lithium 3-((3,4-dicyanophenyl)sulfonyl)propane-1-sulfonate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 09 August 2012 and 11 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

yes

Remarks:

Minor deviations to temperature and humidity were considered not to affect the study. Due to a technical oversight the pre-dose behavioural assessments were not performed but this error was considered to have no impact on the validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: S193308
Description: White powder
Batch number: 422 (Bag 3)
Date received: 02 May 2012
Storage conditions: Room temperature in the dark.
Expiry date: Not supplied

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: Approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 298 to 345g, the females weighed 197 to 229g.
- Fasting period before study: None.
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. 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 (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was available ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage, available ad libitum.
- Acclimation period: The animals were acclimatised for five days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperatures controls were set to achieve target values of 21 ± 2°C.
- Humidity (%): The relative humidity controls were set to achieve target values of 55 ± 15 %.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: The in-life phase of the study was conducted between 22 August 2012 (first day of treatment) and 05 October 2012 (final necropsy).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not specified
- Mixing appropriate amounts with (Type of food): Not applicable
- Storage temperature of food: Not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable as the vehicle was distilled water.
- Concentration in vehicle: 0, 6, 60 or 100 mg/mL.
- Amount of vehicle (if gavage): The teatment volume was 5 mL/kg.
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD OF ANALYSIS
Summary
The concentration of S193308 in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Samples
The test item formulations were diluted with water to give a final, theoretical test item concentration of approximately 0.1 mg/mL.

Standards
Standard solutions of test item were prepared in water at a nominal concentration of 0.1 mg/mL.

Procedure
The standard and sample solutions were analysed by HPLC using the following conditions:

HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: ACE 3 (100 x 3 mm id) at 40°C
Mobile phase: Water: methanol (90:10 v/v)
Flow-rate: 1 mL/min
UV detector Wavelength: 249 nm
Injection volume: 25 μL
Retention time: ~ 1.5 mins
Homogeneity Determinations
The test item formulations were assessed visually.

Stability Determinations
The test item formulations were sampled and analysed initially and then after storage at approximately +4 ºC in the dark for twenty one days.

Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within two days of preparation.

Duration of treatment / exposure:

The male dose groups were killed on treatment Day 43.
At Day 5 post partum, all surviving females and surviving offspring were killed.

Frequency of treatment:

Daily throughout the study.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
30 mg/kg bw/day (equivalent to 6 mg/mL)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day (equivalent to 60 mg/mL)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg bw/day (equivalent to 100 mg/mL)
Basis:
actual ingested

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on range finder study.
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable

Examinations

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. 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.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Urination Lachrymation
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Bizarre/Abnormal/Stereotypic behaviour

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity.
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20 % of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength.
An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Vocalisation Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Touch escape Finger approach
Grasp response

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.
Normal range data for body weight changes in pregnant and lactating females are presented in Addendum 3.

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 on Days 1 and 4 post partum.
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 for females during gestation and lactation.

Water Consumption
Water intake was measured gravimetrically for the first fifteen days of the study. Daily visual inspection of the water bottles was conducted thereafter.

Reproduction Screening
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Laboratory Investigations
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids

Sacrifice and pathology:

Pathology
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced, as necessary, by staining the uteri with a 0.5 % ammonium polysulphide solution (Salewski 1964).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from five selected males and females from each dose group that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Prostate
Brain Seminal vesicles
Epididymides Spleen
Heart Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix)
Pituitary (post fixation)
The following organs, removed from all remaining animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Seminal vesicles, Epididymides, Testes, Prostate, Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

Histopathology
Samples of the following tissues were removed from five selected males and females from each dose group and any animal dying during the study and preserved in buffered 10% formalin, except where stated.
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Eyes*, Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions, Heart, Spleen, Ileum (including peyer’s patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes•, Lungs (with bronchi) #, Thymus, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary gland, Uterus/Cervix, Muscle (skeletal), Vagina

• = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
* = eyes fixed in Davidson’s fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Tissues below were taken from all remaining animals including any animal which fails to produce a pregnancy, and preserved in buffered 10 % formalin, except where stated. The tissues from the remaining control and 500 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined:
Ovaries, pituitary, prostate, coagulating gland, epididymides, seminal vesicles, mammary gland, testes, uterus/cervix, vagina.

The tissues from five selected control and 500 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination.
Since there were indications of treatment-related changes in the adrenals, examination was subsequently extended to include similarly prepared sections of adrenals from five animals per sex from the low and intermediate groups (or all twelve animals per sex in the case of reproductive tissues).

Other examinations:

Reproductive Indices

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = [number of animals mated/number of animals paired] x 100
Pregnancy Index (%) = [number of pregnant females/number of animals mated] x 100


Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = [number of females delivering live offspring/number of pregnant females] x 100

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss = [(number of corpora lutea - number of implantation sites)/number of corpora lutea] x100
Post–implantation loss = [(number of implantation sites - total number of offspring born)/number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = [number of offspring alive on Day 1/number of offspring born] x 100
Viability Index (%) = [number of offspingalive on Day 4/number of offspring alive on Day 1] x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter on Days 1 and 4 post partum, using the following formula: [number of male offspring/total number of offspring] x 100

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring.
For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.
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. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See results

Mortality:

mortality observed, treatment-related

Description (incidence):

See results

Body weight and weight changes:

no effects observed

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Adult responses:
Mortality
One 500 mg/kg bw/day female from Day 29 developed piloerection, stained fur around the snout and eyes. This was accompanied over the following days by decreased respiration, some swelling around the head, hunched posture, soiled ano-genital region, lethargy and dehydration at which time (Day 32) a decision was taken to terminate this animal on humane grounds. Macroscopic examination revealed fluid with or without gaseous distension along the intestinal tract and reddened lungs. It was suspected the deterioration in this animal may have been a result of a dosing procedural injury that only became evident after the event. There was no convincing supporting evidence to suggest it was directly related to test item toxicity. This hypothesis was further supported by histopathological evidence of lung alveolar oedema and haemorrhage, findings commonly associated with an intubation error.
There were no further unscheduled deaths.

Clinical Observations
There were no toxicologically significant clinical signs detected in treated animals.
One male treated with 500 mg/kg bw/day had increased salivation post dose on Day 25. One female treated with 300 mg/kg bw/day had increased salivation post dose on Day 43. The transient and isolated nature of these findings would suggest they were associated with the gavage procedure and not test item toxicity.
One male treated with 30 mg/kg bw/day had noisy respiration from Day 37 onwards. In the absence of similar effects in animals treated with 500 mg/kg bw/day, this abnormality was considered to be an isolated incident of no toxicological significance.

Functional Observations
Behavioural Assessments
There were no treatment-related changes in the behavioural parameters measured.
All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests
There were no convincing treatment related changes detected in the functional performance measurements.
Statistical analysis of the data revealed an increase (p<0.05) in one of the three forelimb grip strength measurements in all female test groups in comparison with the concurrent control. However, there was no dose dependent trend or any other evidence to suggest these changes were associated with test item toxicity. It is therefore reasonable to assume the isolated nature of this finding has arisen fortuitously (possibly as a result of biological variation).

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and the differences were of no toxicological importance.

Body Weight
There were no toxicologically significant effects detected in body weight development.
Males treated at 300 and 500 mg/kg bw/day showed low weight gains in comparison with controls after three weeks of treatment with evident recovery (p<0.01) by Week 4. During the final week of treatment 500 mg/kg bw/day males again showed low weight gains (p<0.05) when compared to controls. Adverse changes in female test groups were confined to the week of lactation when 500 mg/kg bw/day females were noted to have impaired weight gains (p>0.05) in comparison with the control females.
Body weight development in either sex at treated at 30 mg/kg bw/day and females that received 300 mg/kg bw/day showed similar group mean values to that of the controls throughout the study.

Food Consumption
No adverse effect on food consumption or food efficiency was detected in treated animals in comparison with controls throughout the study.
There were occasions when 300 and 500 mg/kg bw/day males showed increased appetite with a similar trend seen on occasion in 500 mg/kg bw/day females. However, increased dietary intake is not commonly associated with adverse treatment-related charges in studies of this nature, these findings were therefore considered to be of no toxicological relevance.

Water Consumption
There were no effects on water intake in test animals in comparison with controls that were considered toxicologically significant.
Gravimetric measurement of water intake undertaken during the maturation study phase revealed an increase in consumption in high dose males (500 mg/kg bw/day) in comparison with the concurrent control; albeit there were indications by the second week of treatment the difference between these groups was diminishing. As there was no convincing supporting evidence to indicate any toxicological association with treatment; it is reasonable to assume this finding was a compensatory response attributable to the oral gavage administration of an unpleasant tasting or slightly irritant test formulation rather than a result of systemic toxicity.
Water consumption in 500 mg/kg bw/day females and in both sexes treated with 30 and 300 mg/kg bw/day remained similar to that of the controls throughout the treatment period.

Reproductive Performance
Mating
No treatment-related effects were detected in mating performance. All paired animals mated within the first four days of pairing.
Statistical analysis of the pre-coital interval data did not reveal any significant intergroup differences.

Fertility
No treatment-related effects were detected on fertility for treated animals when compared to controls.
One female treated with 500 mg/kg bw/day did not achieve pregnancy following evidence of mating. In the absence of any histopathological correlates in the reproductive organs to elucidate the cause of the non-pregnancy in either the paired female or male partner which did not produce a pregnancy, the intergroup difference was considered to be incidental and of no toxicological importance.

Gestation Length
No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22 to 23½ days.

Litter Responses
In total twelve control females, 11 females from the 30 and 300 mg/kg bw/day dose groups and 9 females from the 500 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
No significant differences were detected for corpora lutea or implants for treated animals when compared to controls. Litter sizes and viability for treated groups were also comparable to controls. One female treated with 500 mg/kg bw/day, one female treated with 300 mg/kg bw/day and one female treated with 30 mg/kg bw/day had a total litter loss. Observations of this nature are occasionally observed in reproductive studies, in absence of any associated changes in litter size and offspring viability, these inter and intragroup differences were considered to be of no toxicological importance.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

Offspring Growth and Development
There were no significant differences in litter weights or mean offspring body weights between control and treated animals.
Statistical analysis of the data did not reveal any significant intergroup differences.
No overt clinical evidence of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, cold and physical injuries were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity.
Litters from females treated with 300 or 30 mg/kg bw/day had a statistically significant reduction (p<0.01) in surface righting reflex. In absence of a true dose response and due to the majority of individual values being within normal range, the intergroup differences were considered not to be toxicologically significant.

Laboratory Investigations
Haematology
No treatment-related effects were detected in the haematological parameters measured.
Females treated with 500 or 300 mg/kg bw/day had a statistically significant increase (p<0.05) in mean corpuscular haemoglobin. All individual values were within normal range for rats of the strain and age used, therefore this isolated intergroup difference was considered to have arisen fortuitously.

Blood Chemistry
No toxicologically significant treatment-related effects were detected in the blood chemistry parameters measured.
Females treated with 500 mg/kg bw/day had a statistically significant increase (p<0.01) in glucose concentration. All individual parameters were within normal range for the strain and age used, therefore this intergroup difference was considered to be of no toxicological importance.

Pathology
Necropsy - Offspring
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Necropsy - Adults
No treatment-related macroscopic abnormalities were observed in any animal at study termination.
One control male had increased right pelvic space at necropsy. This was an isolated finding and considered to be a congenital anomaly commonly referred to as hydronephrosis and, therefore, unrelated to treatment.
One female treated with 500 mg/kg bw/day had a mass in the right salivary gland. The isolated nature and absence of any supporting histopathological findings would suggest this discovery to be of no toxicological significance.

