3-((3,4-dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Endpoint summary

Description of key information

Effects of repeated dose exposure.

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:

Experimental phase: 19 June 2017 to 21 August 2017.

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:

yes

Remarks:

Deviations were considered to not have affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1315
- Expiration date of the lot/batch:01-03-2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Darkness at ambient temperature and humidity
- Solubility and stability of the test substance in the solvent/vehicle: The stability and homogeneity of the test item formulations were determined and showed the formulations to be stable for at least 21 days when stored refrigerated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. Formulations were prepared fortnightly and stored at approximately 4 ºC in the dark.

FORM AS APPLIED IN THE TEST: Liquid suspension

Species:

rat

Strain:

other: Wistar Han: RccHan: WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.
- Females nulliparous and non-pregnant: not stated
- Age at study initiation: At the start of treatment the males weighed 281 to 356g, and were approximately eleven weeks old. The females weighed 204 to 255g, and were approximately fourteen weeks old.
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet and water: The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: The animals were acclimatized for nineteen days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. There were deviations from the target range for temperature and short term deviations from the target range for relative humidity that were considered not to have affected the purpose or integrity of the study. Maximum relative humidity recorded during the study, 80%.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Dose level: 75, 300 and 1000 mg/kg bw/day
Treatment volume: 4 mL/kg
Concentration: 18.75, 75 and 250 mg/ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken on 3 occasions and analysed for concentration of the test material. The results indicate that the prepared formulations were within ± 4% of the nominal concentration.

Duration of treatment / exposure:

Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).

Frequency of treatment:

Daily.

Dose / conc.:

75 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:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of previous toxicity work including a Fourteen Day Repeated Dose Range Finding Study in the Rat, in which there were no mortalities or significant adverse clinical observations at the maximum dose used of 1000 mg/kg bw/day.

- Rationale for animal assignment:The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure.

Positive control:

None.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition).

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION :
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OTHER: Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Sacrifice and pathology:

ORGAN WEIGHTS
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues indicated with # were weighed from all remaining animals:

Adrenals
Brain
Epididymides #
Heart
Kidneys
Liver
Ovaries #
Pituitary (weighed partially fixed) #
Prostate #
Seminal Vesicle (with coagulating gland) #
Spleen
Testes #
Thymus
Thyroid (weighed partially fixed with Parathyroid) #
Uterus & Cervix (weighed with oviducts) #

HISTOPATHOLOGY
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved. Tissues indicated with # were preserved from all remaining animals:

Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Cecum
Colon
Cowpers Glands #
Duodenum
Epididymides #
Esophagus
Eyes
Glans Penis #
Gross lesions #
Heart
Ileum (including peyer’s patches)
Jejunum
Kidneys
LABC (levator ani-bulbocavernous) muscle #
Liver
Lungs (with bronchi)
Lymph nodes (mandibular and mesenteric)
Mammary gland #
Muscle (skeletal)
Ovaries #
Pancreas
Pituitary #
Prostate #
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating gland) #
Skin
Spleen
Stomach
Testes #
Thyroid/Parathyroid
Thymus
Trachea
Urinary bladder
Uterus & Cervix (with oviducts) #
Vagina #

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Animals showed increased post dosing salivation. Such salivation is often observed when administering test formulations via the oral gavage route and is generally considered to reflect palatability or slight irritancy of the test item formulations rather than any systemic effect of treatment.

Noisey respiration was observed in in some animals at all doses although the incidence was higher in the 1000 mg/kg bw/day treated group. However, these occurrences only occurred on single occasions, and the most prolonged periods of noisy respiration observed on the study were from animals belonging to the control groups. These incidence of noisy respiration observed during the study was considered to reflect isolated difficulties with dosing particular animals on occasions rather than any effect of treatment.

At 1000 mg/kg bw/day, one female showed piloerection on Day 29 and Day 40 and again piloerection and hunched posture on Day 42 of the study. During behavioural assessments, this female also showed piloerection and hunched posture on Day 11 of gestation and staining around the eyes on Day 12 of lactation. Although the cause of these clinical signs could not be established, they did not prevent this female successfully maintained a litter to Day 13 of lactation and they were considered to be of no toxicological significance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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):

For both sexes at all dosages, statistically significantly higher reticulocytes were apparent when compared to control. All individual values were within the historical control range and there were no supporting statistically significant differences from control for erythrocyte parameters at any dosage. In the absence of any supporting histopathological change this finding was considered to be incidental and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males changes in glucose, albumin/globulin (A/G) ratio, creatinine, bile acid and mean potassium and calcium levels were observed. While in females changes to alanine aminotransferase and cholesterol were observed. The differences were not statistically significant from control and are therefore considered to be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Isolated incidence of clinical signs were observed during the performance of behavioural ssessments but the incidence and distribution did not indicate any underlying neurological effect and these findings were considered to be incidental and of no toxicological significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, lower absolute and body weight relative uterine weights were statistically significantly lower than control and absolute and body weight relative thymus weights were also statistically significantly lower than control. However, in the absence of any supporting histopathology, this finding was considered to be incidental and unrelated to treatment.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Tested up to the maximum recommended dose with only non-treatment related effects observed.

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Conclusions:

Treatment at dosages of up to 1000 mg/kg bw/day was well tolerated and the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item following repeated exposure. The method was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

 

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to eight weeks (females) at dose levels of 75, 300 and 1000 mg/kg bw/day.  As the study included a reproductive /developmental toxicity component, the dosing period included a two-week pre-pairing phase, pairing, gestation and early lactation for females. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same period.

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals for thyroid hormone Thyroxine (T4) analysis.

Results (adult responses)

Mortality: There were no unscheduled deaths on the study.

Clinical Observations: There were no clinical signs observed that were considered to be indicative of systemic toxicity.

Behavioural Assessment: There was no effect of treatment on behavioural assessments for either sex at 75, 300 or 1000 mg/kg bw/day.

Functional Performance Tests: There was no effect of treatment on functional performance tests for either sex at 75, 300 or 1000 mg/kg bw/day.

Sensory Reactivity Assessments: There was no effect of treatment on sensory reactivity assessments for either sex at 75, 300 or 1000 mg/kg bw/day.

 

Body Weight: There was no effect of treatment on body weight gain for males or females at 75, 300 or 1000 mg/kg bw/day throughout the study.

 Food Consumption: There was no effect of treatment on food consumption or food conversion efficiency for males or females at 75, 300 or 1000 mg/kg bw/day throughout the study.

 Water Consumption: There was no effect of treatment on water consumption for either sex throughout the study at 75, 300 or 1000 mg/kg bw/day.

 Blood Chemistry and Haematology: There were no toxicologically significant effects of treatment on haematology or blood chemistry parameters at 75, 300 or 1000 mg/kg bw/day.

 Thyroid Hormone Analysis: Evaluation of Thyroxine (T4) in adult males did not identify any obvious effect of treatment or indication of endocrine disruption at 75, 300 or 1000 mg/kg bw/day.

 

Pathology

 Necropsy: Offspring Macroscopic necropsy findings for offspring did not indicate any effect of treatment at 75, 300 or 1000 mg/kg bw/day. Macroscopic necropsy findings for adults did not indicate any effect of treatment at 75, 300 or 1000 mg/kg bw/day.

Organ Weights: There was no adverse effect of treatment on organ weights for either sex at 75, 300 or 1000 mg/kg bw/day.  

Histopathology: Histopathological examination of tissues from the control and 1000 mg/kg bw/day animals did not reveal any findings considered to be related to treatment with the test item.

 

Conclusion

Treatment at dosages of up to 1000 mg/kg bw/day was well tolerated and the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat