Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-21 to 2009-06-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 1995-07-27
Deviations:
yes
Remarks:
, justification for vehicle was missing
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SymWhite® 377
- Synonym: SymWhite 377 (158281)
- Supplier: Symrise SA
- Physical state: white to beige powder
- Storage condition of test material: at room temperature and protected from light.
- Container: one opaque plastic container

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Sprague-Dawley rat, Rj Han: SD Indemn Of Organism Pathogen Specific Han (IOPS Han)
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at the beginning of treatment period: approximately 10 weeks old
- Weight at the beginning of treatment period: males: mean body weight of 429 g (range: 405 g to 453 g); females: 263 g (range: 245 g to 285 g)
- Housing: the animals were housed in a barriered rodent unit. The males and females were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage.
Towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Autoclaved wood shavings (SICSA, Alfortville, France) were provided as nesting material, a few days before delivery and during the lactation period.
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet (batch Nos. 1860828 and 7691203 (SSNIFF Spezialdiäten GmbH, Soest, Germany)), which was distributed weekly
- Water (ad libitum): tap water (filtered with a 0.22 μm filter)
- Acclimation period: period of 7 days before the beginning of the treatment period

The males and females were sexually mature and the females were nulliparous.
Upon their arrival at the testing laboratory, all animals were given a clinical examination in order to ensure that they were in good condition.
The animal room was disinfected before the arrival of the animals and cleaned regularly
thereafter.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Relative humidity: 50 ± 20%
- Ventilation: about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: soybean oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle in order to achieve the concentrations of 4, 10 and 25 mg/mL.
- The test item dosage forms were prepared weekly based on stability data generated by the testing laboratory, stored at +4°C, protected from light, prior to use and delivered in brown flasks.

VEHICLE
- Batch no.: 067K0068
- Supplier: Sigma (Saint-Quentin-Fallavier, France)

ADMINISTRATION
- The quantity of the dosage form administered to each animal was adjusted according to the most recently recorded body weight.
- A constant dosage-volume of 5 mL/kg/day was used.
- Control animals (group 1) received the vehicle.
- The dosage forms were magnetically stirred continuously throughout the dosing procedure.
- The dosage forms were left to come to room temperature prior to treatment and were administered at room temperature.
Details on mating procedure:
- Females were paired with males from the same dose-level group. One female was placed with one male, in the latter's cage, during the night.
- Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or of sperm in a vaginal lavage.
- The day of confirmed mating was designated day 0 post-coitum.
- Each female was placed with the same male until mating occurred.
- The pre-coital time was calculated for each female.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The HPLC/UV analytical method for the determination of SymWhite® 377 in dosage form samples was provided by the Sponsor and this method was validated by the testing laboratory prior to dosage form analysis.The validation was based on the ICH Q6A guideline adopted in November 1999.

ANALYTICAL METHOD
PRINCIPLES OF ANALYTICAL METHOD
- Compound analyte: SymWhite 377

- Instrument/detector: High Performance Liquid Chromatography with Ultra-Violet detection at 280 nm (detector: Series 1100 (Agilent Technologies))

- Preparation:
a. Control dosage forms (0 mg/mL): An aliquot (1 mL) of each control dosage form was sampled (by accurate weight) under magnetic stirring into a 200 mL volumetric flask and each flask was made to volume with dilution solvent before injection.
b. Test dosage forms (4, 10 and 25 mg/mL): An aliquot (1 mL) of each test item dosage form was sampled (by accurate weight) under magnetic stirring into a 200 mL volumetric flask (or a 250 mL volumetric flask for 25 mg/mL) and each flask was made to volume with dilution solvent. To achieve a target concentration of 10 μg/mL of SymWhite® 377, an additional dilution (2-fold, 5-fold and 10-fold for 4, 10 and 25 mg/mL, respectively) was carried out with dilution solvent before injection.
From the aliquot of dosage form sampled (weighed accurately), the real volume of the aliquot analyzed was determined (using the density of each dosage form) and therefore the value of the first dilution factor was calculated.

- Chromatographic conditions (HPLC/UV)
a. Pump: Series 1100 (Agilent Technologies)
b. Mobile phase: Mobile phase A: 0.1% Formic acid in Milli-Q water, Mobile phase B: Acetonitrile
c. Gradient:
- 0 min, A: 70 %, B: 30 %
- 10 min, A: 30 %, B: 70 %
- 10.1 min, A: 0 %, B: 100 %
- 15 min, A: 0 %, B: 100 %
- 17 min, A: 70 %, B: 30 %
- 20 min, A: 70 %, B: 30 %
d. Column: YMC ODS-AQ, particle size = 5 μm, length = 150 mm, inner diameter = 3 mm
e. Flow rate: 0.6 mL/min
f. Temperature: 40°C
g. Oven: Series 1100 (Agilent Technologies)
h. Detector: Series 1100 (Agilent Technologies)
i. Wavelength: 280 nm
j. Injector: Series 1100 (Agilent Technologies)
k. Injected volume: 10 μL
l. Software: Empower 2 (Waters)
m. Retention time of test item: approx. 8.2 min
n. Analysis time: 20 min.

- Quantification: the concentration of the test item in administered dosage forms was determined from the mean response factor of SymWhite® 377 in standard solutions.
The dosage form concentrations were determined using the following:
[Concentration dosage form] = Area sample x Mean response factor X multiplication factor
where:
Area sample = Area of sample
Mean response factor = Mean response factor of standard solution (n = 10)
Multiplication factor = dilution factor (also including conversion between units if required)

- Assay: diluted samples of dosage form were analyzed by High Performance Liquid Chromatography with Ultra-Violet detection.
One dilution was prepared for each sample and one injection was performed for each final dilution.
The test item peak area was determined for each sample and the corresponding concentration was calculated using the equation obtained from the calibration data. All the results are expressed as mg/mL of SymWhite® 377.

VALIDATION OF THE ANALYTICAL METHOD
The analytical procedure for determination of the SymWhite® 377 concentration was developed at the testing laboratory and was based on the analytical method provided by the Sponsor. The purpose of the study was to verify the following criteria: specificity, linearity, sensitivity, injection repeatability, accuracy and precision, and stability of standard and sample solutions. The solutions were prepared appropriately, and then analyzed by High Performance Liquid Chromatography with Ultra-Violet detection (HPLC-UV).
-Specificity: a vehicle sample, as well as 2 and 40 mg/mL formulation samples were prepared and analyzed. No interfering peaks were observed for the vehicle sample at the analyte retention time. No interfering peaks were observed for the formulation samples around the analyte retention time.
- Specificity in presence of degradation products: Samples of a 2 mg/mL dosage form in soybean oil were diluted in 0.1M HCl, 0.1MNaOH and 1% DMSO, stored for 24H at 50-60°C, and then analyzed by HPLC-DAD. No interfering peaks were observed under the main peak. One interfering peak partially co-eluted in the sample degraded by NaOH. As this peak is only partially co-eluting, it could be detected by HPLC-UV alone. Consequently, the method is still considered to be stability indicating.
- Linearity: standard solutions ranging from 1 to 15 μg/mL were prepared and analyzed.
The calibration curve was linear over a concentration range of from 1 μg/mL to 15 μg/mL (six concentration levels) as the obtained (r²) value was ≥ 0.99, the residuals of measured responses against response at nominal concentration were within ± 3%, and the intercept value was within ± 5% of response at nominal concentration.
- Accuracy and precision: working sample solutions made of vehicle, SymWhite® 377 and diluent were prepared, in triplicates, and at 80%, 100% and 120% of the nominal concentration. These samples were then analyzed.
Accuracy was demonstrated at each level, as the recovery was equal to or within 95-105% in each case.
Precision was demonstrated at each level, as the Coefficient of Variation (CV) was equal to or below 5% in each case.
- Injection repeatability: the repeatability of the assay method was tested with six successive injections of a standard solution at nominal concentration.
Injection repeatability was demonstrated as the CV was equal to or below 3%.
- Sensitivity: A standard solution at 0.1 μg/mL was prepared and analyzed. The obtained Signal-to-Noise (S/N) was superior to 10.
The control group dilution factor before injection is 200. Therefore, the absence of active material in a control group sample could be demonstrated at a level of 0.02 mg/mL.
- Stability in solutions: standard solutions and 2 and 40 mg/mL sample working solutions were analyzed on the day of dilution and after 24 hours and 4 days storage at room temperature, protected from light. Recovery was equal to or between 97-103% in each case.