Organ Weights
There were no toxicologically significant changes detected in either sex of test animals in comparison with controls.
Males treated with 500 mg/kg bw/day had a statistically significant reduction (p<0.05) in pituitary weight, both absolute and relative to terminal body weight. However, the great majority of individual values were within the expected historical ranges for animals of this age and strain and in the absence of any histopathological correlates, this isolated finding was considered to have arisen fortuitously and therefore to be of no toxicological significance.
All treated females had a statistically significant increase (p<0.05) for adrenal weight (both absolute and relative to terminal body weight). Microscopic examination of the adrenal glands revealed increased incidence and mean grade of diffuse hypertrophy of adrenal zona glomerulosa, however, a clear dose relationship was not evident and therefore the findings were considered not to be toxicologically significant.

Histopathology
There were no microscopic findings representing a test item-related effect.
Incidental findings involved an increased incidence of cortical fatty change in 300 mg/kg bw/day males and females together with a diffuse hypertrophy of the adrenal zona glomerulosa. On the basis of these findings the adrenal glands of animals from the low and mid dose groups were examined with results confirming no convincing dose-effect. The findings in the adrenal glands were therefore considered devoid of toxicological relevance.
All other findings were within the range of normal background changes which may be recorded in animals of this strain and age.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Discussion

The oral administration of S193308 to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation period for females) at dose levels of 30, 300 and 500 mg/kg bw/day resulted in treatment related effects detected in animals of either sex treated with 500 mg/kg bw/day. Although there was no evidence of toxicological consequence in this study, animals treated at 500 mg/kg day did show findings indicative of a minimal adverse response to treatment characterized by transient impairment of body weight in both sexes together with a minimal disruption in blood chemistry in the females. There was also a nominal effect on adrenal weight in females that may have had an associated histopathological relationship. However, these findings were no more than minimal and of no toxicological consequence. However, they do preclude citing the No Observed-Effect-Level (NOEL) at 500 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance to rats by gavage, at dose levels of 30, 300 and 500 mg/kg bw/day, resulted in treatment-related changes in either sex at 500 mg/kg bw/day, therefore the ‘No Observed-Effect-Level’ (NOEL) can only be claimed for either sex treated with 300 mg/kg bw/day with the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity considered to be 500 mg/kg bw/day.
No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is 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 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

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 up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

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 4post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality.One female treated with 500 mg/kg bw/day was killedin extremison Day 32 as a result of a suspected mal-dose.

Clinical Observations.No toxicologically significant clinical observations were detected.

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

Body Weight.There was no convincing evidence of any toxicologically important changes in body weight development between test animals of either sex in comparison with controls throughout the study.

Food Consumption.No treatment-related adverse effect on food consumption or food efficiency (the ratio of body weight gain to dietary intake) was detected in test animals of either sex in comparison with controls throughout the study.

Water Consumption.There were no effects on water intake in test animals in comparison with controls that were considered toxicologically significant.

Reproductive Performance:

Mating.There were no treatment-related effects on mating for treated animals.

Fertility.There were no treatment-related effects on conception rates for treated animals.

Gestation Lengths.There were no differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ days.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Of the litters born, litter size at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Offspring Growth and Development.Offspring body weight gain and litter weights at birth and subsequently on Day 1 and Day 4post partumwere comparable to controls. Sex ratio were also comparable to controls.

Laboratory Investigations:

Haematology.No treatment-related effects were detected in the haematological parameters examined. 

Blood Chemistry.No toxicologically significant effects were detected in the blood chemical parameters examined.

Pathology:

Necropsy.No treatment-related macroscopic abnormalities were observed in any animal at study termination.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Histopathology.There were no microscopic findings representing a test item-related effect.

Conclusion.

The oral administration of S193308 to rats by gavage, at dose levels of 30, 300 and 500 mg/kg bw/day, resulted in treatment-related changes in either sex at 500 mg/kg bw/day, therefore the ‘No Observed-Effect-Level’ (NOEL) can only be claimed for either sex treated with 300 mg/kg bw/day with the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity considered to be 500 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.