Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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:
reference to same study
Reference
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
Reason / purpose:
reference to same study
Qualifier:
according to
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
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 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
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
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
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).
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.
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
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)
Key result
Critical effects observed:
no

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.

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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 exposure:
Test item dosing formulations (w/w) are homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations are prepared daily and dosed within 6 hours after adding the vehicle to the test item. 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear, referred to as day 0 of pregnancy
- In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male of proven fertility of the same group for a maximum of 7
days (if possible).
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
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 / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
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

Examinations

Parental animals: Observations and examinations:
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.

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, two surplus pups per litter
- 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, two surplus pups per litter. 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), Th
yroxine (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
Oestrous cyclicity (parental animals):
Estrous cycle is evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage continued during mating until evidence of copulation is observed. On the day of necropsy, a vaginal lavage was taken from all Main and Recovery females to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in Main male parental generations: testis weight, epididymis weight.

For males that fail to sire, a detailed qualitative examination is made, taking into account the tubular stages of the spermatogenic cycle. The examination is conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were used for blood collection, killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, clinical observations, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, serum T4 levels.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; if possible. The cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Main Males: after a minimum of 28 days of administration
- Main Females: PND 14-16
- Recovery Males: 14 days after end of treatment
- Recovery Females: at least 14 days after the first scheduled necropsy of Main females

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.
Postmortem examinations (offspring):
SACRIFICE
- On PND 4, the surplus pups (> 8 pups per litter) was euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups are euthanized on PND 14-16.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Sex is determined both externally and internally. Descriptions of all external abnormalities are recorded. Particular attention is paid to the external reproductive genitals to examine signs of altered development. External abnormalities are collected and fixed in 10% buffered formalin at discretion of the Study Director. In addition, the thyroid is collected from two pups per litter.
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.
Reproductive indices:
Mating index (%), precoital time, number of implantation sites, fertility index (%).
Offspring viability indices:
Gestation index (%) and duration, Post-implantation survival index (%), Live birth index (%), Percentage live males at First Litter Check (%), Percentage live females at First Litter Check (%), Viability index (%), Lactation index (%).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Extended di-estrus during pairing was noted in three females (2 control group, 1 high-dose) with normal litters. The extended di-estrus for one animal could be explained by the continuation of vaginal lavages after mating (mating was overlooked in the initial assessment of the vaginal lavages). The extended di-estrus at 50 mg/kg was not attributed to treatment due to its incidental occurrence.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
An unusually high number of implantation sites (i.e. 19) was noted in one female of the high dose group. This finding was regarded as unrelated to treatment due to its incidental occurrence. The non-pregnancy of one mated female of the lowest dose group, without related histopathology changes in reproductive organs, was regarded as unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.

Effect levels (P0)

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 (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
It was noted that mean body weights of male and female pups at 50 mg/kg were about 10% lower than those of control pups at PND 1 and 4. Except for the combined weight of male and female pups at PND 1, statistical significance was not achieved. Moreover, the relative differences from controls became smaller after culling and mean body weights of 50 mg/kg pups were close to the historical control group means. Therefore, pup body weight was considered not to be affected by treatment with the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were not affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect on areola/nipple retention in male pups
- The statistically significantly higher mean normalized anogenital distance noted in male pups at 15 mg/kg was regarded as unrelated to treatment due to the lack of a dose-related trend.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Gestation index and duration, post-implantation survival index, litter size, live birth index, viability index, lactation index and sex ratio were not affected by treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
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 (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, no reproductive or developmental toxicity effects were observed and the NOAEL for both endpoints was established to be >50 mg/kg bw/day (highest dose tested). Therefore, the substance does not need to be classified for reproduction/developmental toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The reproduction and developmental 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 for the P1 generation. For the F1 generation mortality, clinical signs, body weights, macroscopic changes, anogenital distance, areola/nipple retention and total T4 levels.

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). This includes reproductive parameters and indices. Also no treatment-related effects were noted in the offspring, including limited developmental parameters. Based on these results, the NOAEL for both endpoints (reproduction and developmental) was established to be >50 mg/kg bw/day (highest dose tested). Therefore, the substance does not need to be classified for reproduction/developmental toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Under the conditions of this study, no reproductive or developmental toxicity effects were observed and the NOAEL for both endpoints was established to be >50 mg/kg bw/day (highest dose tested). Therefore, the substance does not need to be classified for reproduction/developmental toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).