Reaction mass of dipotassium 3,3'-sulphonylbis(benzenesulphonate) and potassium 3-(phenylsulphonyl)benzenesulphonate

Endpoint summary

Description of key information

In a GLP Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, conducted in accordance with OECD guideline 422 with male and femal Wister (Han) rats by oral gavage at doses of 0 (vehicle only), 100, 300, and 1000 mg/kg-bw/day, a NOAEL of 300 mg/kg was established for male rats based increased liver weights and minimal to slight microscopic findings at 1000 mg/kg and a NOAEL of 1000 mg/kg was established in female rates based on the absence of toxicologically relevant adverse findings at the highest dose tested.

At 1000mg/kg, increased liver and kidneys weights were observed in both sexes. Histopathological examination of the liver revealed a suspected correlating hepatocellular hypertrophy also in both sexes, which was accompanied by single cell necrosis in males only. Therefore, the changes in the liver in high dose males were considered to be adverse, whereas that in females were considered to be non-adverse, mainly because of the absence of any degenerative findings. In the kidneys, hyaline droplet accumulation, a finding not relevant for humans, was observed in male rats without evidence of tubular damage in the kidneys. No histopathological findings were observed in the female kidneys which could be correlated with the increased weights. In the absence of indicators of kidney damage in both sexes the findings in the kidneys were considered to be non-adverse.

A relation of the changes in body weights and total bilirubin levels with the effects in the liver and/or kidneys in males and of the changes in total bilirubin and creatinine with those in females could not be established from the results in this study. However, based on the magnitude of the change each individual effect was considered to be non-adverse under the conditions of the current study, except for the changes in the liver of the males at 1000 mg/kg. No treatment-related changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, food consumption, haematology and coagulation parameters, T4 thyroid hormone levels and macroscopic examination).

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:

15 November 2017 - 17 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: Reaction mass of dipotassium 3,3’-sulphonylbis(benzenesulphonate) and potassium 3-(phenylsulphonyl)benzenesulphonate
Acronym: KSS-FR
CAS number: Reaction mass of CAS# 63316-33-6 and CAS# 63316-43-8
Molecular formula: C12H8O8S3.2K (Di-KSS); C12H9O5S2.K (Mono-KSS)
Molecular weight: 454.581 (334 = Di-KSS); 336.428 (264 = Mono-KSS)
Appearance: White powder
Batch: KSS-1708-10
Purity/Composition: 92.6
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (expiry date)
pH: 6.95 (water solution)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The test laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age of Males (on Treatment Day 1): Approximately 10 - 12 weeks old
Age of Females (on Treatment Day 1): Approximately 12 - 14 weeks old
Weight of Males (on Treatment Day 1): 271 - 299 grams
Weight of Females (on Treatment Day 1): 198 - 248 grams
Health Status: All males and females were inspected prior to dosing and found to be healthy
Animal Identification: Earmark and Tattoo; Reserve females were randomly selected and numbered R1 through R8 with indelible marker. Pups were randomized and identified by injection of Indian Ink and, as needed tatoo on the feet.
Acclimitization: 7 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males)
Housing: Appropriate depending on stage of the study. Refer to full study report.
Env. Conditions: Temp: 20 - 22 deg Celsius; Relative Humidity: 43 - 57%; Lighting: 12-hour light/dark cycle; Air Changes: >=10/hour with 100% fresh air in the room.
Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
Water: Municipal tap water was freely available to each animal via water bottles
Nesting Material: Paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom)

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Frequecy: Once during week 1 of dosing
Dose Groups Tested: Concentration: All Groups; Homogeneity: Groups 2 and 4
Method: LC-DAD (Method Validated by Analytical Biochemical Laboratory B.V. (ABL)
Stability of Test Substance in Vehicle: Verified to be stable during method development

Duration of treatment / exposure:

Males: 29 days up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females that failed to deliver: 41-51 days
Females that delivered: 50 - 62 days; i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.

Frequency of treatment:

Once daily, 7 days per week.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle only

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose Level Selection: Based on the results of a 10-day dose range finder with oral gavage administration in an attempt to produce graded responses to the test item.
Dose Volume: 5 ml/kg

Positive control:

No

Observations and examinations performed and frequency:

F0 Generation:
Mortality/ moribundity: Twice daily, in the morning and at the end of the working day
Clinical signs: Once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy
Functional observations (for 5 selected animals/sex/group; hearing, pupillary reflex, static righting reflex, strength, locomotor activity): Males: week 4 of treatment; Females: during last week of lactation (PND 6 - 13).
Body weight: Weekly
Food consumption: Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal day (PND) 1, 4, 7, and 13
Estrous cycle determination: All femails daily begining 13 days prior to treatment, the first 14 days of treatment, and during mating until evidence of copulation was observed.
Clinical pathology (for 5 selected animals/sex/group): Blood was collected once on the day of scheduled necropsy and included typical hematology and clinical chemistry parameters, and measurement of thyroid hormone T4 (F0-males).

F1 Generation:
Mortality/ moribundity: Daily
Clinical signs: At least once daily
Body wieght: Live pups were weighed individually on PND 1, 4, 7 and 13
Sex: Sex was externally determined for all pups on PND 1 and 4
Anogenital distance (AGD): Measured for all live pups on PND 1
Areola/Nipple Retention: All male pups in each litter were examined for the number of areola/nipples on PND 13
Culling: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup).
Clinical pathology: Blood of F1-animals was collected on PND 4 and PND 14-16, if possible in included typical hematology and clinical chemistry parameters, and measurement of thyroid hormone T4 (2 pups per litter)

Sacrifice and pathology:

All animals were subjected to a full post mortem examination.

Organ Weights (paired together, as appropriate: brain, cervix, epididymis, adrenal gland, coagulation gland, parathyroid gland, prostate gland, seminal vesicle gland, thyroid gland, heart, kidney, liver, ovaries, spleen, testes, thymus, and uterus.

Tissue Collection/Preservation/Examination: Animal ID, aorta artery, nasopharynx body cavity, bone marrow, femur, sternum, brain (seven levels), cervic, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal glan, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, larynx, liver, lung, mandibular and meenteric lymph nodes, skelatal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, duoenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, tongue, tracea, urinary bladder, uterus. amd vagina.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On the day of scheduled necropsy, one female (no.48, control group) died as a result of a blood sampling-related injury. This death was considered not to be test item-related. No findings were observed at macroscopic examination of this female.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at 1000 mg/kg, slightly reduced body weight gain (up to maximally 6%) was observed during the treatment period, in comparison with controls. No effects on body weights were observed in females up to 1000 mg/kg.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased levels of total bilirubin were observed in both sexes at 1000 mg/kg and decreased creatinine in females at 1000 mg/kg. Lower bilirubin levels were considered to be test item related but the toxicological relevance was doubted as the opposite effect would be expected in case of target (liver) organ toxicity. Furthermore, based the magnitude of the decrease in creatinine in females at 1000 mg/kg, which was possibly related to the histopathological findings in the liver (see below), was considered to be non-adverse. Serum levels of T4 in F0 males were considered not to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver and kidneys weights were observed in both sexes at 1000 mg/kg. Histopathological examination of the liver revealed a suspected correlating hepatocellular hypertrophy also in both sexes, which was accompanied by single cell necrosis in males only. Therefore, the changes in the liver in high dose males were considered to be adverse, whereas that in females were considered to be non-adverse, mainly because of the absence of any degenerative findings. In the kidneys, hyaline droplet accumulation, a finding not relevant for humans, was observed in male rats without evidence of tubular damage in the kidneys. No histopathological findings were observed in the female kidneys which could be correlated with the increased weights. In the absence of indicators of kidney damage in both sexes the findings in the kidneys were considered to be non-adverse.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of the liver revealed a suspected correlating hepatocellular hypertrophy also in both sexes, which was accompanied by single cell necrosis in males only. In the kidneys, hyaline droplet accumulation, a finding not relevant for humans, was observed in male rats without evidence of tubular damage in the kidneys. No histopathological findings were observed in the female kidneys which could be correlated with the increased weights. In the absence of indicators of kidney damage in both sexes the findings in the kidneys were considered to be non-adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No toxicologically relevant adverse effects observed at the highest dose tested.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Organ Weights (Mean Percent Organ Weight Differences from Control Groups)

	Males			Females
Dose Level	100	300	1000	100	300	1000
KIDNEY
- Absolute	-1	-1	7	4	9	17^**
- Relative to bw	0	4	16^**	0	4	13^**
Liver
- Absolute	-3	-1	9	6	12	13
- Relative to bw	-2	4	19^**	3	7	9

^**P<0.01

Histopathology (Summary of Test Item-Related Microscopic Findings - Liver)

	Males				Females
Dose Level:	0	100	300	1000	0	100	300	1000
LIVER	5	5	5	5	5	5	5	5
Hepatocellular centrilobular hypertrophy
- Minimal	---	---	---	4	---	---	---	1
- Slight	---	---	---	1	---	---	---	---
Single cell necrosis
- Slight	---	---	---	1	---	---	---	---

Histopathology (Summary of Test Item-Related Microscopic Findings - Kidney)

	Males
Dose Level:	0	100	300	1000
KIDNEYS	5	5	5	5
Hyaline droplet accumulation
- Minimal	2	1	2	1
- Slight	---	1	---	3
- Moderate	---	---	---	1

Conclusions:

A NOAEL of 300 mg/kg was established in male rats based on increased liver weights and minimal to slight hepatocellular centrilobular hypertrophy at 1000 mg/kg and a NOAEL of 1000 mg/kg was established in female rats based on the absence of any toxicologically relevant adverse effects at the highest dose tested. The relevance of the male rat findings to humans is unknown. Given the adverse effects observed, incidence, dose level, study duration, and unknown relevance to humans, the GHS criteria for classification under specific target organ toxicity (STOT) have not been met.

Executive summary:

In this GLP Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, conducted in accordance with OECD guideline 422 with male and femal Wister (Han) rats by oral gavage at doses of 0 (vehicle only), 100, 300, and 1000 mg/kg-bw/day, a NOAEL of 300 mg/kg was established for male rats based increased liver weights and minimal to slight microscopic findings at 1000 mg/kg and a NOAEL of 1000 mg/kg was established in female rates based on the absence of toxicologically relevant adverse findings at the highest dose tested.

At 1000 mg/kg, increased liver and kidneys weights were observed in both sexes. Histopathological examination of the liver revealed a suspected correlating hepatocellular hypertrophy also in both sexes, which was accompanied by single cell necrosis in males only. Therefore, the changes in the liver in high dose males were considered to be adverse, whereas that in females were considered to be non-adverse, mainly because of the absence of any degenerative findings. In the kidneys, hyaline droplet accumulation, a finding not relevant for humans, was observed in male rats without evidence of tubular damage in the kidneys. No histopathological findings were observed in the female kidneys which could be correlated with the increased weights. In the absence of indicators of kidney damage in both sexes the findings in the kidneys were considered to be non-adverse.

A relation of the changes in body weights and total bilirubin levels with the effects in the liver and/or kidneys in males and of the changes in total bilirubin and creatinine with those in females could not be established from the results in this study. However, based on the magnitude of the change each individual effect was considered to be non-adverse under the conditions of the current study, except for the changes in the liver of the males at 1000 mg/kg. No treatment-related changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, food consumption, haematology and coagulation parameters, T4 thyroid hormone levels and macroscopic examination).

Given the adverse effects observed, incidence, dose levels, study duration, unknown relevance to humand, and the resulting NOAELs of 300 mg/kg (males) and 1000 mg/kg (females), the GHS criteria for classification under specific target organ toxicity (STOT) were not met.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Adequate for the volume being registered.

System:

hepatobiliary

Organ:

liver

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

Mode of Action Analysis / Human Relevance Framework

The adverse effects noted for the livers of male rats only is of unknown relevance to humans but is suspected as not being specifically relevant given that the effects were only seen in male rats at the highest dose tested.

Justification for classification or non-classification

Given the adverse effects observed, incidence, dose levels, study duration, unknown relevance to humand, and the resulting NOAELs of 300 mg/kg (males) and 1000 mg/kg (females), the GHS criteria for classification under specific target organ toxicity (STOT) were not met.