Registration Dossier

Administrative data

Description of key information

Under the conditions of a combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422 ), the oral administration of the test item via the drinking water to Wistar rats revealed adverse findings at 284 mg/kg bw/d (whole product) regarding clinical signs, body weight, food and water consumption. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 245 mg/kg bw/d (whole product) [BASF, 2019]. With regard to the content of Disodium (sulphonatothio)acetate and sodium chloride (38% of whole product), this corresonds to a NOAEL for general systemic toxicity of 93 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Mar 2018 - 05 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted at a concentration of ca. 38 % test substance in an aqueous solution.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
Jul 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz
Limit test:
no
Specific details on test material used for the study:
The concentration of the dry content in the test substance was 38.8% (including sodium and chloride) plus 61.2% water (see Certificate of Analysis according to DIN 55350-18-4.2.2).
Batch identification: 91236868E0.
Homogeneity: Given (visual).
Stability: Expiry date 07 Apr 2021. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
Storage conditions: Ambient (room temperature).
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: age at supply about 10-11 weeks (males) and 9 weeks (females).
- Weight at study initiation: mean premating weight about 384 g (males) and 217 g (females).
- Fasting period before study: no.
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany. During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Lignocel Block Large) were supplied by J. Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany, and used for environmental enrichment.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 28 days.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65% for relative humidity.
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 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)

IN-LIFE DATES: From: 13 Mar 2018 To: 14 May 2018 (males) and 06 Jun 2018 (females).
Route of administration:
oral: drinking water
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
water
Details on oral exposure:
Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride 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 and mixed by shaking. The test substance preparations were produced twice weekly, at least.

After the acclimatization period, the test substance was administered orally via drinking water to the F0 generation parental animals. The animals of the control group were treated in the same way with the vehicle only (drinking water).
Analytical verification of doses or concentrations:
yes
Remarks:
The concentrations of Reaction mass of Disodium (sulphonatothio)acetate and sodium in the samples were calculated by means of their sulfur content.
Details on analytical verification of doses or concentrations:
The stability of Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride in drinking water over a period of 7 days at room temperature was proven (study No. 18L00022). As the mixtures were stored no longer than this time period, the stability was guaranteed.
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, additionally one sample from the mid concentration was taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed.

Concentration and Homogeneity control analyses: Regarding the samples taken at the beginning of the administration period with expected values of 750 ppm and 2500 ppm (Nos. 3-6), the observed concentrations corresponded with 97% to 109% to the expected concentrations. Regarding the sample Nos. 8, 9, 7R, 8R and 9R with an expected value of 7500 ppm, the observed concentration did not correspond precisely. A higher concentration, i.e. 120% to 134%, was determined. Sample No. 7 can be excluded as an outlier. Based on these results, the Study Director considered that the test item was homogeneously distributed in the vehicle. Regarding the precision of the analytical method, the concentrations found for the samples taken at the end of the administration period a good accordance with the expected values was observed. Concerning the sample Nos. 44-46 (375 ppm), 48-50 (3000 ppm) and 51-53 (6000 ppm), the recoveries indicated a homogenous distribution of Reaction mass of Disodium (sulphonatothio)acetate and sodium in the vehicle since they were found to be in a range of 89% to 116%.
Duration of treatment / exposure:
males: 34 days; females: 57 days
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to the day of schedule sacrifice of the animals.
Frequency of treatment:
daily
Dose / conc.:
750 ppm
Remarks:
whole product: 47 (male), 70 (female) mg/kg bw/day;
Disodium (sulphonatothio)acetate and sodium chloride: 17.7 (male), 26.7 (female) mg/kg bw/day
Dose / conc.:
2 500 ppm
Remarks:
whole product: 139 (male), 230 (female) mg/kg bw/day;
Disodium (sulphonatothio)acetate and sodium chloride: 52.8 (male), 87.4 (female) mg/kg bw/day
Dose / conc.:
7 500 other: ppm (day 0-13)
Remarks:
whole product: 284 (male), 387 (female) mg/kg bw/day;
Disodium (sulphonatothio)acetate and sodium chloride: 107.9 (male), 147.1 (female) mg/kg bw/day
Dose / conc.:
6 000 other: ppm (day 13 onwards)
Remarks:
whole product: 245 (male), 453 (female) mg/kg bw/day;
Disodium (sulphonatothio)acetate and sodium chloride: 93.1 (male), 172.2 (female) mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A test study (BASF project No. 10C0543/17S080) was performed beforehand to evaluate the administration route and to select proper concentrations/dose levels for the present OECD 422 study. Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride was administered via drinking water for 2 weeks to groups of each 4 male and 4 female Wistar rats. The concentrations were 0, 8000 and 12000 ppm. Because of severe clinical findings at 12000 ppm, i.e. reduced food and water consumption as well as continuous loss of body weight, the animals of this test group were sacrificed ahead of schedule on study day 7. Treatment
at 8000 ppm was continued as described. Again, continuously reduced food and water consumption as well as mean body weight loss in male and female animals during the first week and recovery during the second week of application were observed. In addition, significantly decreased terminal body weight and significantly increased relative kidney weights were found in male animals, only. No significant changes occurred in female animals.
Based on these results, the following concentrations in drinking water were selected for the present study: 7500 ppm as high concentration, 2500 ppm as mid concentration, 750 ppm as low concentration.
Due to reduced food and water consumption and lower body weight/body weight change values in animals of test group 3 (7500 ppm), the concentration for this test group was reduced to 6000 ppm from study day 13 onwards. Due to naturally occurring increased water consumption of female animals during lactation and, consequently, a much higher test substance intake, the concentrations for all test groups were reduced on purpose by 50% from delivery until sacrifice.
- Fasting period before blood sampling for clinical biochemistry: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals for clinical pathology.
Positive control:
no.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: at least daily.
- A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. Abnormalities and changes were documented for each animal.A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: 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 in handling, 2. Fur, 3. Skin, 4. Posture, 5. Salivation, 6. Respiration, 7. Activity/arousal level, 8. Tremors, 9. Convulsions, 10. Abnormal movements, 11. Gait abnormalities, 12. Lacrimation, 13. Palpebral closure, 14. Exophthalmos, 15. Assessment of the feces discharged during the examination (appearance/ consistency), 16. Assessment of the urine discharged during the examination, 17. Pupil size.

BODY WEIGHT: Yes.
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration body weight of the male and female parental animals was determined twice a week.
- The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• During the mating period, the females was weighed on the day of positive evidence of sperm (GD 0) and on GD 4, 7, 10, 14 and 20.
• Females with litter was weighed on the day of parturition (PND 0), PND 4, PND 7 PND 10 and PND 13.
• Females showing no positive evidence of sperm in the vaginal smear was weighed once a week during this mating interval as will be the males
• Females without litter and after weaning (PND 13) was weighed once a week.

FOOD CONSUMPTION: Yes.
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1- 4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes.
- Time schedule for examinations: Generally, water consumption was determined twice a week for the male and female parental animals
• Additionally, after the 2nd test week water consumption of the females with evidence of sperm was determined for GDs 0-1, 4-5, 7-8, 10-11, 14-15, 17-18 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter, was determined for PNDs 1-2, 4-5, 7-8, 10-11 and 12-13.
• Water consumption of females showing no positive evidence of sperm in the vaginal smear was determined once a week during this mating interval as will be the males
• Water consumption of females without litter was determined once a week.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at termination / study day 34 (males); at PND 14 / study day 50 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at termination, study day 34 (males); at PND 14, study day 50 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. Because of technical error the FOB for male animals started about 10.25 h. Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB
was performed in the first five parental males and the first five surviving females with litter in order of delivery) per group.
- Dose groups that were examined: all.
FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: 1. Posture, 2. Tremors, 3. Convulsions, 4. Abnormal movements, 5. Impairment of gait, 6. Other findings
- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: 1. Behavior on removal from the cage, 2. Fur, 3. Skin 4. Salivation, 5. Nasal discharge, 6. Lacrimation, 7. Eyes/pupil size, 8. Posture, 9. Palpebral closure, 10. Respiration, 11. Tremors, 12. Convulsions, 13. Abnormal movements/stereotypes, 14. Gait abnormalities, 15. Activity/arousal level, 16. Feces (consistency/color) excreted during examination (two minutes), 17. Urine excreted within 2 minutes (amount/color), 18. Rearing within 2 minutes, 19. Other findings
- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: 1. Reaction to an object being moved towards the face (approach response), 2. Touch sensitivity (touch response), 3. Vision (visual placing response), 4. Pupillary reflex, 5. Pinna reflex, 6. Audition (auditory startle response), 7. Coordination of movements (righting response), 8. Behavior during handling, 9. Vocalization, 10. Pain perception (tail pinch), 11. Grip strength of forelimbs, 12. Grip strength of hindlimbs, 13. Landing foot-splay test, 14. Other findings
MOTOR ACTIVITY (MA) ASESSMENT
- The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

OTHER:

ESTROUS CYCLE
- For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period (the estrous cycle data of these individuals were not reported and can be found in the raw data). In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

MALE REPRODUCTION DATA:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.