Determination of dosage form homogeneity and stability:
Before the start of treatment the suitability of the proposed preparation process was confirmed by analysis of the homogeneity and stability. A range of dosage forms were prepared at levels which covered the lowest and highest concentrations proposed for use in the study, and then stored at +4°C and protected from light.
Duplicate samples were taken from three levels of the container (top, middle and bottom) on the day of preparation and on each sampling occasion. All samples were analyzed for test item content.
Samples taken just after preparation were analyzed immediately and the homogeneity/stability test was continued only when satisfactory initial results had been obtained.
Samples taken on each sampling occasion were analyzed immediately. On each occasion, the mean (n = 6) concentration was determined and compared to the nominal concentration, and the Coefficient of Variation (CV %) was calculated.
Acceptance criteria at each time-point:
- mean concentration = nominal concentration ± 15%,
- CV% < 10%.
The frequency of sampling was documented.

Determination of SymWhite®377 concentration in dosage forms:
The dosage form samples were assayed using a validated method, which was authorized by the Study Director prior to use in this study.
The test item concentration in samples of each control and test item dosage form prepared for use in weeks 1 and 6 was determined. Whenever possible these analyses were performed prior to administration of the dosage forms to the animals.
Acceptance criterion:
- measured concentration = nominal concentration ± 15%

RESULTS
Homogeneity and stability:
The dosage forms containing SymWhite® 377 in soybean oil at 2 mg/mL and 40 mg/mL were found to be homogeneous and stable after 11 days storage at +4°C, protected from light. They are therefore considered suitable for routine administration in GLP toxicology studies for up to 10 days after preparation.

Concentration:
The test item concentrations in the administered dosage forms analyzed in weeks 1 and 6 remained within an acceptable range of [-4.5% to +1.0%] of variation compared to the nominal values (please refer to "Any other information on materials and methods incl. tables" below for concentrations of SymWhite® 377 in administered dosage forms (table 1)).
Duration of treatment / exposure:
Each animal was given the appropriate dosage form according to the following schedule:
1. males:
- 15 days before mating,
- during the mating period (up to 13 days depending on the time taken to mate),
- until sacrifice (i.e. at least 5 weeks in total),
2. females:
- 15 days before mating,
- during the mating period (up to 13 days depending on the time taken to mate),
- during pregnancy,
- during lactation until day 4 post-partum inclusive,
- females with no delivery were treated until the day prior to sacrifice.
Day 1 corresponds to the first day of treatment period.

Frequency of treatment:
once a day, at approximately the same time each day, 7 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (group 1), 20 (group 2), 50 (group 3) and 125 mg/kg/day (group 4)
Basis:
other: actual dose received
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: the dose-levels were selected in agreement with the Sponsor, following the results of previous studies:
- a 7-day toxicity study (please refer to Section 7.5.1 Repeated dose toxicity: oral: k_Leuschner_2006) in which dose-levels of 300 and 1000 mg/kg/day elicited mortality and clinical signs of piloerection and hypoactivity. A decreased body weight was also observed,
- a 4-week toxicity study (please refer to Section 7.5.1 Repeated dose toxicity: oral: k_Leuschner_2006) in which the dose-level of 200 mg/kg/day elicited a lower body weight gain and increased urea levels but no mortality or severe clinical signs
- a preliminary prenatal development study (please refer to Section 7.8.2 Developmental toxicity/teratogenicity: k_Davies_2009) in which the dose-level of 200 mg/kg/day resulted in one female found dead and another prematurely sacrificed. Clinical signs of salivation and loud breathing were observed in a dose-related manner at 60 and 200 mg/kg/day and females treated at 200 mg/kg/day also showed ventral decubitus and half-closed eyes. At 200 mg/kg/day a mean body weight loss was recorded over the first 3 days of treatment and mean food consumption was statistically significantly lower for the first 6 days of treatment,
- a 13-week toxicity study which was in week 3 of treatment at the beginning of the present study (please refer to Section 7.5.1 Repeated dose toxicity: oral: k_Davies_2009) in which the dose-level of 200 mg/kg/day was eliciting clinical signs of salivation and loud breathing and a slight lower mean body weight gain.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Morbidity and mortality: each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period, including weekends and public holidays, and once a day during the acclimation period.
- Clinical signs: from arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Time schedule for examinations:
- Males: on the first day of treatment (day 1), then once a week until sacrifice.
- Females: on the first day of treatment (day 1), then once a week until mating (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.
- Two females of group 3 and two females of group 4 prematurely sacrificed or sacrificed because of no delivery, were weighed prior to sacrifice.

FOOD CONSUMPTION
The quantity of food was recorded as follows:
- Males: once a week, over a 7-day period, from the first day of treatment until sacrifice.
- Females: once a week, over a 7-day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice.
- During the pairing period, food consumption was not recorded for males or females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HEMATOLOGY: Yes (prematurely sacrificed animals only: one female of the group 3 on day 39 (day 23 post-coitum) and one female of group 4 on day 42 (day 2 post-partum))
- Blood samples were taken from the orbital sinus of the animals under isoflurane anesthesia, into tubes containing the appropriate anticoagulant.
- Parameters determined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and large unstained cells and monocytes) and prothrombin time.

CLINICAL CHEMISTRY: Yes (prematurely sacrificed animals only: one female of the group 3 on day 39 (day 23 post-coitum) and one female of group 4 on day 42 (day 2 post-partum))
- Blood samples were taken from the orbital sinus of the animals under isoflurane anesthesia, into tubes containing the appropriate anticoagulant.
- Parameters determined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase


Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the females had mated.
Sperm parameters (parental animals):
Not evaluated. Parameters examined in P male parental generations: testis weight and epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Litter size: the total litter size and numbers of pups of each sex were recorded as soon as possible after birth.
- The litters were observed daily in order to note the number of live, dead and cannibalized pups.
- Any gross malformation in pups was noted.
- Clinical signs: the pups were observed daily for clinical signs.
- Body weight: the weight of each pup was recorded on days 1 and 5 post-partum.
Postmortem examinations (parental animals):
All surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

SACRIFICE
- Male animals: after the end of the mating period (after at least 5 weeks of treatment).
- Maternal animals: on day 5 post-partum.
- Females which had not delivered: on day 25 post-coitum (after body weight recording to check for a possible unnoticed delivery).
- Mothers with litter dying entirely: as appropriate,
- Animals prematurely sacrificed (prematurely sacrificed pregnanat female): one female of group 3 was sacrificed by inhalation of carbon dioxide gas followed by cervical dislocation. The pregnancy status was confirmed and the numbers of corpora lutea, implantation sites and implants were recorded.

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
- The numbers of corpora lutea and implantation scars were also recorded for females sacrificed on day 5 post-partum.
- The number of corpora lutea was recorded for one female of group 3 and for one females of group 4 sacrificed on day 25 post-coitum due to no delivery. For these apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The body weight of each animal was recorded before sacrifice on completion of the treatment period.
- The following organs were weighed wet as soon as possible after dissection of males: epididymides and testes.
- The ratio of organ weight to body weight was calculated.
- For all study animals, the following tissues were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, if retained because of abnormalities, and the testes and epididymides which were fixed in Davidson's fixative): macroscopic lesions, epididymides, esphagus, lungs (with bronchi), ovaries (including oviducts), prostate (dorso-lateral and ventral), seminal vesicles (including coagulation gland and secretions), testes, uterus (horns and cervix) and vagina
- All tissues required for microscopic examination were trimmed based on the RITA guidelines (Ruehl-Fehlert, et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).

A microscopic examination was performed on all macroscopic lesions, epididymides, ovaries (including oviducts) and testes:
- all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period,
- for one female of group 3 and one female of group 4 that were sacrificed prematurely,
- for one female of group 3 and one female of group 4 sacrificed because of absence of delivery,
Postmortem examinations (offspring):
Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital

SACRIFICE
- surviving pups: on day 5 post-partum.

GROSS NECROPSY
- All pups, including those found dead or prematurely sacrificed, were carefully examined for gross external abnormalities.
- Pups were then discarded with no post-mortem examination. There was no preservation of tissues.

Statistics:
Data were expressed as group mean values ± standard deviation (body weight, food consumption, corpora lutea, implantations, fetuses, resorptions, pups, gestation length) or as proportions (pre-implantation loss, post-implantation loss, fetal observations, gestation index, live birth index, viability index). Whenever necessary, the experimental unit of comparison was the litter. Data of non-pregnant females are excluded from group mean calculations.

Body weight, food consumption and reproductive data: mean values were compared by one-way variance analysis and Dunnett test, (mean values being considered as normally distributed, variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test.

Organ weights: PathData software (version 6.2b5) was used for the statistical analysis of organ weight data (level of significance: 0.05 or 0.01), which includes the following tests: Kolmogorov test, Bartlett test / F-test, Kruskal-Wallis, one-way analysis of variance, Dunn test, Wilcoxon test, Dunnett test and t-test. These test are choosen depending on the kind of data.
Reproductive indices:
The calculations were performed for each group as follows:
- Pre-implantation loss: (number of corpora lutea - number of implantation sites / number of corpora lutea) X 100
- Post-implantation loss: (number of implantation sites - number of live concepti / number of implantations) X 100
- Mating index: (number of mated animals / number of paired animals) X 100
- Fertility index: (number of pregnant female partners / number of mated pairs) X 100
- Gestation index: (number of females with live born pups / number of pregnant females X 100
Offspring viability indices:
The calculations were performed for each group as follows:
- Live birth index: (number of live born pups / number of delivered pups) X 100
- Viability index on day 5 post-partum: (number of surviving pups on day 5 post-partum / number of live born pups) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality:
- One female treated at 125 mg/kg/day was sacrificed on day 2 post-partum because of dead litter. The female presented no unusual clinical signs during pregnancy or lactation however cannibalised all five of the nine pups left on day 2 post-partum (the other four pups were found dead on day 1 post-partum).
- One female treated at 50 mg/kg/day was prematurely sacrificed on day 23 post-coitum due to clinical signs of pallor of extremities and eyes, locomotory difficulties, piloerection and half-closed eyes. Brownish vaginal discharge and blood in the bedding had been observed from/on day 21 post-coitum. At necropsy, the right uterine horn was twisted and contained two dead fetuses. The right overy had a cyst. This was considered to be either a congenital problem or the uterine horn twisted during the pregnancy, neither of which were caused by treatment with the test item.

Clinical sings:
- Animals treated at 50 or 125 mg/kg/day tended to present loud breathing at the beginning of the treatment period and hypersalivation throughout the treatment period.
- Males treated at 20 mg/kg/day generally presented hypersalivation at the end of the treatment period although three males did show this sign in the first or second week of treatment.
- A similar number of females showed these clinical signs during the gestation and lactation periods.
- Hypersalivation and loud breathing may be related to the oily nature of the vehicle however the incidences were dose-related therefore they are considered to also be related to treatment with the test item but non-adverse.

BODY WEIGHT (PARENTAL ANIMALS)
Males:
- At 125 mg/kg/day, a mean body weight loss over the first week of treatment and a lower mean body weight gain over the second week of treatment was observed, resulting in statistically significantly lower body weight than that of the controls during the premating period. Thereafter, body weight performance improved although the difference was only partially recouped.
- There were no effects of treatment on mean male body weight gain at 20 or 50 mg/kg/day.
Females:
- At 125 mg/kg/day, a lower mean body weight gain than the controls was seen during the first week of treatment; four females lost weight but none more than one control female (-12 g).
- During the gestation period, the mean body weight gain of this group remained lower, particularly. During the second half of gestation (day 14 to day 20 post-coitum: -14% when compared with the controls).
- Mean body weight was statistically significantly lower on the first day of lactation but due to a higher mean body weight gain over the next 4 days some of this was recouped.
- There were no effects of treatment on mean female body weight gain at 20 or 50 mg/kg/day.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
- At 125 mg/kg/day, statistically significantly lower mean food consumption was observed over the first week of treatment only.
- Mean food consumption at 20 or 50 mg/kg/day was comparable with the controls throughout the study.
Females:
- Mean food consumption of all female groups treated with test item was comparable with the controls throughout the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- None of the females treated with the test item had abnormal estrous cycles between the end of the premating phase and mating.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating data: there were no effects of treatment with the test item on mating; all pairs at all dose-levels mated and the mean number of days of pairing before mating was less than the controls at all dose-levels due to one control pair which took an abnormally long 13 days to mate.
- Fertility data: one female from each of the groups treated at 50 or 125 mg/kg/day was non-pregnant. This was considered to be within normal range and not related to treatment with the test item.
-Delivery data: there were no effects on the mean numbers of corpora lutea, implantations or pups at any dose-level, nor on the duration of gestation or the extent of pre-implantation loss. Post-implantation loss was increased at all dose-levels when compared with the controls, but no dose-relationship was observed and a relationship to treatment is doubtful.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- When compared with controls, there was a slight decrease in the terminal body weight of males (-12%) at the end of the treatment period.
- There were no test item-related variations in organ weights.
- The statistically significant changes in testes relative weights at the high-dose were considered to be a reflection of reductions in body weight and not due to test item-related organ toxicity.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Female sacrificed moribund on day 39 (day 23 post-coitum): the torsion of a pregnant uterine horn was the cause of the moribund condition of this female, and was most likely incidental and unrelated to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- There were no test item-related microscopic findings.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on effects on body weights in male and females of the high dose group treated with 125 mg/kg bw/d.
Dose descriptor:
NOEL
Remarks:
reproduction
Effect level:
>= 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on mating, fertility and litter parameters were observed in animals of the high dose group treated with 125 mg/kg bw/day. There were no treatment-related effects on pup mortality, clinical signs, body weight or sex ratio.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
not considered treatment-related
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:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- Four, three, two and 20 pups from the groups treated at 0, 20, 50 or 125 mg/kg/day, respectively, died between post-natal day 1 and post-natal day 5. This increase at 125 mg/kg/day was due almost entirely to two litters which had nine (all pups) and ten (71% of pups) pups found dead or cannibalized.
- Between the remaining seven litters there was only one dead pup, therefore the deaths in the two mentioned litters are considered not to be related to treatment with the test item.

CLINICAL SIGNS (OFFSPRING)
- Isolated clinical signs (wounds, hematomas, scabs) were observed in a few animals in most groups, including the controls.
- It was considered that there were no effects related to the treatment with the test item.

BODY WEIGHT (OFFSPRING)
- There were no effects of treatment with the test item at any dose-level on mean pup body weight or body weight gain.

GROSS PATHOLOGY (OFFSPRING)
- No treatment-related external abnormalities were observed.

OTHER FINDINGS (OFFSPRING)
- Sex ration: the percentage of male pups was similar between all groups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The dose-level of 125 mg/kg/day elicited clinical signs of loud breathing and hypersalivation in males and females. Animals treated at 20 or 50 mg/kg/day presented hypersalivation generally throughout the treatment period (at 20 mg/kg/day this was limited to the males) and two males in each group also showed loud breathing. However, these findings were related to treatment with the test item, but not considered as adverse.

At the high dose level (125 mg/kg/day), males showed an initial mean body weight loss (days 1 to 8) followed by body weight gains which were sometimes lower than the controls. The mean body weight remained statistically significantly lower throughout the study. Mean male food consumption was statistically significantly lower for the first week of treatment. HIgh dose females had a lower mean body weight gain over the first week of treatment and over the gestation period, but effects were less marked and more transient than in the males and food consumption was unaffected. No effects on body weight were seen in animals of the 20 and 50 mg/kg/day groups.

In parental animals, there were no effects on mating and fertility or litter parameters, and no treatment-related effects on organ weights and no relevant macroscopic or microscopic abnormalities were found. Pup mortality (high dose only) and clinical signs were considered to be unrelated to treatment and there were no effects on pup body weight gain.

Under the experimental conditions of this screening study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 50 mg/kg/day and the No Observed Effect Level (NOEL) for mating, fertility and litter parameters was considered to be equal to or greater than 125 mg/kg/day.