Reaction mass of sodium prop-2-enesulphonate and sodium chloride

Endpoint summary

Description of key information

rat, 35d males, 49/53d females, gavage: NOAEL systemic toxicity >= 1000 mg/kg bw/d (no adverse effects observed; GLP, OECD 422, BASF SE 2010f)

rat, 90d, male/females, gavage: NOAEL systemic, female = 1000mg/kg bw/d, male = 300 mg/kg bw/d, effects on testis and liver (GLP, OECD 408, BASF SE 2017)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: W(C3H5SO3Na) 26.2 g/100 g (study No. 15L00527)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species: Rat
Strain: Crl:WI(Han)
Supplier: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
Reason for the selection: The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Crl:WI(Han)
Supplier: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
Sex: Males / females
Age when supplied: 36 ± 1 days
Age at the beginning of the
administration period: 42 ± 1 days
Animal identification: Ear tattoo (animal number)

HOUSING AND DIET
The animals were housed together (5 animals per cage) in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, and large play tunnels (Art. 14153) supplied by PLEXX B.V., Elst, The Netherlands, were added for environmental enrichment.
The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24°C, a range of relative humidity of 30-70% and 15 air changes per hour. The day/night cycle was 12 hours (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h). There were no or only minimal deviations from these limits.
The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
2-Propene-1-sulfonic acid, sodium salt was applied as a solution. To prepare this solution the appropriate amount of test substance was weighed out depending on the desired
concentration. Then, drinking water was filled up to the desired volume, subsequently mixed with a magnetic stirrer. The test-substance preparations were produced at least weekly and
stored at room temperature. The administration volume was 10 mL/kg body weight

As the product itself was already a aqueous solution the dosage of the whole product was
0 mg/kg
382 mg/kg
1145 mg/kg
3817 mg/kg

This corresponds to a.i. of
0 mg/kg
100 mg/kg
300 mg/kg
1000 mg/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Details can be found in "Concentration Control Analysis of “2-Propene-1-sulfonic acid, sodium salt” in “Drinking water”; Study No. 16L00334"

Concentration control was perfomed using Capillary electrophoresis (CD) with internal standard quantification. Expected concentration could be varified with recovery rates between 101-103%.

Duration of treatment / exposure:

90d

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

active ingredient
Testitem is containing 73,8g water. Applied volume is increased to achieve active ingredient as depicted above.

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

active ingredient
Testitem is containing 73,8g water. Applied volume is increased to achieve active ingredient as depicted above.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

active ingredient
Testitem is containing 73,8g water. Applied volume is increased to achieve active ingredient as depicted above.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: according to OECD 422 screening study 95R0505/09074
- study conducted according to guideline

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:
1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Impairment of gait
12. Lacrimation
13. Palpebral closure
14. Exophthalmus
15. Feces (appearance/ consistency)
16. Urine
17. Pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION :
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: water consumption was determined weekly from week 6 onwards and calculated as mean water consumption in grams per animal and day

OPHTHALMOSCOPIC EXAMINATION: Yes
Prior to the start of the administration period on day -3 the eyes of all animals and on study day 91 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
Parameters checked in table:
Leukocyte count
(WBC)
Erythrocyte count
(RBC)
Hemoglobin
(HGB)
Hematocrit
(HCT)
Mean corpuscular
volume
Mean corpuscular
hemoglobin
(MCH)
Mean corpuscular
hemoglobin
Platelet count
(PLT)
Differential blood
count
Reticulocytes (RET)
Prothrombin time (Hepato
Quick’s test)
(HQT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- Parameters checked in table
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
γ-Glutamyltransferase
Sodium
(NA)
Potassium
(K)
Chloride
(CL)
Inorganic phosphate
(INP)
Calcium
(CA)
Urea
(UREA)
Creatinine
(CREA)
Glucose
(GLUC)
Total bilirubin
(TBIL)
Total protein
(TPROT)
Albumin
(ALB)
Globulins
(GLOB)
Triglycerides
(TRIG)
Cholesterol
(CHOL)


URINALYSIS: Yes
For urinalysis, the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
- Parameters checked in table
pH
Protein (PRO)
Glucose (GLU)
Ketones (KET)
Urobilinogen (UBG)
Bilirubin (BIL)
Blood
Specific gravity
Sediment
Color, turbidity (COL, TURB)
Volume (VOL) [mL]


NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests.

