to necropsy. On the day of necropsy, an additional clinical observation will be performed. Animals will be observed within their cage unless necessary for identification or confirmation of possible findings. Cage debris will be examined to detect premature birth, if applicable.
BODY WEIGHT: Yes F0-generation.
- Time schedule for examinations: Animals were individually weighed on Days 3, 7, 9, 12, 15, 18, 21, 24, 27 and 29 post coitum
FOOD CONSUMPTION (if feeding study): Yes, F0-generation, Daily from Day 3 post coitum onwards.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Time schedule for examinations: Daily from Day 3 post coitum onwards. Water consumption will be determined by weighing the individual drinking bottles. Test material intake will be calculated as concentration of test material in drinking water against relative water consumption.
- T- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 post-coitum. Animals will be euthanized by intravenous injection of sodium pentobarbital. If necessary, the animal will be refrigerated to minimize autolysis.

- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) will be subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

Ovaries and uterine content:

Ovaries and uterine content
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
For Female No. 46 that was sacrificed before planned necropsy, these findings are reported in the individual data tables only.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Blood sampling:

- Plasma: No
- Serum: No
- Volume collected : not applicable
- Other:

Fetal examinations:

Terminal Procedures – F1-Generation Method of Euthanasia – F1-Generation Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube. Fetal Examinations (scheduled) – F1-Generation Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations, as described in the following sections. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). External Examinations – F1-Generation Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for fetuses of animals sacrificed before planned necropsy). Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions, a gross external examination was performed. Visceral Examinations – F1-Generation All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. Abnormalities were not collected and fixed in 10% buffered formalin. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice. All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons. Skeletal Examinations – F1-Generation All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses from all groups. All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
 
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and at least 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Indices:

Reproduction and Developmental Variables
For each group, the following calculations were performed:
Preimplantation loss (%): ([(number of corpora lutea - number of implantation sites)] / [number of corpora lutea]) x 100

Postimplantation loss (%): ([(number of implantation sites - number of live fetuses)] / [number of implantation sites]) x 100

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ([number of viable fetuses affected/litter] / [number of viable festuses/litter]) x 100

Historical control data:

Historical Control Data are available (Appendix 17)

Clinical signs:

no effects observed

Description (incidence and severity):

No test material-related clinical signs were noted during the treatment period.table 1
Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Dermal irritation (if dermal study):

not specified

Description (incidence and severity):

Oral drinking water study.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights, body weight gain and weight gain adjusted for gravid uterus of test material-treated animals were considered to be unaffected.Table 2,3,4 and Figure 1

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test material-related changes in food consumption were recorded. Table 5 and Figure 2

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

No test material-related changes in water consumption were recorded at 1671 ppm.
At 5013 ppm, mean water consumption was higher during the majority of the treatment period (not reaching statistical significance), resulting in an overall mean water consumption that was 7.7% above control (not reaching statistical significance).
At 16711 ppm, mean water consumption was higher during the complete treatment period, reaching statistical significance over Days 7-15 post-coitum, resulting in an overall mean water consumption that was 23.8% above control (statistically significant).Table 6 and figure 3

Mean test material intake over the study period was as follows:

Test Material Intake:
Groep 2: dose Dose level 1671 ppm, Anticipated Dose level 100 mg/kg/day, Mean over Means Intake (mg test material/kg body weight/day) 99 (88-113 range)
Groep 3: dose Dose level 5013 ppm, Anticipated Dose level 300 mg/kg/day, Mean over Means Intake (mg test material/kg body weight/day) 326 (302-358 range)
Groep 4: dose Dose level 16711 ppm, Anticipated Dose level 1000 mg/kg/day, Mean over Means Intake (mg test material/kg body weight/day) 1256 (1039-1468 range) Table 7

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Weight of gravid uterus was considered not to be affected. tabel 4.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alternations . table 8

Number of abortions:

no effects observed

Description (incidence and severity):

he numbers of pregnant females, corpora lutea and implantation sites, and pre- and postimplantation loss in the control and test material-treated groups were considered unaffected by treatment with the test material. table 9,10.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre- and postimplantation loss in the control and test material-treated groups were considered unaffected by treatment with the test material.(table 10)

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre- and postimplantation loss in the control and test material-treated groups were considered unaffected by treatment with the test material. (Table 9)

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre- and postimplantation loss in the control and test material-treated groups were considered unaffected by treatment with the test material. (Table 10)

Dead fetuses:

no effects observed

Description (incidence and severity):

table 10

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 256 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related effects on fetal body weights (both sexes) noted up to 5013 ppm.
At 16711 ppm, mean male fetal body weights were 6% lower compared to control. Comparable mean female fetal body weights were comparable to the control group. (table 10)

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment with the test material. (appendix 15)

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related effects on litter size in any group.
The slightly higher litter size in the 16711 ppm group was attributed to the slightly higher number of corpora lutea and hence slightly higher number of implantations at this highest dose level when compared to control (all not reaching statistical significance).(Appendix 15)

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test material-related external malformations and variations were observed.
In this study, external malformations were observed in 1 (1), 0 (0), 1 (1) and 2 (2) fetuses (litters), from the control, 1671, 5013 and 16711 ppm groups, respectively.
Fetus No. 08-L4 (control) presented with a domed head, cleft palate, forepaw hyperflexion and hindpaw malrotation. As these findings were observed in the control group, they were spontaneous in origin.
The malformations “hindpaw malrotation”, “omphalocele” or “herniated umbilicus” were observed in single fetuses at 5013 and 16711 ppm only (Nos. 66-L3, 85-L7 and 78-L12, respectively). As these malformations were observed in a single fetus and/or at comparable incidences as in the concurrent control or available historical control data , they were considered not to be related to treatment with the test material.
The external variation “distended abdomen” was observed for Fetus Nos. 08-L4 (control) and 61-L4 (5013 ppm). Both fetuses presented with visceral malformations likely to be related to the distended abdomen. Based on the low incidence and/or the exclusive occurrence in a control fetus, this was not indicative of a relationship to the treatment with the test material.Table 11,12

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test material-related skeletal malformations and variations were recorded.
In this study, skeletal malformations were observed in 1 (1), 2 (2), 1 (1) and 1 (1) fetuses (litters), from the control, 1671, 5013 and 16711 ppm groups, respectively. Malformations concerned the frontal skull bone, ribs, sternebra and vertebra with the most common findings being fused frontal and (severely) fused sternebra, both observed in a single litter at 1671 and 5013 ppm each in this study. However, due to the infrequent occurrences and the lack of a dose related incidence, they were considered not to be related to treatment with the test material.
Skeletal variations occurred in the skull, hindlimb, pelvic girdle, (supernumerary) ribs, sternebra and vertebra. Amongst skeletal variations a statistically significantly higher mean incidence of short cervical supernumerary ribs was observed at 1671 ppm when compared to the control group. However, it was concluded that the lack of a dose-related incidence ruled out a relationship to the treatment with the test material.
At 16711 ppm, more full thoracolumbar supernumerary ribs were observed than when compared to the control group. The mean fetal incidence remained within the available historical control data2 and was therefore considered unrelated to treatment with the test material.
In all other cases, variations were observed either infrequently, in incidences comparable to the control group and/or in the absence of a dose-related incidence trend. Therefore, they were considered not to be related to treatment with the test material.Table 11,12

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related visceral malformations and variations were recorded.
In this study, visceral malformations were observed in 7 (5), 2 (2), 3 (2) and 2 (2) fetuses (litters), from the control, 1671, 5013 and 16711 ppm groups, respectively.
Most malformations concerned the kidney that was malpositioned, misshapen and/or small. These occurred in two control fetuses (Nos. 11-L3 and -R11), and in one fetus at 1671 and 5013 ppm each (Nos. 26-L2 and 61-L4, respectively). As the preponderance of kidney malformations were observed in the control group coupled with a less severe observation at 5013 ppm and absence at 16711 ppm, it was deemed not to be related to the test material.
Another notable visceral malformation was retroesophageal right subclavian artery, observed in each group treated with the test material once or twice. This was concluded not to be related to treatment with the test material as a dose-response was lacking and the mean fetal incidence fell within the range of historical control data . All other visceral malformations observed in test material-treated fetuses were observed only once (twice in the case of ventricular septum defect) without a dose response and were therefore deemed to be chance occurrences.
Visceral variations in this study were observed across all groups in a wide range of structures, including heart, innominate artery, kidney, liver, lung, spleen, gonads and ureter. In all cases, due to infrequent observation, observation in the control group (only) and/or a lack of dose-response, it was considered that these findings were not related to treatment with the test material.
One incidental finding of diverticulum (stomach) was observed in the high dose group. However, at the isolated incidence this was considered a chance discovery.Table 11,12

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

no test material-related teratogenic effects observed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 256 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable because of methodological limitations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study in time mated female New Zealand White rabbits, the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Reaction Mass of Disodium 2,2’-oxydiethanesulfonate and Sodium Ethenesulfonate were established:
Maternal and Developmental NOAELs: > 16711 ppm, corresponding to an actual mean test material intake of 1256 mg/kg bw/day.

