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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

The objective of this study was to obtain information on the toxicity of the test item (Reaction mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate ) when administrated in the drinking water to Wistar rats for 90days. The study has been designed for the characterization of the test item toxicity, for an indication of the dose relationship and the determination of the No observed Adverse Effect Level( NOAEL). the dose levels and and the ,contractions in the drinking water were selected based on the DRF study ( CR 203322259)

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

Specific details on test material used for the study:

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 %

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rat as rodent in one of the standard strains for repeat-dose toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Crl: WI(Han)
Animal Condition: Outbred, SPF-Quality
Animal Supplier: Charles River Deutschland, Sulzfeld, Germany or Charles River
Number of Males: 40 (plus 2 alternates)
Number of Females: 40 (plus 2 alternates; nulliparous and non-pregnant)
Target Age at Initiation of Drinking Water Administration: At least 6 weeks
Target Weight at Initiation of Drinking Water Administration: 100 to 300 g (males) and 100 to 200 g (females)

Fasting period before study ;Animals were not fasted before the start of the study. Animals were fasted overnight before the necropcy.

Housing
Polycarbonate cages P2000 cages with height 21.5 cm, containing sterilized wooden fibers as bedding material (Lignocel S8-15 JRS- J Rettenmaier & Sohne Gmbh+CO. KG, Rosenberg, Germany) equipped with water bottles.
During locomotor activity monitoring animals are housed individually in a Hi temp polycarbonate cage ( Ancare corp ., USA : dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Cage Identification: Color-coded cage card indicating at least Test Facility Study nr., group animal identification numbers.

Diet: SM R/M-Z from SSNIFF specialdiaten GmbH, Soest, Germany.
Type: Pellets
Frequency: Ad libitum expect during designated procedurers.
Analysis: Results of analysis for nutritrional components and environmental contaminants are available at the Test Facility. It is considered that there' are no known contaminants in the feed that would interfere with the objectives of the study.

Environmental conditions
The actual daily mean temperature during the study period was 21 to 22°C with an actual daily mean relative humidity of 42 to 74%. The values that were outside the targeted mean humidity range occurred for 9 days and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.

In Life Dates
Study Initiation Date: 23 Jun 2022
Initiation of Dosing: 04 Jul 2022
Completion of In-life: 04 Oct 2022
Experimental Start Date: 23 Jun 2022
Experimental Completion Date: 15 Nov 2022

Administration / exposure

Route of administration:

oral: drinking water

Details on route of administration:

Drinking water study.
The oral route was selected as the most relevant route of human exposure.

Vehicle:

water

Details on oral exposure:

drinking water study

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis
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
Interval Concentration
(M) Homogeneity
(TMB) Sampling From
Week 1
(range 04 Jul 2022 – 10 Jul 2022) All groups
2 x approximately 500 mg Groups 2 and 4
2 x approximately
500 mg Dosing container
Week 6
(range 8 Aug 2022 – 14 Aug 2022) All groups
2 x approximately 500 mg Groups 2 and 4
2 x approximately
500 mg Dosing container
Week 12
(range 19 Sep 2022 – 25 Sep 2022) All groups
2 x approximately 500 mg Groups 2 and 4
2 x approximately
500 mg Dosing container
M = sample collected from approximately Middle; TMB = sample collected from approximately Top, Middle and Bottom
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.
Residual samples will be discarded after completion of the sample analysis.
Analytical Method
Analyses described below will be performed by using a validated analytical procedure (Test Facility Study No. 20343523).
Concentration and Homogeneity Analysis
Storage Conditions: Room temperature set to maintain 21°C
Acceptance Criteria: For concentration: mean sample concentration results within or equal to ± 10% of theoretical concentration.
For homogeneity, relative standard deviation (RSD) of concentrations of  10% for each group.
Stability Analysis
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 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.

Duration of treatment / exposure:

90 days

Frequency of treatment:

at libitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L drinking water

Doses / concentrations 2

Dose / conc.:

700 mg/L drinking water

Remarks:

In the drinking water: Mean achieved dose level was 93 mg/kg bw/day for the males and 103 mg/kg bw/day for females

Doses / concentrations 3

Dose / conc.:

2 308 mg/L drinking water

Remarks:

in the drinking water : mean achieved dose levels was 277 mg/kg bw/day for the mails, and 315 mg/kg bw/day for the females

Doses / concentrations 4

Dose / conc.:

7 692 mg/L drinking water

Remarks:

in the drinking water: mean achieved dose level was 975 mg/kg bw/day for the males and 1148 mg/kg bw/day for the females

No. of animals per sex per dose:

10 males , and 10 females.

Control animals:

yes, concurrent no treatment

Details on study design:

Dose selection.

The dose levels were selected based on the results of a (14 day repeated dose toxicity study with oral exposure of Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate in drinking water in rats, Test Facility Study No. 20332259), and in an attempt to produce graded responses to the test material. In this study administration of 1000 mg/kg/day via drinking water resulted in slightly lower body weight gain and food consumption over the whole administration period.
The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from day 1
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly; pretreatment and from Week 1 and throughout the study, and on the day of necropsy.

BODY WEIGHT: Yes (figure 1)
- Time schedule for examinations: Every 4 days; from at least Day 1 and throughout the study, In order to monitor the health status, animals may be weighed more often.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption : Yes, (table 4, fig 2) Weekly; from at least Day 1 and throughout the study
Quantitatively measured per cage.
- Compound intake calculated : not applicable

FOOD EFFICIENCY: YES. (table 3)
Weekly; from at least Day 1 and throughout the study
- 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:.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes (table 5, and figure 3)
- Time schedule for examinations: Every two days, Quantitatively measured per cage.
The actual test material intake will be estimated based on the body weight and food consumption values. (table 6)



OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment Period - All Main Study animals once (including spare animals)
- Dose groups that were examined: Dosing Period - All Group 1 and 4 Main Study animals during Week 13. If treatment-related findings are noted, the other animals will also be examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropcy on Day 90.
A blood smear will be prepared from each hematology sample. Blood smears are labeled, stained, and stored. These smears will not be examined, but may be evaluated when required to confirm analyzer results. The smears may be subsequently evaluated and this evaluation will be described in a Study Plan amendment.

White Blood Cell (WBC)
Neutrophils (absolute)
Lymphocytes (absolute)
Monocytes (absolute)
Eosinophils (absolute)
Basophils (absolute)
Large unstained cells (LUC) (absolute)
Red Blood Cell (RBC) Reticulocytes (absolute)
Red Blood Cell Distribution Width (RDW)
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelets

- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Not specified
- How many animals:
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes (table 11)
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 90.

Alanine aminotransferase (ALT) Triglycerides
Aspartate aminotransferase (AST) HDL and LDL Cholesterol
Alkaline Phosphatase (ALP) Sodium
Total protein Potassium
Albumin Chloride
Total Bilirubin Calcium
Urea Inorganic Phosphate (Inorg. Phos)
Creatinine Triiodothyronine (T3)
Glucose Thyroxine (T4)
Cholesterol Thyroid-Stimulating Hormone (TSH)

After receipt of the serum for T3, T4 and TSH analysis it will be divided in two aliquots.
One aliquot will be used for measurement of thyroid hormones TSH using the IMMULITE® 1000 analyser. The aliquot for TSH will be stored in an ultra-low freezer set to maintain 80°C until analysis. Any remaining sample after TSH analysis will be discarded.
The other aliquot will be used for measurement of T3 and T4 using LC MS. The aliquot for T3 and T4 will be collected in uniquely labelled clear 1.4 mL V-bottom Micronic polypropylene tubes and stored in a freezer set to maintain -20°C until analysis. Measurement of T3 and T4 will be performed according to the bioanalytical method validated in Test Facility Study No. 20213516. Any samples remaining after the LC-MS analysis will be returned to storage for the retention period.
The LC-MS analysis will be based on the following guidelines:
European Medicines Agency (EMA). Guideline on Bioanalytical Method Validation. EMEA/CHMP/EWP/192217/2009, 01 February 2012.
Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2018.
- Animals fasted: yes
- How many animals: all
- Parameters checked in table [No.3] were examined.
End of Treatment - on day

SERUM HORMONES/LIPIDS: Yes

Plasma/ Estrous Stage Determination
Estrous stage determination; YES ( Appendix 15)
Frequency: End of Treatment - on the day of necropsy, a vaginal smear will be taken to determine the stage of estrus from all Main Study animals. This will be done for all females, except for females that have to be euthanized in extremis or die spontaneously

Procedure: Estrous stage will be evaluated by examining the vaginal cytology of the samples obtained by vaginal smears procedures.
- How many animals: all females