Sacrifice and pathology:

The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

GROSS PATHOLOGY: Yes (see table)

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix

The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or
in modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testes
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

HISTOPATHOLOGY: Yes (see table )

Histopathological evaluation of the HE-stained slides and special stains was performed by the study pathologist.
Organs
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Female mammary gland
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Liver
20. Lung
21. Lymph nodes
(mesenteric and axillary lymph nodes)
22. Ovaries
23. Pancreas
24. Parathyroid glands
25. Peyer’s patches
26. Pituitary gland
27. Prostate
28. Rectum
29. Salivary glands
(mandibular and sublingual glands)
30. Sciatic nerve
31. Seminal vesicles
32. Skin
33. Spinal cord
(cervical, thoracic and lumbar cord)
34. Spleen
35. Stomach (forestomach and glandular stomach)
36. Testes
37. Thymus
38. Thyroid glands
39. Trachea
40. Urinary bladder
41. Uterus
42. Vagina

Statistics:

Statistics of clinical pathology
Means, medians and standard deviations of each test group were calculated for several parameters.
Statistical analyses used in this report are
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse signs of toxicity were observed.

Slight salivation shortly after test-substance administration was observed in 6 male and 6 female animals of test group 3 (1000 mg/kg bw/d) on several study days from study day 32 onwards. From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both kind of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations.

Mydriasis was observed in the left eye of male animal No. 25 of test group 2 (300 mg/kg bw/d) from study day 85 until sacrifice. The finding was assessed to be incidental and not related to treatment as the eye was already affected before treatment.

Respiration sounds were observed in female animal No. 60 of test group 1 (100 mg/kg bw/d) from study day 83 until sacrifice. The same animal showed slightly reduced nutritional condition from study day 88 onwards. The reason for these findings could not clearly be described but a dilation of the esophagus was found at necropsy (see also pathology table IIC-64). Thus, a gavage error might have been occurred and small amounts of the test substance preparation could have been applied into the trachea or the thoracic cavity. These changes were not of toxicological concern.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Significant deviations to the control were only observed for single body weight change values of female animals in test group 3 (1000 mg/kg bw/d), i.e. on study day 70 (+14%), in test group 2 (300 mg/kg bw/d), i.e. on study day 49 (+17%), and in test group 1 (100 mg/kg bw/d), i.e. on study day 14 (-17%). These changes were assessed to be incidental and not related to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

From study day 42 onwards, increased water consumption was observed in all cages of test groups 2 and 3 (300 and 1000 mg/kg bw/d). In test group 3, the maximum deviations of +77% in male and +32% in female animals were observed on study day 70. In test groups 2, maximum deviation of +42% occurred in male animals on study day 56 and of +24% in female animals on study day 49. The increase in water consumption was assessed to be related to adaptive and physiological responses to the test substance administration, i.e. the clearance and elimination of 2-Propene-1-sulfonic acid, sodium salt the effects were not considered to be adverse.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The left eye of male animal No. 25 of test group 2 (300 mg/kg bw/d) showed isolated blood vessels in the ocular fundus before and at the end of the administration period. This finding was assessed to be incidental and not related to treatment as it was already observed before treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

After the administration period in males of test group 3 (1000 mg/kg bw/d) total white blood cell (WBC) counts were significantly higher compared to controls, but the mean was within the historical control range. Therefore, this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After the administration period in males of test group 3 (1000 mg/kg bw/d) glucose, sodium and chloride levels were significantly lower compared to controls, but all three parameter values were within historical control ranges. Therefore, these changes were regarded as incidental and not treatment-related.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

After the administration period in rats of both sexes of test group 3 (1000 mg/kg bw/d) urine pH values were significantly decreased. The effect was due to the administration of the alkaline salt of a sulfonic acid as compound. Therefore, the metabolic alkalosis was compensated by an increased respiratory rate, resulting in a decrease of bicarbonate and carbon dioxide blood levels. The lower bicarbonate blood levels were partly compensated by a resorption of bicarbonate in the renal tubules and an increased excretion of H+-ions. Consequently, urine pH values were decreased. This procedure reflects the normal function of the kidneys and was regarded as an adaptive and non-adverse effect.
In males of test group 2 (300 mg/kg bw/d) urine volume was significantly higher and urine specific gravity was significantly lower compared to controls, but the change was not dosedependent. Therefore, these alterations were regarded as incidental and not treatment related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increase in absolute and relative kidney weights in the highest dosegroup (1000 mg/kg bw/d) (males and females)
Increase in absolut kidney weight at 300 and 1000 mg/kg bw/d (females), 1000 mg/kg bw/d (males) and liver weight at 1000 mg/kg bw/d (females)

The significant absolute kidneys’ weight increase of test group 3 (1000mg/kg bw/d) males (2.567 g) was minimally above the historical control range values, whereas the significant relative weight increase (0.691%) was within the historical control values. In females, the significant absolute and relative weight increase of the kidneys in test groups 3 (1000mg/kg bw/d) were within the historical control range. The kidneys weight increases in male and female animals of test group 3 were assumed to be possibly treatment-related but not adverse. The absolute kidneys weight increase in females of test group 2 (300mg/kg bw/d) was minimal and within the historical control range and was, therefore, not regarded as treatment-related. The significant absolute liver weight increase was within the range of the historical control values and was, therefore, regarded as not treatment-related.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Myocardial necrosis (grade 1) was observed at 4/10 male animals at 1000 mg/kg bw/d, whereas in control 1/10 (males) and at 100mg/kg bw/d group 1/10 (males) showed also this effect. The increased incidence in test group 1000mg/kg bw/d suggested a treatment-related effect.

Kidney of male animals showed increase in tubular Cast and lympho-histiocytic Infiltrate for the highest dose group (4/10) compared to control (1-2/10).
This is a minimal increase of basophilic tubules, hyaline tubular casts and lympho-histiocytic infiltrates compared to the control group. This pattern of lesions is characteristic of a spontaneous and incipient chronic progressive nephropathy (CPN), commonly seen as background entity in rodents, with higher incidence and grading in male animals and without relevance to humans (Hard et al. 2009). Therefore, histopathological findings observed in males of test group 3 were judged to represent a treatment-related exacerbation of a CPN characteristic of rodents, which was considered not adverse and without human relevance.

Testis showed an minimal increased severity in the from control compared to highest dose group. Epididymes shows an increase Debris (grade 1) at 1000mg/kg bw day (2/10) and epithelial vacuolation (1/10).
The slight tubular degeneration with multifocal pattern was assumed to be possibly treatment related and adverse. Secondary to the slight multifocal tubular degeneration in test group 3, the epididymides of the two animals with slight multifocal tubular degeneration showed a minimal increase of cellular debris, most notably in the caput. In the epididymides of one male with slight testicular degeneration, a minimal bilateral focal epithelial vacuolation was noted at the cauda adjacent to the corpus. This type of finding together with the debris and the testicular degeneration were considered to be possibly treatment-related and adverse.

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

The administration of 2-Propene-1-sulfonic acid, sodium salt by gavage for 3 months to male and female Wistar rats caused test substance-related, adverse signs of systemic toxicity in male animals only at an effective dose level of 1000 mg/kg bw/d. Therefore, under the conditions of the present study the NOAEL was 300 mg/kg bw/d for male and 1000 mg/kg bw/d female Wistar rats.

Executive summary:

2-Propene-1-sulfonic acid, sodium salt was administered by gavage to groups of 10 male and 10 female Wistar rats at effective dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 3 months. Drinking water served as vehicle. Considering a purity of the test substance of 26.2%, the dose levels of the whole product, i.e. 2-Propene-1-sulfonic acid, sodium salt in water, were 382, 1145 and 3817 mg/kg bw/d, respectively. With regard to clinical examinations, signs of general systemic toxicity were not observed even at a dose level of 1000 mg/kg bw/d of 2-Propene-1-sulfonic acid, sodium salt. The increase in water consumption was assessed to be related to adaptive and physiological responses to the test substance administration, i.e. the clearance and elimination of 2-Propene-1-sulfonic acid, sodium salt. The effects were not considered to be adverse. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of 2-Propene-1-sulfonic acid, sodium salt of 1000 mg/kg bw/d. Regarding pathology, target organs were the kidneys of males and females, and the heart, testes and epididymides of male animals in test group 3 (1000 mg/kg bw/d).

