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Toxicological information

Repeated dose toxicity: oral

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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:
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 for classification 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 occurred during th study. These were not considered to have affected 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:
other: Wistar Han:RccHan:WIST
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: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was available ad libitum.
- Water: 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 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. 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD OF ANALYSIS
Summary
The concentration of S193115 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:
eight weeks
Frequency of treatment:
The animals were dosed daily for eight weeks.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were chosen based on the results of previous toxicity work.

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, soon after dosing, and one hour 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
Bizarre/Abnormal/Stereotypic behaviour
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

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 on each occasion (at least two hours after dosing), 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:
Grasp response
Touch escape
Vocalisation
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until 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. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE:
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 AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing phase).

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 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).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 male & 5 female
- Parameters checked: 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).


CLINICAL CHEMISTRY: Yes
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

URINALYSIS: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone 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.

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 were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group.

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 tissues were weighed from all remaining animals: Epididymides, Prostate, Seminal vesicles, Testes, Ovaries, Pituitary (post fixation), Uterus (weighed with cervix).


HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from five selected males and five selected females from each dose group 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
Gross lesions lumbar)
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

The following tissues were preserved from all remaining animals: Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides, Gross lesions, Mammary gland, Testes, Uterus/Cervix, Vagina

Tissues were despatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). 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 failed to mate or 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. The tissues shown in bold from the remaining control and 500 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

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.

Microscopic examination was conducted by the Study Pathologist (P Millar at Peter Millar Associates Ltd).

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



Statistics:
See 'Any other information on materials and methods incl. tables' section

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
see details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
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:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Animals of either sex treated with 500 and 300 mg/kg bw/day showed increased salivation from Day 7 and Day 27 (respectively) onwards. Two females treated with 30 mg/kg bw/day also showed increased salivation on Day 27. One female treated with 500 mg/kg bw/day had generalised fur loss between Days 35 and 43. Observations of this nature are considered to be low incidence findings observed in females in studies of this type and are considered unrelated to test item toxicity. No such effects were detected in males treated with 30 mg/kg bw/day. There were no unscheduled deaths.

BODY WEIGHT AND WEIGHT GAIN
Females treated with 500 mg/kg bw/day showed a reduction in body weight gain during the final week of gestation and during lactation. Females treated with 300 mg/kg bw/day also showed a reduction in body weight gain during lactation. No such effects were detected in females treated with 30 mg/kg bw/day.
No adverse effects on body weight development were detected in any treated males. Males treated with 500 mg/kg bw/day showed a statistically significant increase in body weight gain during Week 4 (p<0.05). Males from all treatment groups also showed a statistically significant increase in body weight gain during Week 6 (p<0.01). An increase in body weight gain is considered not to represent an adverse effect of treatment therefore the intergroup differences are of no toxicological importance.

FOOD CONSUMPTION
No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

WATER CONSUMPTION
Animals of either sex treated with 500 and 300 mg/kg bw/day showed a substantial increase in water consumption throughout the treatment period. Statistically significant increases in water consumption were evident in females from all treatment groups throughout gestation and in females treated with 500 mg/kg bw/day during lactation.

No such effects were detected in males treated with 30 mg/kg bw/day.

HAEMATOLOGY
No toxicologically significant effects were detected in the haematological parameters examined.

Females treated with 500 mg/kg bw/day showed a statistically significant reduction in total leucocyte count and lymphocytes. All of the individual values were within normal range for rats of the strain and age used, therefore the intergroup differences were considered not to be of toxicological importance. Females treated with 500 mg/kg bw/day also showed a statistically significant increase in eosinophils. Although some individual values were outside of the normal range expected, in the absence of any associated microscopic changes, the intergroup difference was considered not to be of toxicological importance.

CLINICAL CHEMISTRY
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males treated with 500 mg/kg bw/day showed a statistically significant reduction in total protein and albumin. Although some individual values were outside of the normal ranges expected, in the absence of any associated microscopic changes the intergroup differences may have resulted from the over hydration of the animals and do not therefore represent an adverse effect of treatment.

Males from all treatment groups showed a statistically significant increase in alanine aminotransferase. Males treated with 500 mg/kg bw/day also showed a statistically significant increase in aspartate aminotransferase and bile acids. Although some individual values were outside of the normal range expected, in the absence of either a true dose related response or any associated hepatic microscopic changes, the intergroup differences were considered not to be of toxicological importance. Males from all treatment groups showed a statistically significant reduction in chloride concentration. All of the individual values were within normal ranges for rats of the strain and age used and in the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance. Females treated with 500 mg/kg bw/day showed a statistically significant reduction in urea. Females from this treatment group and females treated with 300 mg/kg bw/day also showed a statistically significant increase in glucose. The majority of individual values were within normal ranges for rats of the strain and age used therefore, the intergroup differences were considered not to be of toxicological importance.

NEUROBEHAVIOUR
Behavioural Assessments

Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

All inter and intra group differences in behavioural 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.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Males from all treatment groups showed a statistically significant reduction in mean forelimb grip strength. The intergroup differences were confined to one out of the three tests and in the absence of a true dose related response or any associated clinical signs to suggest neurotoxicity the intergroup differences were considered of no toxicological importance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

ORGAN WEIGHTS
There were no toxicologically significant effects detected in the organ weights measured.

Males from all treatment groups showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. All individual values were with normal range for rats of the strain and age used and in the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance. Males treated with 500 mg/kg bw/day also showed a statistically significant reduction in seminal vesicle weight both absolute and relative to terminal body weight. The majority of individual values were with normal range for rats of the strain and age used and in the absence of any associated histopathological correlates the intergroup difference was considered not to be of toxicological importance. Males treated with 300 mg/kg bw/day showed a statistically significant increase in absolute and relative pituitary weight. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance. Females treated with 500 and 300 mg/kg bw/day showed a statistically significant increase in thyroid weight both absolute and relative to terminal body weight. Although the majority of individual values were outside of the normal range for rats of the strain and age used, in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

GROSS PATHOLOGY
One female treated with 500 mg/kg bw/day had a dark spleen at necropsy. One female treated with 300 mg/kg bw/day and one female treated with 30 mg/kg bw/day had an enlarged spleen. A further female treated with 300 mg/kg bw/day had dark kidneys at necropsy. 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 can not be discounted as an effect of treatment.

No adverse effects were detected in any treated males.

One male treated with 300 mg/kg bw/day had an enlarged left mandibular lymph node and one male treated with 30 mg/kg bw/day had reddened lungs at necropsy. In the absence of any associated histopathology changes or similar effects at 500 mg/kg bw/day the intergroup differences were considered not to be of toxicological significance.


HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment related microscopic abnormalities were detected:

Kidneys: Minimal or mild hypertrophy of the epithelium of the collecting tubules in the renal medulla was present in three males and two females treated with 300 mg/kg bw/day and in all animals of either sex treated with 500 mg/kg bw/day. These inter-group differences might have resulted as an adaptive response to the increase in water intake seen in animals from the intermediate and highest dosage groups.

Spleen: The incidence and severity of minimal to moderate extramedullary haematopoiesis was greater in either sex treated with 300 or 500 mg/kg bw/day and in females treated with 30 mg/kg bw/day than in controls. In females, however, the inter-group differences were not clearly dosage-related and it is possible that the differences reflected a response to greater spontaneous blood loss during parturition rather than a test-item related change.

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 Discussion

The oral administration of the test material 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 can not be discounted as an effect of treatment. Histopathological examination of the kidneys and spleen revealed minimal or mild hypertrophy of the epithelium of the collecting tubules in the renal medulla in animals of either sex treated with 500 and 300 mg/kg bw/day and increased incidence and severity of extramedullary haematopoiesis also in animals of either sex treated with 500 and 300 mg/kg bw/day and in females treated with 30 mg/kg bwday. 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.

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, 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. The effects detected in the kidneys and spleen were not considered to represent an adverse effect of treatment therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore 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 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).

 

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 & Conclusions

The oral administration of the test material to rats by gavage, at dose levels of 30, 300 and 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. The effects detected in the kidneys and spleen were not considered to represent an adverse effect of treatment therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 500 mg/kg bw/day.

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