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
For the motor activity data set parametric (ANOVA) tests on group means will applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, will be used in order to select the best fitting statistical model.
Once during the Dosing Period. The first 5 animals per sex per group during Week 13. These tests will be performed after clinical observations (including arena observation, if applicable).
Each animal will be observed for 2 minutes in an opaque open- field observation box. Parameters will be evaluated per Testing Facility SOP (SOP DIE-H-168) and study specific procedures. The following evaluations will be conducted:


Parameter Observed
Type of Data Rating Sale or Unit of Measurement
Posture/Body carriage Categorical -2 to 0
Convulsions Categorical 0 to 4
Stereotypy Categorical 0 to 4
Tremor Categorical 0 to 3
Palpebral closure/Ptosis Categorical 0 to 2
Ease of removal Categorical -1 to 2
Handling reactivity Categorical -1 to 2
Rearing Continuous Counts
Arousal/Alertness Categorical -2 to 2
Gait/Mobility Categorical 0 to 3
Vocalizations Categorical 0 to 1
Respiration Categorical -1 to 2
Defecation Categorical -1 to 1
Urination Categorical -1 to 1
Appearance Categorical 0 to 2
Lacrimation Categorical 0 to 2
Salivation Categorical 0 to 2
Exophthalmus Categorical 0 to 1
Erected fur Categorical 0 to 1
Touch response/Tactile reflex Categorical -1 to 1
Startle response Categorical -1 to 1
Tail pinch response Categorical -1 to 1
Pupil response Categorical 0 to 2
Body temperature Continuous ℃
Body tone Categorical -1 to 1
Air righting reflex Categorical 0 to 2
Grip strength (forelimb and hindlimb) Continuous g
Locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations will be reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

Immunology: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table 4 [Thymus and spleen organ weights] were examined.

OTHER: OTHER: sperm analysis (table 12)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes ,see table 14 and Appendix 21 and Appendix 22.

HISTOPATHOLOGY: Yes ,see table 15 and appendix 21 and 22

Other examinations:

Sperm analysis see table 12 and Appendix 19

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Animal No. 67 (300 mg/kg/day) was noted with extreme biting on Day 93.

The scab and protruding eyeball noted during the Dosing Period occurred within the range of background findings to be expected for rats 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 item.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effect on body weight was observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable between all groups.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was increased in males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test material related observations were recorded.

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology
No clear test material-related effects on hematology parameters were seen in males.
In females, an increased WBC at 1000 mg/kg/day and increased NEUT at 100, 300 and 1000 mg/kg/day, was observed
See table nr 2.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry
Total protein was decreased in males at 300 and 1000 mg/kg/day
Albumin is decreased in males at 100, 300 and 1000 mg/kg/day
Sodium is decreased in males at 100, 300 and 1000 mg/kg/day
Potassium is decreased in males at 300 and 1000 mg/kg/day
Thyroid stimulating hormone is increased in males at 100, 300 and 1000 mg/kg/day.
Glucose is decreased in females at 1000 mg/kg/day
Chloride is decreased in females at 1000 mg/kg/day

Remaining differences in clinical chemistry parameters were considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, were of a magnitude of change commonly observed in rats under similar study conditions and/or were likely the result of a relatively low control value.
see table nr 3

Endocrine findings:

no effects observed

Description (incidence and severity):

T4 concentrations were higher in males at 300 mg/kg bw/day when compared to concurrent controls (1.24x). Mean values remained within the historical control range .

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No effects on activity and excitability functional observations were noted.
On autonomic functional observation, erected fur was noted in 2/5 females each at 300 and 1000 mg/kg bw/day. A slightly impaired gait/mobility was observed in 1/5 females at 1000 mg/kg bw/day on neuromuscular functional observation. Furthermore, lower body temperature was noted in females at 300 and 1000 mg/kg bw/day on physiological functional observations (38.06°C and 38.04°C, respectively, compared to 38.84°C in controls; not statistically significant at 300 mg/kg bw/day). On sensorimotor functional observations, an exaggerated reaction was noted on the touch response/tactile reflex in 1/5 females at 300 mg/kg bw/day and in 1/5 males and 2/5 females at 1000 mg/kg bw/day and on the tail pinch response in 1/5 males and 2/5 females at 300 mg/kg bw/day and in 2/5 males and 3/5 females at 1000 mg/kg bw/day.
Any other observations noted occurred within the range of background findings to be expected for rats 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.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related alterations in organ weights.
Any differences, including those that reached statistical significance i.e., higher kidney weight in males and higher liver weight in females (both as relative to body weight) at 300 mg/kg bw/day were considered not test material-related as they occurred in the absence of a dose-related trend.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related gross observations at doses up to the highest dose level tested.All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Test material-related microscopic findings after treatment with the test material were noted in the thymus of females at a target dose of 1000 mg/kg bw/day. These findings are summarized in Text Table 7.
Text Table 7
Summary Test Material-Related Microscopic Findings– Scheduled Euthanasia Animals
Females
Target Dose level (mg/kg bw/day): 0 100 300 1000

THYMUS a 10 10 10 10
Apoptosis, increased; lymphoid
Minimal 1 - 1 4
a Number of tissues examined from each group.
Shaded values indicate a test material-related effect.
In the thymus, an increased incidence of minimal lymphoid apoptosis was present in 4/10 females at a target dose of 1000 mg/kg bw/day. The single occurrence of this finding in females at a target dose of 300 mg/kg bw/day was interpreted as not test material-related and similar to that observed in one control female.
There were no other test material-related histologic changes.
Remaining microscopic changes were considered to be incidental findings or 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.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

> 975 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Effect level:

> 1 148 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Dose formulation analyses (see appendix3)

No test material was detected in the Group 1 formulations.

For the drinking water preparation sample of Group 4F prepared for use in Week 13, the mean concentration was 112% of target. The mean accuracy of the QC low and QC high samples were above 100% (i.e., 104% and 109% respectively). If the study samples would be corrected for the recoveries of the QC samples, the study samples would be within acceptance criteria. Therefore, the result of Group 4F was accepted.

All other concentrations analyzed in the drinking water preparation samples of Groups 2, 3 and 4 (Week 1), Groups 2, 3F, 3M, 4F and 4M (Week 6) and Groups 2F, 2M, 3F, 3M and 4M (Week 13) were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration).

The formulations of Groups 2 and 4 (Week 1), Groups 2 and 4M (Week 6) and Groups 2F and 4M (Week 13) were homogeneous (i.e., coefficient of variation ≤ 10%).

Applicant's summary and conclusion

Conclusions:

In conclusion, Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate by drinking water in Wistar Han rats was administered for at least 90 days at target levels of 100, 300 and 1000 mg/kg bw/day.
The average test material intake was 93, 277 and 975 mg/kg bw/day for males and 103, 315 and 1148 mg/kg bw/day for females for target dose levels of 100, 300 and 1000 mg/kg bw/day, respectively.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate of at least 1000 mg/kg bw/day was established.

Executive summary:

The objective of this study was to determine the potential toxicity of Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate, when given via drinking water for 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The study design was as follows: see table  experimental design

Chemical analyses of formulations were conducted three times during the study and confirmed that for formulations of the test material in drinking water were prepared accurately and homogeneously.

The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observation tests, body weights, food consumption, water consumption, ophthalmology, stage of estrous, sperm analysis, clinical pathology parameters (hematology, coagulation and clinical chemistry), macroscopic examination, organ weights and microscopic examination.

No test material-related effects were noted at 100 mg/kg bw/day.

At 300 mg/kg bw/day, higher water consumption in females throughout the whole duration of the study was noted. At functional observations, erected fur, lower body temperature, and an exaggerated reaction on the touch response/tactile reflex in females and on the tail pinch response in males and females was noted. Changes in clinical chemistry comprised a higher T4 concentration in males. All these findings were considered to be non-adverse.

At 1000 mg/kg bw/day, higher water consumption in males and females was noted throughout the whole duration of the study. At functional observations, erected fur, slightly impaired gait/mobility and lower body temperature in females and an exaggerated reaction on the touch response/tactile reflex and tail pinch response in males and females was noted. At clinical chemistry, a lower glucose concentration in females was noted. All these findings were considered to be non-adverse.
On histopathology, a non-adverse, slightly higher incidence of minimal increased lymphoid apoptosis was noted in the thymus of females.

No mortality and no test material-related changes were noted in any of the remaining parameters investigated in this study (i.e., clinical signs, functional observations (activity and excitability), body weight, food consumption, ophthalmoscopy, hematology, coagulation, sperm analysis, macroscopic examination and organ weights).

In conclusion, Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate by drinking water in Wistar Han rats was administered for at least 90 days at target levels of 100, 300 and 1000 mg/kg bw/day.

The average test material intake was 93, 277 and 975 mg/kg bw/day for males and 103, 315 and 1148 mg/kg bw/day for females for target dose levels of 100, 300 and 1000 mg/kg bw/day, respectively.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate of at least 1000 mg/kg bw/day was established.