Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
CAS Number: 15337-18-5
Appearance: Viscous yellow liquid
Purity: 96.6%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) rat
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males 69 to 76 days old; females 83 to 90 days old
- Weight at study initiation: Males 332 to 405 g; females 240 to 304 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Males: six days prior to the commencement of treatment; females: 20 days prior to the commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24ºC
- Humidity: 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 hours light : 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Method of preparation
The required amount of test item was weighed into a suitable container. Starting with the lowest concentration, approximately 40 to 50 % of the final volume of vehicle was added to the test item and it was magnetically stirred until uniformly mixed. A further amount of vehicle was added to make up to the required volume and further mixing, using a magnetic stirrer, was performed until the formulation was homogeneous. The remaining concentrations were then formulated in ascending order of concentration.

Frequency of preparation
Daily

Storage of formulation
Formulations were prepared daily and administrated within four hours of preparation.
Details on mating procedure:
- Pairing commenced: After a minimum of two weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups.
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical procedure was successfully validated with respect to specificity, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity was confirmed for the test item in arachis oil formulations at nominal concentrations of 5 mg/mL and 300 mg/mL. Storage stability of the test item in arachis oil could not be confirmed; formulations were prepared daily and used within four hours.
The mean concentrations of the test item in dose formulations analyzed for the study (study week 1, study week 4, and lactation day 12) were within +10/-15% of nominal concentrations, confirming accurate formulation. The difference between the samples was within 3%, confirming precise analysis.
Duration of treatment / exposure:
Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Frequency of treatment:
Once daily
Details on study schedule:
Time of Necropsy
- F0 males: After final investigations completed (after at least five weeks of treatment).
- F0 females failing to produce a viable litter: Day 25 after mating.
- F0 females: Day 14 of lactation (following terminal blood sampling)
- F1 offspring:
>Selected offspring for thyroid hormone analysis - Day 4 of age.
>Scheduled kill - Day 13 of age.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
28 mg/kg bw/day (nominal)
Dose / conc.:
85 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based upon a range-finding study (please see RSS section 7.5.1 Supporting study, Envigo, 2018 (range-finding)).

Examinations

Parental animals: Observations and examinations:
MORTALITY
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

CLINICAL AND BEHAVIORAL OBSERVATIONS
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

SIGNS ASSOCIATED WITH DOSING
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
- F0 males: Week 1 – daily, Week 2 onwards - once each week
- F0 females: Week 1 – daily, Week 2 – once, Gestation phase - Days 0, 7, 14 and 20 and Lactation phase - Days 1, 6 and 13.
Detailed observations were recorded at the following times in relation to dose administration:
- Pre-dose observation
- One to two hours after completion of dosing
- As late as possible in the working day

DETAILED PHYSICAL EXAMINATION
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examinations and observations were performed on each animal. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.
-Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and at Day 7-9 of lactation for females. The following measurements, reflexes and responses were recorded: approach response, pinna reflex, auditory startle reflex, tail pinch response, and grip strength.
-Motor activity: During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing).

BODY WEIGHT
The weight of animals was recorded as follows:
F0 males: Weekly during acclimatization, before dosing on the day that treatment commenced , (Day 1) and weekly thereafter and on the day of necropsy.
F0 females: Weekly during acclimatization, before dosing on the day that treatment commenced, (Day 1) and weekly before pairing, Days 0, 7, 14 and 20 after mating, Day 1, 4, 7, 13 and 14 of lactation and on the day of necropsy.

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Weekly before pairing, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Day 15-22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows: Days 0-6, 7-13 and 14-19 after mating, and Days 1-3, 4-6 and 7-13 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

BLOOD CHEMISTRY
Blood samples were collected at the following occasion:
At termination: The five lowest numbered surviving males per group and the first five lactating females with a litter per group.
Animals were held under light general anesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Hematology, Peripheral Blood
Blood samples were collected at the following occasion:
At termination: the five lowest numbered surviving males per group, and the first five lactating females with a litter per group.
Animals were held under light general anesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)
Mean cell hemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

THYROID HORMONE ANALYSIS
Blood samples were collected as follows:
At termination:
- All adults
- Day 4 of age: Offspring: two females per litter (where possible) and no pups were allocated to these procedures on Day 4 of age if the resultant live litter size would fall below 10 pups.
> one for T4 (serum)
> one for TSH (plasma- optional analysis)
- Day 13 of age: Offspring: two males and two females per litter (where possible):
> two for T4 (serum); where possible one male and one female
> two for TSH (plasma – optional analysis); where possible one male and one female

In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: No overnight deprivation of food.
Anesthetic: Adults: Isoflurane, Offspring: None.
Blood sample site: Adults: Sublingual vein, Offspring: Decapitation.
Parameter: Thyroid stimulating hormone (TSH); (anticoagulant: K2EDTA with no separator gel) and Thyroxine (T4); (anticoagulant: none).
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:
- Dry smears: From beginning of treatment until animals were paired for mating using cotton swabs
- Wet smears: Using pipette lavage during the following phases:
> For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
> After pairing until mating.
> For four days before scheduled termination (nominally Days 11 to 14 of lactation).
Sperm parameters (parental animals):
For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
- Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
- Individual offspring body weights: Days 1, 4, 7 and 13 of age.
- Ano-genital distance: Day 1 - all F1 offspring.
- Nipple/areolae count: Day 13 of age - male offspring.
Postmortem examinations (parental animals):
Necropsy
- F0 males: After final investigations completed (after at least five weeks of treatment)
- F0 females failing to produce a viable litter: Day 25 after mating
- F0 females Day 14 of lactation (following terminal blood sampling)

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
- Testes: Initially in modified Davidson’s fluid.
- Eyes: In Davidson’s fluid.

Histology
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned. For bilateral organs, sections of both organs were prepared.
- Full List:
>All adult animals killed prematurely.
>At scheduled termination, the five lowest numbered surviving F0 males and females with a surviving litter in Groups 1 and 4.
- Abnormalities: All F0 animals.
- Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified.

Light Microscopy
Tissues preserved for examination were examined as follows:
- Premature deaths: F0 males and females from Groups 1 and 4 only.
- Scheduled kill: The first five F0 males and first five lactating F0 females in Groups 1 and 4 and All F0 animals from all groups.
Postmortem examinations (offspring):
SACRIFICE
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled Kill: Day 13 of age.

GROSS NECROPSY and HISTOPATHOLOGY
- Performed on the five lowest numbered surviving males and first five lactating females with a surviving litter in Groups 1 and 4.
- Hematology, Peripheral Blood, Blood Chemistry: Performed at termination on the five lowest numbered surviving males and the first five lactating females with a surviving litter iin each dose group.
- Thyroid Hormone Analysis:
> Blood samples were collected as follows:
- Day 4 of age: F1 offspring, two females per litter
- Day 13 of age: F1 offspring, two males and two females per litter (where possible).
- All surviving F0 adult males and females (except the female which failed to litter) at termination.
Reproductive indices:
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Group mean values were calculated from individual litter values.
Offspring viability indices:
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Detailed Physical Examinations and Observations
One female receiving 250 mg/kg/day was observed to have piloerection, partially closed eyelids and abnormal staining of the muzzle on Days 12-15 of treatment. This female showed no body weight gain between Days 1-8 of treatment, and showed body weight loss between Days 8 15 of treatment. Chromodacryorrhea was also observed in one female receiving 28 mg/kg/day.
No other signs were observed during the weekly detailed physical examination for any animals that were considered to be treatment-related.

Signs associated with dosing
There were no signs associated with the dosing procedure.
Mortality:
no mortality observed
Description (incidence):
One female rat receiving 250 mg/kg/day was sacrificed for reasons of animal welfare on Day 12 of treatment, due to general poor clinical condition, including a decrease in activity, piloerection, an abnormal gait, tremors and hunched posture. There were no macroscopic findings at necropsy, and histopathological examination revealed minimal mineralization of the kidney papilla, minimal unilateral tubular basophilia in the kidney, minimal ulceration in the glandular region of the stomach, and minimal atrophy in the uterus but these findings did not account for the poor condition of this female. This death is considered unlikely to be related to administration of the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight and body weight gain for males during Days 1-36 of treatment was similar to Controls across all groups.
Slight body weight loss was observed in females receiving 28 and 250 mg/kg/day from Days 1-8 of treatment; while body weight gain in females receiving 85 mg/kg/day during this period was the same as Controls. Body weight gain for females receiving 85 or 250 mg/kg/day was slightly low between Days 8-15 when compared with Controls. However, these changes were considered incidental because there was no statistical significance and no similar differences were observed in the male rats.
The body weight gain of females receiving 250 mg/kg/day was similar to that of the Control during Days 0-7 of gestation, slightly lower than that of the Control during Gestation Days 14-20 and . The bodyweight gain of females receiving 28 or 85 mg/kg/day was similar to that of the Control throughout gestation.
The bodyweight gain of females receiving 28, 85 or 250 mg/kg/day was generally similar to that of the Control during Days 1-14 of lactation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption for males during Days 1-15 and 22-36 of treatment was similar to Controls across all groups.
The food consumption of females receiving 250 mg/kg/day was slightly lower than that of the Control during Days 1-15 of treatment and throughout gestation and Days 1-7 of lactation, while the food consumption of females receiving 28 or 85 mg/kg/day was similar to that of the Control during Days 1-15 of treatment and throughout gestation and Days 1-14 of lactation. However, these changes were considered non-adverse because there were no correlated body weight changes observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological investigations in Week 6 for males and on Day 14 of lactation for females did not reveal any findings that were considered related to treatment.
The investigations in Week 6 for males revealed slightly but statistically low haematocrit counts in males receiving 28, 85 or 250 mg/kg/day when compared with the Control, although a dose response was not apparent. In addition, mean cell volumes (MCV) were slightly but statistically lower than Control for males receiving 85 or 250 mg/kg/day. These differences were marginal and within the historical control data (HCD) range, so no effect of treatment was inferred.
The investigations on Day 14 of lactation for females revealed slightly but statistically high red blood cell (RBC) counts when compared with the Control. This difference was marginal and within the HCD range, so no effect of treatment was inferred.
All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical examination of blood plasma in Week 6 for males and on Day 14 of lactation for females did not reveal any findings that were considered related to treatment.
The investigations in Week 6 for males revealed no statistically significant differences from Control in males receiving 28, 85 or 250 mg/kg/day.
The biochemical examination of the blood plasma on Day 14 of lactation for females revealed, when compared with the Controls, statistically significantly low bilirubin concentrations in females receiving 85 or 250 mg/kg/day. This difference was marginal and within the HCD range, so no effect of treatment was inferred.
All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity observations and grip strength were similar for animals treated at 28, 85 or 250 mg/kg/day, when compared with the Controls. Motor activity, assessed by low and high beam breaks over a 60 minute period was unaffected by treatment with the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item related effects were observed.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cyclicity was unaffected by treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One control female, one female received 85 mg/kg/day, and one female received 250 mg/kg/day failed to litter and were found to be not pregnant at macroscopic examination on Day 25 of gestation.
Nine, ten, nine, and eight females receiving the test item at 0, 28, 85 and 250 mg/kg/day, respectively; achieved pregnancy and gave birth to a live litter which survived until scheduled termination on Day 14 of lactation.

Details on results (P0)

All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
not specified

Results: F1 generation

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a combined repeat dose toxicity study (OECD 422), oral administration of the test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 28, 85 or 250 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was well-tolerated in the adult animals with no treatment related adverse effects observed. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and for reproductive/developmental toxicity of the test item was considered to be 250 mg/kg/day, the highest tolerable dose tested.
Executive summary:

The purpose of this study was to assess the potential systemic toxicity in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by oral administration for at least five weeks.

 

Three groups of ten male and ten female Crl:CD(SD) rats received the test item at doses of 28, 85 or 250 mg/kg/day by oral gavage administration. The dose levels were selected based on the results of a 14-day preliminary study in which 100, 250, 500 and 1000 mg/kg/day of the test item were administered to male and female Crl:CD(SD) rats and the dose levels of 500 or 1000 mg/kg/day were not tolerated and resulted in the early termination of these groups on Day 3 of study. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, arachis oil, at the same volume dose as treated groups.

 

During the study, clinical condition, detailed physical examination, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone (T4) analysis, estrous cycles, pre coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Thyroid hormone (T4) analysis was performed for the Day 13 offspring. Nipple counts were performed on male offspring on Day 13 of age. 

 

There was no premature animal death related to the test item treatment. One female was sacrificed for reasons of animal welfare on Day 12 due to general poor clinical condition, however this death was considered not related to treatment. 

Clinical condition, behavior in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment. There were no signs seen in association with dosing.

Slight variations of mean body weights and body weight gains were observed in females during the dosing period when compared with Controls. However, these variations were considered incidental because there were no statistical significance and no similar differences were observed in the male rats.

The food consumption of females received 250 mg/kg/day was slightly lower than that of the Control during Days 1-15 of treatment and throughout gestation and Days 1-7 of lactation, but it was considered non-adverse because there were no correlated body weight changes observed.

 

Estrous cycles, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. There was no effect of treatment on the number of implantations or litter size.

Haematological investigations of the plasma and biochemical examinations of the blood did not reveal any findings that could be attributed to treatment.

 

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 offspring.

 

The evaluation of organ weights of males after 5 weeks of treatment and of females on Day 14 of lactation revealed no significant differences when compared with the Controls.

The macroscopic and microscopic examination of adult males and females did not reveal any findings related to treatment.

 

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment.

 

In conclusion, oral administration of the test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 28, 85 or 250 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was well-tolerated in the adult animals with no treatment related adverse effects observed. 

 

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations, litter size or the growth of the offspring.

In the context of this study, the test item showed no evidence of being an endocrine disruptor.

 

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and for reproductive/developmental toxicity was considered to be 250 mg/kg/day, the highest tolerable dose tested.