THYROID HORMONES (males only)
- Time schedule for collection of blood: at termination, study day 34.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: all surviving males at termination.
- Parameters checked in table 3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
See table 5.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations for males and females: During the pre-mating period, a single treatment-related finding was observed in test group 3, i.e. piloerection was observed for male animal No. 31 of test group 3 (7500 ppm) from study days 9 to 12. The finding was assessed to be related to impaired water and food consumption during the pre-mating phase and treatment at a concentration of 7500 ppm. The animal recovered after reducing the test substance’s concentration in drinking water to 6000 ppm from study day 13 (last day of premating) onwards. No additional clinical findings of toxicological concern occurred in pre-mating, mating and post-mating periods.

Clinical observations for females during gestation: No treatment-related, adverse findings were observed. Female animal No. 137 of test group 3 (7500/6000 ppm) showed an injury at the left ear from gestation day (GD) 0 onwards. This finding was assed as spontaneous in nature and not related to treatment. Note: Female animal No. 116 of test group 1 (750 ppm) was not pregnant.

Clinical observations for females during lactation: No treatment-related, adverse findings were observed. Female animal No. 137 of test group 3 (7500/6000 ppm) showed an injury at the left ear from lactation day (PND) 0 onwards. Female animal No. 107 of test group 0 (control group) lost its complete litter one day after delivery. Until sacrifice, no other findings became evident. Both findings were assed as spontaneous in nature and not related to treatment.

Detailed clinical observations: During detailed clinical observations (DCO), no treatment-related findings occurred. Female animal No. 137 of test group 3 (7500/6000 ppm) showed an injury at the left ear from study day 21 onwards. This finding was assed as spontaneous in nature and not to treatment.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight loss was observed in male and female animals of test group 3 during the first week of treatment (7500 ppm). In male animals of test group 3 (7500/6000 ppm) significantly lower body weight was detected from study day 7 onwards. Female animals of test group 3 showed a significantly decreased mean body weight on pre-mating days 3 and 7 (7500 ppm) and between GD 7 and GD 17 (6000 ppm). Consequently, in male animals of test group 3 (7500/6000 ppm) the mean body weight change value between pre-mating days 0-3 and 3-7 as well as 0-13 and between mating days 7-14 were significantly lower. In female animals of test group 3 (7500 ppm) the mean body weight change value was significantly lower between pre-mating days 0-3 and 0-13. No treatment-related changes in mean body weights and mean body weight change values were observed in male and female animals of test groups 1 and 2 (750 and 2500 ppm) during the entire study.
Males of test group 3 showed a significant decrease of the final body weight, which was considered treatment-related (see table 7).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the pre-mating phase, significantly decreased food consumption was observed in male animals of test group 3 (7500 ppm) with a maximum by -22% on study day 7. In female animals of test group 3 (7500 ppm) significantly decreased food consumption was observed during pre-mating and on GDs 7 and 14 with a maximum deviation of -21% on pre-mating day 7. No treatment-related changes in food consumption were observed in male and female animals during the entire study test groups 1 and 2 (750 and 2500 ppm).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In male animals of test group 3 (7500/6000 ppm) significantly decreased water consumption was observed over the entire study period with a maximum by -45% on study day 7. In female animals of test group 3 (7500/6000 ppm) significantly decreased water consumption was observed during the pre-mating and gestation periods with a maximum of -48% on pre-mating day 3. During lactation, water consumption in female animals of test group 3 (7500/6000 ppm) was only marginally affected as the concentration in drinking water was reduced by 50%. No treatment-related, adverse findings were observed in test groups 1 and 2 (750 and 2500 ppm).

