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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium ethylenesulphonate

EC Number:

221-242-5

EC Name:

Sodium ethylenesulphonate

Cas Number:

3039-83-6

Molecular formula:

C2H4O3S.Na

IUPAC Name:

sodium ethenesulfonate

Test material form:

liquid

Details on test material:

25% solution in water

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

1 671 ppm

Remarks:

Corresponding to 99 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

5 031 ppm

Remarks:

Corresponding to 326 mg/Kgbw/day

Doses / concentrations 4

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).

Examinations

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)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

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