In male animals of test group 3, the heart showed a slight increase in the incidence of focal myocardial necrosis compared to the control. The severity and type of lesion as well as the localization were consistent with the so called rodent progressive cardiomyopathy (Chanut et al 2013). This lesion is highly frequently reported in rat strains as a spontaneous finding without causing functional alterations. Since the severity and type of lesions (necrosis with macrophage infiltration) found in test group 3 were comparable to the lesion found in the control animal, the increased incidence in test group 3 was assumed to be possible treatment-related and adverse. The kidneys had a minimal statistical significant increase of the absolute weight (+16%)

above the historical control values, with a relative weight increase (+16%) that was within the historical control values. This change was considered possibly treatment-related but not adverse. Histopathology revealed a minimal increase of basophilic tubules, hyaline tubular casts and lympho-histiocytic infiltrates compared to the control group. This pattern of lesions is characteristic of a spontaneous and incipient chronic progressive nephropathy (CPN), commonly seen as background entity in rodents, with higher incidence and grading in male animals and without relevance to humans (Hard et al. 2009). Therefore, histopathological findings observed in males of test group 3 were judged to represent a treatment-related exacerbation of a CPN characteristic of rodents, which was considered not adverse and without human relevance. In the testes, the incidence of tubular degeneration did not differ between the control and test group 3 animals. However, the severity was minimally increased in two animals (grade

2) of test group 3 and showed a multifocal pattern distribution, which differed from the minimal (grade 1) and focal pattern seen in the control group. Secondary to the slight multifocal tubular degeneration in test group 3, the epididymides of the two animals with

slight multifocal tubular degeneration showed a minimal increase of cellular debris and additionally, one of them showed a focal epithelial vacuolation at the cauda. Although the incidence of affected males with slight multifocal tubular degeneration and associated

epididymidal findings is low, this change was assumed to be possibly treatment-related and adverse. In female animals of test group 3, the kidneys exhibited significant increases of the absolute (+16%) and relative (+11%) weights that were within the historical control range and without histopathological changes. The weight deviations were regarded as possibly treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

CONCLUSION

The administration of 2-Propene-1-sulfonic acid, sodium salt by gavage for 3 months to male and female Wistar rats caused test substance-related, adverse signs of systemic toxicity in male animals only at an effective dose level of 1000 mg/kg bw/d.

Therefore, under the conditions of the present study the NOAEL was 300 mg/kg bw/d for male and 1000 mg/kg bw/d female Wistar rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

other: different organs

Organ:

heart

testes

Additional information

In a GLP conform study according to OECD guideline 422, the test item was given daily as an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) (BASF SE, 2010f). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (drinking water). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 1 week post-mating for males, and the entire gestation period as well as 4 days of lactation and approximately 1 week thereafter for females.The female animals were sacrificed 49 or 53 days after the beginning of the administration, and examined. The male animals were sacrificed 35 days after the beginning of the administration, and examined.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinalysis were performed in 5 parental animals of either sex towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation,under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The various analytical analyses confirmed compound identity using IR- UV- and NMR-spectroscopic methods, demonstrated the stability of the test substance solutions in drinking water at room temperature for a period of 7 days and confirmed the overall accuracy of the prepared concentrations.

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000mg/kg bw/d)

F0/ Parental animals: No test substance-related adverse findings

Test group 2 (300 mg/kg bw/d)

F0/ Parental animals: No test substance-related adverse findings

Test group 1 (100 mg/kg bw/d)

F0/ Parental animals: No test substance-related adverse finding

Therefore under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats Golpanol ALS wasserfrei had no adverse effects on fertility of the F0 parental animals of both sexes at 100; 300 and 1000 mg/kg bw/d. The test substance caused also no impairments of the reproductive performance. Mating behaviour, conception, gestation, parturition, as well as sexual organ weights and gross and histopathological findings of these organs were not influenced up to 1000 mg/kg bw/d. No general systemic toxicity was noted in the F0 parental animals at all dose levels tested. Golpanol ALS wasserfrei caused no indicationsof test substance-induced effects in low-, mid- and high-dose rats regarding considering food consumption and body weights/ body weight gain as well asdetailed clinical examinations in an open field, in a functional observational battery (FOB) and measurements of motor activity.

Concerning clinical and gross pathology as well as histopathology all findingsoccurred either singly or were biologically equally distributed over the control group and the treatment groups. They are considered to be incidental or spontaneous in origin and without any relation to treatmentup to 1000 mg/kg bw/d.