The concentration in drinking water had to be reduced after study day 13 of administration. The actual test substance intakes are given in table 6. The mean values of the test substance intake, either for the whole product (38% of Disodium (sulphonatothio)acetate and sodium chloride as well as about 62% of water) or Disodium (sulphonatothio)acetate and sodium chloride, only, for male and female animals were calculated and related to the days of treatment.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At the end of the administration period, in females of test group 3 (7500/6000 ppm) mean corpuscular volume (MCV) was significantly increased. MCV is a calculated red blood cell index, derived from the measured parameters red blood cell (RBC) counts and hematocrit. Both measured parameters were not changed. Therefore, the only changed MCV value in females of test group 3 was regarded as incidental and not treatment-related.
In males of test groups 1 and 3 (750 and 7500/6000 ppm) absolute monocyte counts were significantly increased, but the values were within the historical control range (males: absolute monocytes 0.07-0.14 Giga/L). In females of test group 2 (2500 ppm), relative neutrophil cell counts were significantly increased, and relative lymphocyte counts were significantly decreased, but the changes were not dose-dependent. Therefore, the changes
in this paragraph were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period, in males of test group 3 (7500/6000 ppm) alkaline phosphatase (ALP) activities and chloride levels were significantly decreased, whereas urea and cholesterol values were significantly increased. Urea levels were significantly higher compared to study controls already in males of test groups 1 and 2 (750 and 2500 ppm) and ALP activity were also significantly decreased in males of test group 1. However, all values were within historical control ranges (males: ALP 1.07-1.71 μkat/L; chloride 97.9- 104.1 mmol/L; urea 3.96-7.20 mmol/L; cholesterol 1.60-2.27 mmol/L). Therefore, the mentioned alterations in this paragraph were regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute weights: When compared to control the group 0, mean absolute weight of ovary was significantly increased in parental females of test group 1 (750 ppm) (see table 7). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative weights: When compared to control the group 0, mean relative weight of ovary was significantly increased in parental females of test group 2 (2500 ppm) (see table 8). All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The significant decrease of the final body weight in males of test group 3 was considered treatment-related, whereas the significant increase of the absolute and relative weight of the ovaries in test group 1 and 2, respectively, was regarded as incidental and without relation to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related gross lesions were noted. A single individual macroscopic focus in the skin of a female of test group 3 was observed at necropsy but did not show a histopathological correlate.
Fertility: Female animal No. 116, which was not pregnant, as well as the male mating partner No. 16 did not show relevant gross lesions.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery: Deviations from "zero values" were obtained in quantitative parameters in male and female animals. Without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
- Home cage observations: No test substance-related effects were observed.
- Open field observations: No test substance-related effects were observed.
- Sensorimotor tests/reflexes: No test substance-related effects were observed.
- Quantitative parameters: No test substance-related effects were observed.

Motor activity measurement: Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals. Single interval No. 7 was significantly decreased in male animals of test group 3 (7500/6000 ppm) and single interval No. 12 was significantly increased in male animals of test group 2 (2500 ppm) and test group 3 (7500/6000 ppm). Since these findings were isolated events and no further significant changes were observed in the respective sum of all intervals, these changes were regarded incidental and spontaneous in nature.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The increased mucification of the vaginal and cervical epithelium (see table 9) was comparable in each animal. It was characterized by the presence of a low stratum germinativum (2-3 layers thick) and variable overlaying cuboidal to columnar mucified cells (2-3 layers thick). Specially in the vagina, additional polymorphonuclear leukocytes were seen infiltrating the lamina propria and epithelium and were also present in the lumen intermingled with desquamated cells and mucus. 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. The stages of spermatogenesis in the testes of males of the high-dose test group were comparable to those of the controls. In high-dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
Fertility: Female animal No. 116, which was not pregnant, as well as the male mating partner No. 16 did not show relevant histopathological findings.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cycle:
- Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups was 3.9 to 4.0 days.

