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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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:

other: OECD Guideline 422

Deviations:

no

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

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 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.

Details on mating procedure:

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

On completion of the pre-pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.

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:

The animals were dosed daily throughout the study

Duration of test:

56 days

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.

Examinations

Maternal examinations:

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

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:
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

Fetal examinations:

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.

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.

Indices:

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 animals mated / Number of pregnant females )x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Fertility effects:
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.

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.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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.

Effect levels (fetuses)

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

changes in litter size and weights

changes in postnatal survival

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Offspring Growth and Development:

Group values for total litter weights, offspring body weight and body weight change, surface righting reflex and a summary incidence of clinical signs are given in Tables 13, 16 and 17. Individual values and observations are given in Appendices 11, 14 and 15. There were no significant differences in litter weights or mean offspring body weights between control and treated animals. Please see all appendicies and tables in the section attached background material section.

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.

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 for systemic toxicity. No treatment related effects were detected in the reproductive parameters or developmental parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 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.