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Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: Combined repeated dose reproduction and developmental toxicity screening study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase Start Date: 17 July 2013. Experimental Phase End: 04 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets the criteria as reliable without restriction according to Klimisch et al (1997).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

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 from the normal procedure for handling motor activity data ocurred during th study. These were not considered to have afected the validity of the study.)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
lithium 3-((3,4-dicyanophenyl)thio)propane-1-sulfonate
EC Number:
700-787-7
Cas Number:
769953-18-6
Molecular formula:
C11H9N2O3S2Li
IUPAC Name:
lithium 3-((3,4-dicyanophenyl)thio)propane-1-sulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): S193115
- Physical state: White Solid
- Analytical purity: 94.4% w/w
- Lot/batch No.: 398
- Storage condition of test material: Room temperature in the dark.

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 307 to 366g, the females weighed 200 to 234g.
- 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 seven days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperatures controls were set to achieve target values of 22 ± 3°C.
- Humidity (%): The relative humidity controls were set to achieve target values of 50 ± 20 %.
- 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 17 July 2013 (first day of treatment) and 08 September 2013 (last day of 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. Results showed the formulations to be stable for at least 21 days and therefore formulations were prepared fortnightly for the first four weeks and weekly thereafter 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 as active.
- Amount of vehicle (if gavage): The teatment volume was 5 mL/kg.
- Lot/batch no. (if required): No data
- Purity: No data
Details on mating procedure:
- M/F ratio per cage: Animlas were paired on a 1 male : 1 female basis within each group.
- Length of cohabitation: 14 days
- Proof of pregnancy: 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).
- After successful mating each pregnant female was caged (how): Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: No





Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD OF ANALYSIS
Summary
The concentration of the test item in the test item formulations was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique.
Samples
The test item formulations received were diluted with water. Where necessary, sample solutions were further diluted with water to achieve a working concentration.
Standards
The test item was also used as the analytical standard. 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/UV using the following conditions:

HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: ACE 3 C18 (125 x 3 mm id)
Mobile phase: Eluent A: 0.1% acetic acid in water
Mobile phase: Eluent B: 0.1% acetic acid in methanol
Flow-rate: 0.5 mL/min
UV detector Wavelength: 250 nm
Injection volume: 10 μL
Retention time: ~ 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.

Variation
The results indicate that the prepared formulations were within ± 4% of the nominal concentration.
Duration of treatment / exposure:
The male dose groups were killed on treatement Day 43.
At day 5 post partum, all surviving females and offspring were killed.
Any females which failed to achieve pregnancy or produce a litter were killed on or after day 25 post coitum.
Frequency of treatment:
The animals were dosed daily throughout the study.
Details on study schedule:
i) 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). The first day of dosing was designated as Day 1 of the study.

ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

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

iv) 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.

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

vi) 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.

vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.

ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg/bw/day (as active)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/bw/day (as active)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/bw/day (as active)
Basis:
actual ingested
No. of animals per sex per dose:
12 per sex per 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

Parental animals: Observations and 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
Toe pinch
Tail pinch
Touch escape
Grasp response
Finger approach
Pupil reflex
Blink reflex
Startle reflex

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.

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.

Reproductive Performance

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
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

Litter observations:
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.
Postmortem examinations (parental animals):
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
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Pituitary (post fixation)
Prostate
Seminal vesicles
Spleen
Testes
Thymus
Thyroid (weighed post-fixation with Parathyroid)
Uterus (weighed with Cervix)

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.

Since there were indications of treatment-related kidney and spleen changes, examination was subsequently extended to include similarly prepared sections of the kidneys and spleen from animals in the low and intermediate groups.
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. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water 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.

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module or SPSS statistical package.

Reproductive 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 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
Offspring viability indices:
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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
(See results)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(See results)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
(See results)
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(See results)
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
(See results)

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Sex:
female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The oral administration of the test item to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of up to 500 mg/kg bw/day, resulted in treatment related microscopic effects in animals of either sex treated with 500 and 300 mg/kg bw/day and in females treated with 30 mg/kg bw/day.

 

Clinical signs were confined to increased salivation detected in animals of either sex treated with 500 and 300 mg/kg bw/day and in females treated with 30 mg/kg bw/day. Water consumption was also significantly increased for these animals throughout the treatment period. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and do not necessarily represent an adverse effect of treatment.

 

No adverse effect was apparent for body weight or food consumption in treated males throughout the treatment period or in treated females during maturation. A reduction in body weight gain was evident however in females treated with 500 and 300 mg/kg bw/day during the final week of gestation and during lactation. Food consumption was not adversely affected during this period. Parental body weight on Days 0, 7 and 14 of gestation were comparable to control females therefore the reduction in body weight gain during the final week of gestation is most likely to be a result of the reduced litter size at these dosages. Body weight on Days 1 and 4 of lactation for females treated with 500 and 300 mg/kg bw/day was actually higher than control females therefore a lower body weight gain during this period would not be unexpected.

 

Although there were some statistically significant differences in treated animals from controls for the hematological and blood chemical parameters measured, these differences were considered not to be of toxicological significance. Macroscopic findings detected at necropsy were confined to isolated findings in either the kidneys or spleen of treated females. Although some of these findings were not directly associated with a specific microscopic finding, histopathology examinations did reveal changes in the spleen and kidneys of these groups therefore the macroscopic abnormalities cannot be discounted as an effect of treatment. 

 

The effects detected in the kidneys may have resulted as an adaptive response to the increase in water intake seen in animals from the intermediate and highest dosage groups and therefore in the absence of any degenerative changes are not considered to represent an adverse effect of treatment. The microscopic splenic changes in females were not clearly dosage-related and it is possible that the differences reflected a greater response to spontaneous blood loss during parturition and the subsequent blood sampling rather than a test-item related change. There were no statistically significant differences in adult organ weights that, in the absence of any evidence of histopathological change, were considered to be of any toxicological significance. Due to the changes detected in this study, a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 500 mg/kg bw/day for systemic toxicity.

 

Mating performance was unaffected by treatment however three females treated with 500 mg/kg bw/day and two females treated with 300 mg/kg bw/day were non pregnant following positive evidence of mating. One female treated with 500 mg/kg bw/day also had a total litter loss at birth following a longer gestation period. Corpora lutea counts for 500 mg/kg bw/day females were slightly lower than controls however implantation sites were reduced in females treated with 500 and 300 mg/kg bw/day. Subsequently, pre and post implantation losses were increased and litter size at birth, Day 1 and Day 4 of lactation was lower. A true treatment related effect on embryo lethality can therefore not be excluded. For litters reared to Day 5 of age there was no obvious adverse effect on survival, growth and development of the offspring, although the number of litters available for assessment was quite low and the improved post partum survival may have been due to the lower litter size.

 

Within the confines of this screening study it is not possible to establish the exact cause of the reduced number of implantation sites and subsequent reduced litter size. The increased pre-implantation loss may suggest that viable embryos were not formed or did not successfully implant and the increased post implantation loss may suggest that foetal loss may have occurred in utero prior to delivery or in the immediate period around the time of parturition. It should be noted that for high dose females the difference in overall implant numbers was slightly affected by a smaller mean corpora lutea count. No such effects were evident at 30 mg/kg bw/day therefore based on the equivocal findings observed at 500 and 300 mg/kg bw/day the NOEL for reproductive toxicity was considered to be 30 mg/kg bw/day.

 

 

 

Applicant's summary and conclusion

Conclusions:
The oral administration of the test material to rats by gavage, at dose levels of 30, 300 and 500 mg/kg bw/day, did not result in treatment related effects on mating performance or offspring growth and development. A reduction in the number of implantation sites and subsequent reduced litter size was observed, although it was not possible to determine if this was incidental in nature, or caused by the test item.

The No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 30 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 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 22 March 1996).

Method

 

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar strain rats, for 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).

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group 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 4 post 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 females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

 

Results and Conclusion

 

The oral administration of the test material to rats by gavage, at dose levels of 30, 300 and 500 mg/kg bw/day, did not result in treatment related effects on mating performance or offspring viability. A reduction in the number of implantation sites and subsequent reduced litter size was observed, although it was not possible to determine if this was incidental in nature, or caused by the test item.

 

The No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 30 mg/kg bw/day

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