Executive summary:

The objectives of this study were to determine the potential of Reaction Mass of Disodium 2,2’-oxydiethanesulfonate and Sodium Ethenesulfonate to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via the drinking water to time-mated female New Zealand White rabbits from Days 7 to 29 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 1671, 5013 and 16711 ppm (corresponding to an anticipated test material intake of 100, 300 and 1000 mg/kg/day, respectively), based on the results of the Dose Range Finder (Test Facility Study No. 2032272).

Chemical analyses of drinking water formulations were conducted once during the study and confirmed that drinking water formulations of test material were prepared accurately and homogenously.

The following parameters and end points were evaluated in this study for the F₀-generation: mortality/moribundity, clinical signs, body weights, food consumption, water consumption, test material intake, macroscopic examination, and uterine contents (including corpora lutea, implantation sites, pre- and postimplantation loss and number of live and dead fetuses).

In addition, the following parameters were determined for the F₁-generation: fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Animals were exposed to 1671, 5013 and 16711 ppm, corresponding with a mean test material intake of 99, 326 and 1256 mg/kg/day, respectively.

Maternal findings.

No mortality  occurred during this study.

At 5013 and 16711 ppm, mean water consumption was dose-dependently higher than control, which was considered non-adverse.

No test material-related changes were noted in the remaining maternal parameters investigated in this study (i.e., mortality/moribundity, clinical appearance, body weight, food consumption, macroscopic evaluation, corpora lutea, uterine contents including implantation sites and pre‑and postimplantation loss.

Fetal Findings

At 16711 ppm, body weights of male fetuses were lower than control. Based on the magnitude of change (<10% lower than control) and as this effect was not observed in female fetuses, this was considered to be non-adverse.

No test material-related changes were noted in any of the developmental parameters investigated in this study (i.e., litter size, sex ratio, external, visceral and skeletal malformations and developmental variations).

Administration of Reaction Mass of Disodium 2,2’-oxydiethanesulfonate and Sodium Ethenesulfonate by drinking water was well tolerated up to 16711 ppm.

In conclusion, based on these results the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Reaction Mass of Disodium 2,2’-oxydiethanesulfonate and Sodium Ethenesulfonate were established:

Maternal and  Developmental NOAEL's >16711 ppm, corresponding to an actual material intake  of 1256 mg/kg bw/day

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Specific details
Reaction mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate
Batch lot number:202108300033
Dry weight content : 33.65 %, correction factor is 2.971. The test item is a multi-constituent
Composition.
Constituent1:Sodium ethylene sulphonate cas 3039-83-6: 75.01 %
Constituent 2 : Isethionate bisether, disodium salt cas 63440-92-6: 16.52%
Impurity 1:Isethionate sodium cas 1562-00-1:4.12%
Impurity 2:Sodium ethionate, disodium salt cas1562-03-4:0.62 %
Impurity 3:Sodium sulfate cas 7757-82-6 :2.76 %
Impurity 4:Sodium ethandisulfonate disodium salt cas 5325-43-9:0.64 %

Species:

rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hannover Wistar Rats
- Age at study initiation: 11-15 weeks old
- Weight at study initiation: 2013-316 g
- Fasting period before study: no (pregnant anaimals)
- Housing: individual housing. Polycarbonate cages (Makrolong type MIII, height 18 cm), sterilized wooden fibers as bedding material.(lignocel S8-15 ,JSR J Rettenmaier & Söhne GmbH+Co KG, Rosenberg Germany) Equipped with water bottles.
- Diet (e.g. ad libitum): at libitum, animals were provided with SM R/M from SSNIFF Spezialdiäten GmbH, D-59494 Soest Germany. Pellets
- Water (e.g. ad libitum): ad libitum, Municipal tap water, Periodic analysis of the drinking water is performed. And the results of these analysis are on file on the test facility.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 40-70 %
- Air changes (per hr): At least 10 changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light and 12 hours dark.