Up to 1000 mg/kg bw/dGolpanol ALS wasserfreicaused no developmental toxicity. No alteration of the pre-/postnatal development of the F1 offspring was indicated in number of delivered, liveborn and stillborn pups as well as survival, weight/weight gain, and sex ratio of pups.

Thus, under the conditions of the screening test the NOAEL (no observed adverse effect level) for general, systemic toxicity was ≥ 1000 mg/kg bw/d for the F0 parental rats, the highest dose tested.

Further 2-Propene-1-sulfonic acid, sodium salt was administered by gavage to groups of 10 male and 10 female Wistar rats at effective dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 3 months. Drinking water served as vehicle. Considering a purity of the test substance of 26.2%, the dose levels of the whole product, i.e. 2-Propene-1-sulfonic acid, sodium salt in water, were 382, 1145 and 3817 mg/kg bw/d, respectively. With regard to clinical examinations, signs of general systemic toxicity were not observed even at a dose level of 1000 mg/kg bw/d of 2-Propene-1-sulfonic acid, sodium salt. The increase in water consumption was assessed to be related to adaptive and physiological responses to the test substance administration, i.e. the clearance and elimination of 2-Propene-1-sulfonic acid, sodium salt. The effects were not considered to be adverse. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of 2-Propene-1-sulfonic acid, sodium salt of 1000 mg/kg bw/d. Regarding pathology, target organs were the kidneys of males and females, and the heart, testes and epididymides of male animals in test group 3 (1000 mg/kg bw/d).

In male animals of test group 3, the heart showed a slight increase in the incidence of focal myocardial necrosis compared to the control. The severity and type of lesion as well as the localization were consistent with the so called rodent progressive cardiomyopathy (Chanut et al 2013). This lesion is highly frequently reported in rat strains as a spontaneous finding without causing functional alterations. Since the severity and type of lesions (necrosis with macrophage infiltration) found in test group 3 were comparable to the lesion found in the control animal, the increased incidence in test group 3 was assumed to be possible treatment-related and adverse. The kidneys had a minimal statistical significant increase of the absolute weight (+16%)

above the historical control values, with a relative weight increase (+16%) that was within the historical control values. This change was considered possibly treatment-related but not adverse. Histopathology revealed a minimal increase of basophilic tubules, hyaline tubular casts and lympho-histiocytic infiltrates compared to the control group. This pattern of lesions is characteristic of a spontaneous and incipient chronic progressive nephropathy (CPN), commonly seen as background entity in rodents, with higher incidence and grading in male animals and without relevance to humans (Hard et al. 2009). Therefore, histopathological findings observed in males of test group 3 were judged to represent a treatment-related exacerbation of a CPN characteristic of rodents, which was considered not adverse and without human relevance. In the testes, the incidence of tubular degeneration did not differ between the control and test group 3 animals. However, the severity was minimally increased in two animals (grade

2) of test group 3 and showed a multifocal pattern distribution, which differed from the minimal (grade 1) and focal pattern seen in the control group. Secondary to the slight multifocal tubular degeneration in test group 3, the epididymides of the two animals with

slight multifocal tubular degeneration showed a minimal increase of cellular debris and additionally, one of them showed a focal epithelial vacuolation at the cauda. Although the incidence of affected males with slight multifocal tubular degeneration and associated

epididymidal findings is low, this change was assumed to be possibly treatment-related and adverse. In female animals of test group 3, the kidneys exhibited significant increases of the absolute (+16%) and relative (+11%) weights that were within the historical control range and without histopathological changes. The weight deviations were regarded as possibly treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Concluding the administration of 2-Propene-1-sulfonic acid, sodium salt by gavage for 3 months to male and female Wistar rats caused test substance-related, adverse signs of systemic toxicity in male animals only at an effective dose level of 1000 mg/kg bw/d. Therefore, under the conditions of the present study the NOAEL was 300 mg/kg bw/d for male and 1000 mg/kg bw/d female Wistar rats.

Justification for classification or non-classification

No substance-related effects were observed in a screening test for reprotoxic effects in rats in doses up to 1000 mg/kg bw/d and further in a 90d study up to a dose of 300mg/kg bw/d. Therefore, no indication is given for classification according to the criteria of DSD (67/548/EEC) and CLP (1272/2008/EC), respectively.