Male reproduction data:
- Male mating index: The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups.
- Male fertility index: Fertility was proven in nearly all of the F0 parental males within the scheduled mating interval to produce F1 litter. Male animal No. 16 of test group 1 (750 ppm), which was mated with female No. 116, did not generate F1 pups and no implants were found at necropsy. Thus, the male fertility index was 100% in test groups 0, 2 and 3 and 90% in test group 1. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female reproduction and delivery data:
- Female mating index: The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 2.9 days for test group 0, 1.9 days for test group 1, 2.4 days for test group 2 and 2.6 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Female fertility index: Most sperm positive rats delivered pups with the exception of female animal No. 116 of test group 1 (750 ppm), which was mated with male No. 16, did not deliver pups and showed no implantation site. Female animal No. 133 of test group 3 (7500/6000 ppm), which was mated with male animal No. 33, was not sperm-positive but delivered pups and showed implants. Thus, a defined GD 0 was not generated for this animal. The female fertility index was 100% in test groups 0, 2 and 3 and 90% in test group 1. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Fertility indices for F0 females: The mean duration of gestation was 22.3 days in test groups 0 and 2, 22.2 days in test group 1 and 22.7 days in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Gestation index: The gestation index was 100% in all test groups (0, 750, 2500 and 7500/6000 ppm).
- Live birth indices: The rate live birth indices were 100% in test groups 0 and 1 (control and 750 ppm), 97.7% in test group 2 (2500 ppm) and 98.2% in test group 3 (7500/6000 ppm). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Postimplantation loss: The postimplantation loss was 5.0% in control group 0, 11.3% in test group 1 (750 ppm), 4.5% in test group 2 (2500 ppm) and 12.3% in test group 3 (7500/6000 ppm). The values were still within the normal range of biological variation inherent in the strain of rats used for this study. In addition, a clear dose-response relationship was not observed.

F1 generation litter/pups
- Pup number and status at delivery: The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3. No significant deviations occurred.
- Pup viability index/mortality The viability index indicating pup mortality between PND 0 and 4 was 90.0% in test group 0 (control), 100.0% in test group 1 (750 ppm), 99.1% in test group 2 (2500 ppm) and 98.3% in test group 3 (7500/6000 ppm). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Sex ratio: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Pup clinical observations: Female pup No. 107-14 of control group (0 ppm) showed poor general condition and hypothermia. The pup Nos. 107-01 to 107-13 of test group 0 (control), female pup No. 128-11 of test group 2 (2500 ppm) and female pup No. 137-12 of test group 3 (7500/6000 ppm) were found cannibalized on PND 1. These changes were assessed to be incidental. All other F1 pups in all test groups did not show clinical signs up to scheduled sacrifice on PND 4 and PND 13.
- Pup body weight data: Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group and showed no test-substance related changes. One male runt was found in one litter of test group 0 (control) and 1 female runt was found in one litter of test group 3 (7500/6000 ppm). A relation to dosing was not observed, test substance-related effects did not occur.
- Anogenital distance: No test substance-related effects were observed.
- Anogenital index: No test substance-related effects were observed.
- Nipple/areola anlagen: The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
- Pup necropsy observations: No test substance-related effects were observed. Male pup No. 139-06 of test group 3 (7500/6000 ppm) showed post mortem autolysis. These findings were observed as the pups were found dead and the autolysis was in progress before the daily clinical observations started. The pup Nos. 107-01 to 107-13 of test group 0 (control), female pup No. 128-11 of test group 2 (2500 ppm) and female pup No. 137-12 of test group 3 (7500/6000 ppm) were cannibalized on PND 1. These findings were assessed to be incidental and not related to the test substance. Note: Inadvertently, for male pup Nos. 131-09, 131-10, 131-11 and female pup Nos. 128-10 and 131-16 the pup necropsy observation was not documented. This mistake does not have an influence on the validity of the present study.

Thyroid hormones:
- In parental males (test groups 1, 2 and 3; 750, 2500 and 7500/6000 ppm) and in male and female pups at PND13 (test groups 11, 12 and 13; 750, 2500 and 7500/6000 ppm), no treatment-related alterations of T4 and TSH levels were observed.
Dose descriptor:
LOAEL
Remarks:
general systemic toxicity
Effect level:
7 500 ppm
Based on:
other: whole product
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: whole product: 284 (male), 387 (female) mg/kg bw/day
Remarks:
Disodium (sulphonatothio)acetate and sodium chloride: 107.9 (male), 147.1 (female) mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
6 000 ppm
Based on:
other: whole product
Sex:
male/female
Basis for effect level:
other: No treatment-related, adverse effects were observed at this dose.
Remarks on result:
other: whole product: 245 (male), 453 (female) mg/kg bw/day
Remarks:
Disodium (sulphonatothio)acetate and sodium chloride: 93.1 (male), 172.2 (female) mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
6 000 ppm
Based on:
other: whole product
Sex:
male/female
Basis for effect level:
other: No treatment-related, adverse effects were observed up to the highest tested dose.
Remarks on result:
other: whole product: 245 (male), 453 (female) mg/kg bw/day
Remarks:
Disodium (sulphonatothio)acetate and sodium chloride: 93.1 (male), 172.2 (female) mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
6 000 ppm
Based on:
other: whole product
Sex:
male/female
Basis for effect level:
other: No treatment-related, adverse effects were observed up to the highest tested dose.
Remarks on result:
other: whole product: 245 (male), 453 (female) mg/kg bw/day
Remarks:
Disodium (sulphonatothio)acetate and sodium chloride: 93.1 (male), 172.2 (female) mg/kg bw/day
Critical effects observed:
no