IN-LIFE DATES: From: To: 17Juni 2022 to 15 Juli 2022.

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

Municipal tap water, Periodic analysis of the drinking water is performed. And the results of these analysis are on file on the test facility.

Details on exposure:

The test item was formulated in the vehicle (water).
Drinking water preparations will be divided into aliquots where required to allow to be dispensed on each dosing occasion.
Adjustment will be made for specific gravity of the test material. A correction will be made for the purity/composition of the test material.A factor of 2.971 will be used to correct for purity/composition of the test material.

Preparation Details

Dose formulation:Test material
Procedure:Diluted in the required amount of municipal water
Frequency of preparation: At least weekly
Storage conditions set to maintain: 21 °C

Stability in the vehicle:

Stability analyses performed previously in conjunction with the method development and validation (Test Facility Study No. 20343523) demonstrated that the test material is stable in the vehicle ( water) when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20343523.


Stability for at least 8 days at room temperature (15 to 25°C) under normal laboratory light conditions in drinking water is confirmed over the concentration range 250 to 25550 ppm (solutions, 5000 mL bulk volume), project 20343523.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Drinking water preparation samples will be collected for analysis as indicated in the following table. Additional samples may be collected and analyzed at the discretion of the Study Director.

Drinking Water Preparation Sample Collection Schedule

Occasion: Week 1 of treatment: (range: 27 Jun 2022 – 03 Jul 2022)
Concentration (M):All groups, 2 x approximately 500 mg
Homogeneity (TMB): Groups 2 and 4, 2 x approximately 500 mg
Sampling form :Dosing container

M = sample collected from approximately Middle; TMB = sample collected from approximately Top, Middle and Bottom; t = time
a Sampling from dosing containers drinking water batches that will be used in Week 1 of administration.
All samples to be analyzed will be transferred (at room temperature) to the analytical laboratory at the Test Facility for same day analysis, where possible or stored for analysis within known formulation stability period.
Analyses described below will be performed using a validated analytical procedure (Test Facility Study No. 20343523).

Results:
Accuracy
The concentrations analysed in the drinking water preparation samples og group2,Group 3, and group 4 were in agreement with the target concentration (i.e. mean sample concentrartions results were within or equal to 90-110 % of target concentration. No terst material was detected in Group 1drinking water preparation samples.

Homogenicity.
The drinking water preparation samples of Group 2and Group4 were homogeneous (i.e coefficient of variation <=10%)

Details on mating procedure:

- Impregnation procedure: purchased pregnant females (arrived at CR on Day 0/1 post coitum)
- If cohoused: not cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: not known , this was done at the supplier
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: both Han Wistar rats
- Proof of pregnancy: vaginal plug
- Any other deviations from standard protocol:non

Duration of treatment / exposure:

From gestation Day 6 to Day 21 post-coitum.

Frequency of treatment:

Ad libitum drinking water study

Dose / conc.:

0 ppm

Remarks:

control group

Dose / conc.:

750 ppm

Remarks:

target dose level 100 mg/kg bw/day

Dose / conc.:

2 000 ppm

Remarks:

target dose level 300 mg/kg bw/day

Dose / conc.:

5 500 ppm

Remarks:

target dose level 1000 mg/kg bw/day

No. of animals per sex per dose:

22 females per dose group.

Control animals:

yes

Details on study design:

Dose selection rationale:
The dose levels in this study were selected to be 0, 750, 2000 and 5500 ppm (corresponding to a target test material intake of 0, 100, 300 and 1000 mg/kg/day, respectively), based on the results of the Dose Range Finder (Test Facility Study No. 20332264).
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: day 21 post-coitum
- Other:

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/viability all parental animals. At least twice daily beginning upopn arrival trough termination/release . Except on day of receipt and necropsy where frequency will be at least once daily.
Animals will be observed within their cage unless necessary for identification or co,confirmation of possible findings.
- Cage side observations checked in table [1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on days 2,6,15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
On days 2,6,9,12,15,18 and 21 post-coitum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes table 5 and figure 2
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No, drink water study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes /
- Time schedule for examinations:Every two days, Quantitatively measured. table 6

Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes, drink water study. table 7


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21post coitum
- Organs examined:

All animals (including animals found dead or sacrificed before planned necropsy and females with delivery prior to necropsy) will be subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities will be recorded, collected and fixed in the appropriate fixative, together with the animal identification; collection of specific macroscopic abnormalities may be omitted only at the discretion of the Study Director.
Each ovary and uterine horn of all animals will be dissected and examined as quickly as possible to determine the following as part of the necropsy procedure:
• The number of corpora lutea.
• The weight of the uterus (not for animals found dead, sacrificed before planned necropsy or that started to deliver).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of early and late resorptions.
• The sex of each fetus based on the anogenital distance, if possible (not for animals found dead, sacrificed before planned necropsy or that started to deliver).
• Placental morphology.
In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique in order to detect any former implantation sites.
For animals found dead, sacrificed before planned necropsy or that started to deliver, these findings will be reported in the individual data tables only.
Necropsy procedures will be performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, will be available.


OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes , table 4
- Number of corpora lutea: Yes , table 13
- Number of implantations: Yes,table 13
- Number of early resorptions: Yes Appendix 18
- Number of late resorptions: Yes Appendix18
- Other:

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected: 1 ml of blood samples is taken on Day 21 post-coitum
- Other:thyroid hormone T3,T4, TSH Appendix 13

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
- Anogenital distance of all live rodent pups:yes

Historical control data:

Historical control data regarding Fetal pathology available in Appendix 21

Clinical signs:

no effects observed

Description (incidence and severity):

No test material-related clinical signs were noted during the treatment period.
Erected fur was noted once for one female at 750 ppm. In the absence of a dose-related trend and at the single incidence observed, this was unrelated to treatment with the test material.
(Appendix 6 and table 1)

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights, body weight gain and gravid uterus adjusted body weight gain of test material treated animals remained in the same range as control over the treatment period.
table 2,3 and 4.Fig 1 and Appendix 5,6,7,8,9.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Mean water consumption over the whole treatment period was 5%, 15% and 50% higher compared to control at 750, 2000 and 5500 ppm, respectively. table 6 Fig 3 Appendix 11.
Mean test material intake over the study period was as follows:
Group 2 Nominal dietary ppm 750 , Mean over mean Intake (mg test material/kg BW/day) 104 (86-115).
Group 3 Nominal dietary ppm 2000, Mean over mean Intake (mg test material/kg BW/day) 295 (239-320).
Group 4 Nominal dietary ppm 5500, Mean over mean Intake (mg test material/kg BW/day) 1110(862-1211).
Water consumption was dose-dependently higher than control at all dose levels. However, since other parameters assessed in this study indicated that animals were in a healthy state, this was considered non-adverse.

Ophthalmological findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

Serum levels of T3, T4 and TSH were considered to be unaffected by treatment with the test material. ( table 8,and Appendix 13).
There were no test material-related alterations in thyroid gland weights.(table 9 and Appendix 14)

Urinalysis findings:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to be related to treatment with the test material.
There were no gross observations in the thyroid glands.
Any findings that were noted among control and/or treated animals were considered to be unrelated to treatment with the test material, as they remained within the range of biological variation for rats of this age and strain, occurred in the control group only and/or lacked a dose relationship.table 10.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test material-related microscopic observations in the thyroid glands.
All the recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations
table 11.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation loss was higher at 2000 ppm (10.67% vs 6.62% in control, not statistically significant). As treatment with the test material started after implantation (Day 6 post-coitum), this was considered not related to treatment with the test material.
Postimplantation loss was higher at 5500 ppm (8.60% vs. 7.09% in control, not statistically significant). As this was mainly attributed to only one female (Female No. 80 with a post-implantation loss of 45%) and no dose-response was noted, this effect was considered not related to treatment with the test material.

Dead fetuses:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

he numbers of pregnant females, corpora lutea and implantations sites in control and test material-treated groups were within the same range and in the range of normal biological variation.
At the time of scheduled necropsy all females were found to be pregnant, except Female Nos. 7, 16, 22 (control), 27, 34, 39 (750 ppm) and 46 (2000 ppm).
table 12, 13 and Appendix 17,Appendix 18

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 110 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal body weights (male, female and all) were considered to be unaffected by treatment with the test material. (table 13 and Appendix18, Appendix 20)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Litter size was considered to be unaffected by treatment with the test material.(table 13 Appendix 18)

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered to be unaffected by treatment with the test material. (table 13 Appendix 18)

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean fetal anogenital distance (male, female and all) was considered to be unaffected by treatment with the test material. (table13 Appzndix 18 ,Appendix 20)

