3-methyltetrahydrothiophene 1,1-dioxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Feb 2018 - 31 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

yes

Remarks:

but none influenced validity of the study

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methyltetrahydrothiophene 1,1-dioxide

EC Number:

212-833-9

EC Name:

3-methyltetrahydrothiophene 1,1-dioxide

Cas Number:

872-93-5

Molecular formula:

C5H10O2S

IUPAC Name:

3-methyl-1λ⁶-thiolane-1,1-dione

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl: WI(Han).

Details on species / strain selection:

Outbred, SPF-Quality. 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females: nulliparous and non-pregnant
- Age at study initiation (initiation of dosing): Males: approximately 10-12 weeks. Females: approximately 12-14 weeks.
- Weight at study initiation: Males: 250 to 350 g. Females: 200 to 250 g.
- Fasting period before study: no, except during motor activity measurements, animals will not have access to food for a maximum of 2 hours.
- Housing:
Pretest/pre-mating period (females only), recovery animals: group housed (5 animals, same sex and dosing) in polycarbonate cages
Mating phase, cohabitate Main males and Main females 1:1 in Macrolon plastic cages
Post-mating phase: Main males 5 males/cage, Females individual, Macrolon plastic cages.
Lactation: Main females Macrolon plastic cages, together with pups
The cages will contain appropriate bedding and will be equipped with water bottles.

During locomotor activity monitoring: F0-animals housed individually in a Hi-temp polycarbonate cage without cage enrichment,bedding material, food and water.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days prior to start of the pretest period (females) or at least 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) will be provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals will not have access to food for a maximum of 2 hours. The feed is analyzed by the supplier for nutritional components and environmental contaminants.
Municipal tap water is used Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): At least 10.
- Photoperiod (hrs dark / hrs light): 12/12 (may be interrupted for designated procedures).

IN-LIFE DATES:
Arrival: males 28 Mar 2018 (Males), 14 Mar 2018 (Females)
Completion in life: 15 Jun 2018

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item and vehicle are administered to the appropriate animals once daily oral gavage 7 days a week for a minimum of 28 days.

Vehicle:

water

Remarks:

Elix

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) are homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator for a maximum of 8 days. If not used immediately after preparation, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. Adjustment is made for specific gravity of the test item. No correction was
made for the purity/composition of the test item.

- VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for Analysis: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis).
Sample Volume: Approximately 500 mg accurately weighed.
Acceptance Criteria: For concentration, the criteria for acceptability were mean sample concentration results within or equal to ± 10% for solutions or ±15% for suspensions of target concentration. For homogeneity, the criteria for acceptability were a coefficient of variation (CV) of concentrations of ≤ 10% for each group.

Duration of treatment / exposure:

Males were treated for a minimum of 28 days, up to and including the day before scheduled necropsy. This includes a minimum of two weeks prior to mating and during the mating period.

Females were treated for at least14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering.

Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 (additional 5 for 2 recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on dose range finding study, 50 mg/kg was selected as highest dose level for this study because testing of higher dose levels was considered unfeasible based on dose-limiting effects observed in the dose range finder study (i.e. clinical signs of toxicity and transient weight loss in females treated at 100 mg/kg for 17 days).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females will be weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Weekly, except for Main males and Main females which are housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females is measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Regular basis throughout the study (visual examination)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after end of treatment on scheduled necropsy (main animals), on day of scheduled necropsy (recovery animals)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes (except F0 main females)
- How many animals: all main and recovery animals.
- Parameters examined.: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells platelets, Reticulocyte (absolute), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after end of treatment on scheduled necropsy (main animals), on day of scheduled necropsy (recovery animals)
- Animals fasted: Yes (except F0 main females)
- How many animals: all main and recovery animals. T4 measurements only for F0 males.
- Parameters examined: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein Sodium, Albumin Potassium, Total Bilirubin Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos), Thyroxine (T4).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION / FUNCTIONAL TESTS: Yes
- Time schedule for examinations: males during week 4 of treatment, females during last week of lactation. Recovery animals at the end of the recovery phase.
- Dose groups that were examined: all (5 animals of each group)
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.

IMMUNOLOGY: No

ESTROUS CYCLE: Yes

GENERAL REPRODUCTION DATA: Yes, for females
Parameters examined: male number paired with, mating data, confirmation of pregnancy and delivery day

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Post mortem examination with special attention to reproductive organs. Number of former implantation sites, where no sites were macroscopically visible nongravid uteri were further examined using Salewski staining technique.

ORGAN WEIGHTS: Yes, for all selected main animals and all recovery animals
Tissue examined: Brain, Cervix, Epididymis, Gland, adrenal, Gland, coagulation, Gland, parathyroid, Gland, prostate, Gland, seminal vesicle, Gland, thyroid, Heart, Kidney, Liver, Ovaries, Spleen, Testes, Thymus, Uterus.
For all remaining main animals: Epididymis, Gland, coagulation, Gland, parathyroid, Gland, prostate, Gland, seminal vesicle, Gland, thyroid, Testes.

HISTOPATHOLOGY: Yes, with HE staining
- For selected main animals: Animal identification*, Artery, aorta*, Body cavity, nasopharynx*, Bone marrow, Bone, femur, Bone, sternum, Brain (seven levels), Cervix, Epididymis, Esophagus*, Eye, Gland, adrenal, Gland, coagulation, Gland, harderian*, Gland, lacrimal*, Gland,, mammary, Gland, parathyroid, Gland, pituitary, Gland, prostate, Gland, salivary*, Gland, seminal vesicle, Gland, thyroid, Gross lesions/masses, Gut-associated lymphoid tissued, Heart, Kidney, Large intestine, cecum, Large intestine, colon, Large intestine, rectum, Larynx*, Liver, Lung, Lymph node (mandibular and mesenteric site), Muscle, skeletal, Nerve, optica*, Nerve, sciatic, Ovaries, Pancreas*, Skin*, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach, Testes, Thymus, Tongue*, Trachea, Urinary bladder, Uterus, Vagina. *Collected but not processed for staining
- For males that failed to sire and female that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina
- For remaining animals: Gross lesions/masses
- All remaining main animals (collected but not preserved): Animal identification, Cervix, Epididymis, Gland, coagulation, Gland, mammary, Gland, parathyroid, Gland, pituitary, Gland, prostate, Gland, seminal vesicle, Gland, thyroid, Gross lesions/masses, Ovaries, Testes, Uterus, 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% or 5% levels.
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-parametric: 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. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs of toxicity were noted. Salivation was noted at 50 mg/kg in 9/15 females (lactating and nulliparous), starting after 5 weeks of treatment but was considered a physiological response.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain of males at 50 mg/kg was slightly (statistically significant) lower than that of controls in the first week of the treatment period. As the change was only minor and complete recovery was seen in the subsequent period, it was not regarded as toxicologically relevant. A few statistically significant differences from controls were noted in females at the intermediate dose levels, but without a dose-related trend and therefore not considered related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant difference noted in females was regarded as unrelated to treatment due to its incidental occurrence.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Isolated, statistically significant intergroup differences observed at the end of the treatment period were regarded as unrelated to treatment due to the lack of a dose-related response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some statistically significant effects on ALAT, potassium, chloride, inorganic phosphate were noted for the high-dose groups, but all findings remained within histiorical control values. Statistically significantly higher (11%) creatinine values were observed in males and nulliparous females at the end of the recovery period, but were regarded as unrelated to treatment due to the lack of a test item-related change in creatinine at the end of the treatment period. Serum levels of T4 in F0-males were not affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered not to be affected by treatment and hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Minor statistically significant effects were noted for high-dose females with regard to grip strength and males with regard to motor activity. However, as these findings were within the historical control range, these effects were not considered treatment related. No other statistically significant differences were found.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

All statistically significant changes were considered to be unrelated to treatment as they occurred without dose-relationship, remained within historical control data and no microscopic correlate was observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One female showed a malignant tumor in the mammary gland, but his was considered an incidental finding that can occur in young rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed (highest dose tested)

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Dose formulation analysis: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤10%).

Dose-range finding study: This study tested dose levels of 50 and 100 mg/kg bw/day for 15 and 17 days in 3 female rats/group. Severe clinical signs were noted for the 100 mg/kg bw/day group (hunched posture, flat gait, decrease locomotor activity, piloerection in all rats). In both groups, no clear effects on body weight, food consumption and organ weights were observed and no abnormalities were noted in the macroscopic examination. Based on these findings, the lowest tested level was selected for the main study as the maximum dose, mainly for humane reasons.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL for repeated dose toxicity was determined to be >50 mg/kg bw/day based on the absence of treatment-related effects. Based on this result and the results of the dose-range finding study, the substance needs to be classified for repeated dose toxicity (STOT RE 2 / H373) based on the criteria and guidance values (for a 28-day study) outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The toxicity of 3-methyl sulfolane was evaluated in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in accordance with the OECD TG 422 and under GLP conditions. Dose levels were based on a dose-range finding study and set at 5, 15 and 50 mg/kg bw/day. Doses were analytically verified during the study. Two recovery groups were included to study persistence of possible effects in the dose groups. Effects on clinical signs, mortality, body weight (changes), food consumption, functional performance, hematology, clinical chemistry (including T4 analysis), gross necropsy, organ weights, histopathology and some reproductive parameters were studied.

The study was fully performed in accordance with the guideline and GLP conditions without any deviations that influence the validity. No significant adverse effects that were considered treatment-related were noted up to the highest dose tested in males and females (including recovery groups). Therefore, the NOAEL for repeated dose toxicity was determined to be >50 mg/kg bw/day based on the absence of treatment-related effects. Based on this result and the results of the dose-range finding study, the substance needs to be classified for repeated dose toxicity (STOT RE 2 / H373) based on the criteria and guidance values (for a 28-day study) outlined in Annex I of the CLP Regulation (1272/2008/EC).