Table 6: Mean test substance intake (mg/kg bw/d) of whole product Disodium (sulphonatothio)acetate and sodium chloride (38% of whole product)

 

Test group 1

(750 ppm)

Test group 2

(2500 ppm)

Test group 3

(adjusted ppm)

whole product

F0 males

Pre-mating

(study days 0-13)

47

144

284

(7500 ppm)

Mating

(14 days)

46

134

245

(6000 ppm)

Mean value

47

139

265

F0 females

Premating

(study days 0-13)

63

205

387

(7500 ppm)

Gestation

(20 days)

78

248

426

(6000 ppm)

Lactation

(13 days)

66

(375 ppm)

229

(1250 ppm)

495

(3000 ppm)

Mean values of entire administration period

70

230

433

Disodium (sulphonatothio) acetate and sodium chloride (38% of whole product)

F0 males

Pre-mating

(study days 0-13)

17.9

54.7

107.9

(7500 ppm)

Mating

(14 days)

17.5

50.9

93.1

(6000 ppm)

Mean value

17.7

52.8

100.5

F0 females

Premating

(study days 0-13)

23.9

77.9

147.1

(7500 ppm)

Gestation

(20 days)

29.6

94.2

161.9

(6000 ppm)

Lactation

(13 days)

25.1

(375 ppm)

87.0

(1250 ppm)

188.1

(3000 ppm)

Mean values of entire administration period

26.7

87.4

164.7

 

 

Table 7: Absolute weights

 

 

Male animals

Female animals

Test group (ppm)

1

(750)

2

(2500)

3 (7500/6000)

1

(750)

2

(2500)

3 (7500/6000)

Final body weight

-2.3%

-0.6%

-7.9%**

 

 

 

Ovaries

 

 

 

+11.658%*

+16.471%

-9.733%

*p ≤ 0.05; **p ≤ 0.01

 

 

Table 8: Relative weights

 

Male animals

Test group (ppm)

1

(750)

2

(2500)

3

(7500/6000)

Ovaries

+11.690%

+16.600%*

-7.125%

*p ≤ 0.05; **p ≤ 0.01

 

Table 9: Histopathology

 

Vagina and Cervix

Female animals

Test group (ppm)

0

(0)

1

(750)

2

(2500)

3 (7500/6000)

No. of animals

10

10

10

10

Mucification, increased

1

0

1

5

·        Grade1

 

 

 

2

·        Grade2

1

 

2

3

 

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration via drinking water of Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride to Wistar rats revealed signs of toxicity in male and female animals at a concentration of 7500 ppm (whole product). No adverse signs of toxicity were observed at a concentration of 6000 ppm (whole product).
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 6000 ppm for male (245 mg/kg bw/d) and female Wistar rats (453 mg/kg bw/d). The NOAEL for reproductive performance and fertility was set to 7500/6000 ppm for male and female Wistar rats. The NOAEL for developmental toxicity was 7500/6000 ppm.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
93 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral repeated dose toxicity

key study:

The oral repeated dose toxicity of Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride was investigated by means of a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and GLP.

Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) via the drinking water at concentrations of 0, 750, 2500 and 7500 ppm (whole product consisting of 38% of Disodium (sulphonatothio)acetate and sodium chloride as well as about 62% of water). Control animals were dosed daily with the vehicle only (drinking water). Due to reduced food and water consumption and reduced body weight/body weight change values in animals of test group 3 (7500 ppm), the concentration for this test group was reduced to 6000 ppm from study day 13 onwards (end of premating phase).

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to the day of schedule sacrifice of the animals. Due to naturally occurring increased water consumption of female animals during lactation and, consequently, a much higher test-substance intake, the concentrations for all test groups were reduced on purpose by 50% from delivery until sacrifice.

Observations: The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) 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 regularly once weekly before and after the mating period, as well as in dams during gestation days 0-7, 7-14 and 14-20 and lactation days 1-4, 4-7, 7-10 and 10-13. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 4, 7, 10, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted. At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2 under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing. Towards the end of the administration period, a functional observational battery was performed, and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group towards the end of the administration period. 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 analyses confirmed:

• the stability of the test-substance preparations for a period of 7 days at room temperature,

• the homogeneous distribution of the test substance in the vehicle,

• the correctness of the prepared concentrations in most preparations.

The following test substance-related, relevant findings were noted:

Test group 3: 7500 ppm (between study days 0 to 13)

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• Piloerection was observed in a single male animal between study days 9 to 12.

• Decreased water consumption in male and female animals showing a maximum deviation of -45% on study day 7 in males and -48% on study day 3 in females.

• Decreased food consumption in male and female animals showing a maximum deviation of -22% in males and -21% in females on study day 7.

• Impaired body weight development in male and female animals including body weight loss between study days 0-7 in male animals and between 0-3 in female animals.

Test group 3: 6000 ppm (from study day 13 onwards)

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Test group 2: 2500 ppm

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Test group 1: 750 ppm

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Conclusion: Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration via drinking water of Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride to Wistar rats revealed signs of toxicity in male and female animals at a concentration of 7500 ppm (whole product). No adverse signs of toxicity were observed at a concentration of 6000 ppm (whole product). Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 6000 ppm for male (245 mg/kg bw/d) and female Wistar rats (453 mg/kg bw/d).

With regard to the content of Disodium (sulphonatothio)acetate and sodium chloride (38% of whole product), this corresonds to a NOAEL for general systemic toxicity of 93 mg/kg bw/d and 172 mg/kg bw/d for males and females, respectively.

The NOAEL for reproductive performance and fertility was set to 7500/6000 ppm for male and female Wistar rats. The NOAEL for developmental toxicity was 7500/6000 ppm.

This study was chosen as key study, since compared to the supporting study, higher dosages were tested at which no adverse effects were observed. Moreover this GLP-conform study was conducted according to the newest guideline version.

supporting study

The test substance (ca. 39.5 % test substance preparation) was administered to male and female Wistar rats for 4 weeks in drinking water at nominal concentrations of 0; 300; 2000 and 12000 ppm (0, 11, 62, 287 mg/kg). Based upon analytically found concentrations and recorded water consumption and body weights, these concentrations corresponded to a test substance intake (active ingredient) of 0; 11, 62, and 287 mg/kg body weight/day, respectively.

Five animals per sex of all groups were treated for 4 weeks and sacrificed thereafter; the remaining 5 animals per sex of control and high dose group were maintained for another 2 weeks without administration of the test substance (recovery period).

At the high concentration, water consumption was impaired in both sexes during the administration period. Also food consumption, body weights and food efficiency (day 7 only) were statistically significantly lower than controls, most probably as a consequence of the impaired water consumption. For all parameters, a recovery could be observed, although recovery from the impairment of body weight in high dose males was not complete until the end of two 2-week treatment free period.

The slight increase in serum alanine aminotransferase activities seen in the high dose males is indicative of mild hepatocellular damage at dose levels of 287 mg/kg bw/day.

The increases in urea concentrations in the sera of the high dose males and females are possibly due to mild impairment of kidney function. Dysfunction of the kidneys coupled with increased renal loss of magnesium may be the cause of slight hypomagnesemia seen in the high dose females. On the other hand, the excretion of decreased amounts of concentrated urine demonstrates the ability of the kidneys to concentrate the urine. High urinary specific gravity indicate adequate renal function to maintain normal homeostasis. Since there is no evidence of a nephrotoxic effect, the mechanism of the aforementioned changes in serum and urine parameters are due to reduced water consumption rather than damage to or functional impairment of the kidneys. Moreover, the decreases in serum creatinine and albumin concentrations seen in the high dose females are probably associated with the reduced food consumption and body weight of the animals given 287 mg/kg bw/day of the test compound.

Histopathology revealed no findings which could be attributed to the test substance administration.

Thus the NOAEL in this study was at ca. 62 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.