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No test material-related skeletal malformations and variations were recorded.
Skeletal malformations were only seen in two control fetuses. For fetus 5-R10, the skull abnormalities were associated with external malformations of the face and mouth. Moreover, this fetus presented with several other malformations affecting vertebrae, ribs and sternebra. The other fetus (No. 3-L3) had sternoschisis.
All recorded skeletal variations occurred in a diverse array of skeletal structures across all groups. These variations were either scored infrequently, were comparable to the control group, occurred in the absence of a dose-relationship and/or were within the range of available historical control data. Therefore, these skeletal variations were considered not to be related to treatment with the test material.(Table 14,Table15 and Appendix 19)

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test material-related skeletal malformations and variations were recorded.
Skeletal malformations were only seen in two control fetuses. For fetus 5-R10, the skull abnormalities were associated with external malformations of the face and mouth. Moreover, this fetus presented with several other malformations affecting vertebrae, ribs and sternebra. The other fetus (No. 3-L3) had sternoschisis.
All recorded skeletal variations occurred in a diverse array of skeletal structures across all groups. These variations were either scored infrequently, were comparable to the control group, occurred in the absence of a dose-relationship and/or were within the range of available historical control data. Therefore, these skeletal variations were considered not to be related to treatment with the test material.(Table 14,Table15 and Appendix 19)

Description (incidence and severity):

No test material-related visceral malformations and variations were recorded.
Besides a single case of situs inversus in a control fetus (No. 3-L6) which was considered spontaneous in origin, no other visceral malformations were observed in this study.
Visceral variations were limited to supernumerary liver lobes, supernumerary spleens and convoluted and/or dilated ureters. The low incidences and group distribution of these findings did not indicate a relationship to treatment with the test material.
Incidental visceral findings, not otherwise classified as malformations or variations, were not observed in this study.(Table 14, Table 15, and Appendix 19)

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 110 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study in time mated female Wistar Han rats the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate were established:
Maternal and Developmental NOAELs: At least 5500 ppm, corresponding to an actual mean test material intake of 1110 mg/kg/day. ( NOAEL's >1110 mg/kg/day)

Executive summary:

The objectives of this study were to determine the potential of Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via drinking water to time-mated female Wistar Han rats from Days 6 to 21 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 750, 2000 and 5500 ppm (corresponding to a target test material intake of 0, 100, 300 and 1000 mg/kg/day, respectively), based on the results of the Dose Range Finder (Test Facility Study No. 20332264).

The study design was as follows:

Text Table 1
Experimental Design

Group No.	Test Material Identification	Dose Level ^a (ppm)	Corrected Dose Level (ppm)	Target Dose Level (mg/kg/day)	Number of Females
1	-	0^b	0	0	22
2	Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate	750	2228	100	22
3		2000	5942	300	22
4		5500	16340	1000	22

^a    The test material has a purity of 33.65%, dose calculations were corrected for purity (correction factor 2.971).

^b    Municipal tap water without test item.

Chemical analyses of municipal tap water preparation samples were conducted once during the study to assess accuracy and homogeneity and confirmed that drinking water preparations containing the test material were prepared accurately and homogenously.

The following parameters and end points were evaluated in this study for the F₀-generation: mortality/viabilities, clinical signs, body weights, food consumption, water consumption, test material intake, thyroid hormone levels (T3, T3, TSH), organ weights (thyroid gland), macroscopic examination, microscopic examination (thyroid gland) and uterine contents (including corpora lutea, implantation sites, pre- and postimplantation loss and number of live and dead fetuses).

In addition, the following parameters were determined for the F₁-generation: fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

Animals were exposed to 750, 2000 and 5500 ppm in drinking water, corresponding with a mean test material intake of 104, 295 and 1110 mg/kg/day, respectively.

At all dose levels, mean water consumption was dose-dependently higher than control, which was considered non-adverse.

No test material-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e., mortality/moribundity, clinical appearance, body weight, food consumption, thyroid hormone levels (T3, T4, TSH), thyroid gland weights, macroscopic evaluation, microscopic evaluation of the thyroid gland and uterine contents including corpora lutea implantation sites and pre- and postimplantation loss).

 

No developmental toxicity was observed in the 750, 2000 and 5500 ppm groups

In conclusion, based on the results in this prenatal developmental toxicity study the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate were established:

Maternal and Developmental NOAELs:

At least 5500 ppm, corresponding to an actual mean test material intake of 1110 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed