Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2008-09-24 to 2009-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study 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

Principles of method if other than guideline:
Preliminary dose-range finding study in pregnant females to establish a dose level for a OECD 414 Developmental toxicity study
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
480-070-0
EC Name:
-
Cas Number:
85-27-8
Molecular formula:
C14H14O2
IUPAC Name:
4-(1-phenylethyl)benzene-1,3-diol
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
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: 10 - 11 weeks old
- Weight at the beginning of treatment period: mean body weight of 283 g
- Housing: the animals were housed in a barriered rodent unit. The animals were individually housed 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.
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet (batch No. 1860828; SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly
- Water (ad libitum): tap water (filtered with a 0.22 μm filter)
- Acclimation period: at least 4 days before beginning of the treatment period (arrival of half of the females on day 1 post coitum and the other half on day 2 post coitum)

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, 12 and 40 mg/mL.
- The test item dosage forms were prepared approximately weekly based on 11-day stability proven by the testing laboratory. They were 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 alone.
- The dosage forms were 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosage form samples were assayed using a validated HPLC/UV method, which was authorized by the Study Director prior to use in this study. This method was validated at 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: dual λ absorbance detector 2487 (Waters), Series 1100 (Agilent Technologies))

- Preparation:
a. Control dosage forms (0 ng/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, 12 and 40 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 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, 6-fold and 20-fold for 4, 12 and 40 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), 2695 Alliance (Waters)
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), 2695 Alliance (Waters)
h. Detector: Dual λ absorbance detector 2487 (Waters)
i. Wavelength: 280 nm
j. Injector: Series 1100 (Agilent Technologies), 2695 Alliance (Waters)
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 on the first and last days of treatment 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
The test item concentrations in the administered dosage forms analyzed on the first and last day of treatment remained within an acceptable range of [-4.2% to +1.3%] 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)).
Standard solutions at 10 μg/mL are stable for 4 days when stored at room temperature, protected from light. Analytical working solutions from dosage
forms of 2 mg/mL to 40 mg/mL in soybean oil are stable for 4 days when stored at room temperature, protected from light.
Details on mating procedure:
- Impregnation procedure: the females were mated at the breeder's facilities.
- Proof of pregnancy: the day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum.
Duration of treatment / exposure:
The dosage forms were administered from day 6 to day 20 post-coitum inclusive.
Frequency of treatment:
once a day, approximately at the same time
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (group 1), 20 (group 2), 60 (group 3) and 200 (group 4) mg/kg bw/day
Basis:
other: actual dose received
No. of animals per sex per dose:
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:
- 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,
- 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.


Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule and cage side observations : each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekend and public holidays. One prematurely sacrificed female (group 4), showing signs of poor clinical condition was weighed, blood sampled for laboratory investigations and then humanely sacrificed.
- From arrival, each animal was 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: the body weight of each female was recorded on days 2, 4, 6, 9, 12, 15, 18 and 21 post-coitum, and prior to premature sacrifice for one female (group 4).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes
The quantity of food consumed by each female was recorded for the following intervals: days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21: females were sacrificed on day 21 post-coitum.
- Females prematurely sacrificed and found dead: one female (group 4), in poor clinical condition, was sacrificed by exposure to carbon dioxide
gas followed by cervical dislocation.
Prematurely sacrificed female (group 4) and one female found dead (group 4) were submitted for a complete macroscopic post-mortem examination and the numbers of corpora lutea and implantation sites were recorded.
-Other females: females were sacrificed by exposure to carbon dioxide gas followed by cervical dislocation and were submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
- The left kidney, urinary bladder and ureter and lymph nodes from one female of group 3, lung of one female of group 4 and vagina from the prematurely sacrificed female and found dead female (group 4) were sampled and kept preserved in 10% buffered formalin.
- In addition, the liver and kidneys of both unscheduled death females were retained in 10% buffered formalin.

HEMATOLOGY: Yes
- A complete hematological examination was performed on one female of group 4 which was prematurely sacrificed.
- The following parameters were determined for this female: erythrocytes (RBC), hemoglobin (HB), 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**
* blood smears were prepared from the samples taken and archived after May Grünwald Giemsa staining.
** sample was kept at -20°C for one day until analysis.

BLOOD BIOCHEMISTRY: Yes
- A complete hematological examination was performed on the one female of group 4 which was prematurely sacrificed.
- The following parameters were determined for this female: lithium heparin tubes (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)
Ovaries and uterine content:
Prematurely sacrificed female (group 4) and one female found dead (group 4) were submitted for a complete macroscopic post-mortem examination and the numbers of corpora lutea and implantation sites were recorded.

Other females:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; the weight of the gravid uterus was recorded for each pregnant female at hysterectomy.
- Number of corpora lutea: Yes
- Number and distribution of dead and live fetuses: Yes
- Number and distribution of implantations: Yes
- Number and distribution of early resorptions: Yes
- Number and distribution of late resorptions: Yes
- Number and distribution of uterine scars: Yes

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with discernible digits.

Uterine horn(s) without visible implantation site were immersed in an aqueous solution of ammonium sulphide (Salewski) to reveal the presence of uterine scars, which were counted.
A gross evaluation of placentas was also undertaken.
Fetal examinations:
- The body weight of each fetus was recorded.
- The sex of the fetuses was determined by visual assessment of anogenital distance at the time of hysterectomy.
- The fetuses were sacrificed by a subcutaneous injection of thiopental sodium.
- The fetal findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformations or variations:
- malformation refers to a permanent structural change that is likely to adversely affect the survival or health,
- variation refers to a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development).

- External examinations: Yes
- Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. All fetuses were then discarded.
- Photographs of the gastroschisis of one fetus of group 2 and the exencephaly of one fetus of group 1 were taken to document findings and archived with the study data.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:
Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by the Fisher exact probability test.
Indices:
For each litter was calculated:
- total number of resorptions,
- total number of live fetuses,
- % of live fetuses per litter,
- % of pre-implantation loss,
- % of post-implantation loss,
- average fetal body weight,

For each group:
- percentage of animals affected: fetal observations (% of fetuses, % of litters),
- mean % per litter of fetuses affected,
- % of pre-implantation loss relative to the number of corpora lutea (mean of pre-implantation, loss per litter),
- % of live fetuses and % of dead fetuses (relative to total number of fetuses),
- % of male fetuses (relative to total number of live and non autolyzed dead fetuses),
- mean, standard deviation and % relative to the number of implantation sites: resorptions plus scars, scars, early resorptions, late resorptions,
- % of post-implantation loss relative to the number of implantation sites (mean of post-implantatin loss per litter, % of dams affected.

Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: for further information please refer to the "Details on maternal toxic effects" below

Details on maternal toxic effects:
CLINICAL EXAMINATIONS
- Pregnancy status: 4, 0, 0 and 1 non-pregnant females in the groups treated at 0, 20, 60 or 200 mg/kg/day, respectively.

Mortality:
- One female treated at 200 mg/kg/day was prematurely sacrificed on day 14 post-coitum with clinical signs of piloerection, loss of balance, ventral decubitus, abdominal breathing, half-closed eyes and reddish vaginal discharge. Hypersalivation and loud breathing had been observed since the beginning of the treatment period. A body weight loss of -41 g was recorded for this female between day 12 and day 14 post-coitum. At necropsy, reddish-colored contents were found in the vagina and the female had 13 implantation sites.
- Another female treated at 200 mg/kg/day was found dead on day 15 post-coitum. Hypersalivation had been observed since day 7 post-coitum, loud breathing since day 8 post-coitum, emaciated appearance since day 10 post-coitum and reddish vaginal discharge on day 14 post-coitum. At necropsy, reddish-colored contents were found in the vagina and the female had 13 implantation sites.

Clinical signs:
- All females treated at 200 mg/kg/day showed hypersalivation and loud breathing during the study. Hypersalivation generally started during the first few days of dosing and lasted until the end of the treatment period. Loud breathing started later than the hypersalivation and was observed intermittently until the end or near the end of the treatment period. Incidences of reddish vaginal discharge (5/10 females, including the unscheduled deaths), yellow-colored fur (2/10 females), ventral decubitus (4/10 females), half-closed eyes (4/10 females) and piloerection (2/10 females) were also observed mid to late gestation.
- At 60 mg/kg/day, 8/10 females showed hypersalivation from midway through the treatment period, one female showed loud breathing from day 14 to day 16 post-coitum and there were incidences of brownish or reddish vaginal discharge (6/10 females) at the end of the treatment period. In addition, one female had yellow-colored fur from day 10 post-coitum.
- At 20 mg/kg/day, two females were observed to have hypersalivation from mid to late gestation.
- Two control females also showed hypersalivation towards the end of the treatment period and another control female showed reddish vaginal discharge and blood in the bedding on day 21 post-coitum.
- Hypersalivation may be related to the oily nature of the vehicle since it was observed in two control females but the incidences were noticeably higher in the 60 and 200 mg/kg/day dose groups therefore it is considered to also be related to treatment with the test item but non-adverse.
- The test item did evoke severe clinical signs including ventral decubitus, half-closed eyes and reddish vaginal discharge.

Body weight and body weight change:
- Females treated at 200 mg/kg/day had a mean body weight loss over the first 3 days of treatment; 6/9 pregnant females lost weight, ranging between -2 g and -35 g. From day 9 post-coitum until the end of the study, mean body weight gains were comparable with the controls but the initial body weight loss resulted in a -19% difference in overall body weight gain when compared with the controls.
- Both the low- and intermediate-dose groups had a lower mean body weight gain between day 6 and day 9 post-coitum when compared with the controls (-17% and -22%, respectively). Thereafter, body weight gains were comparable with those of the controls, or were slightly higher, and overall body weight gain was unaffected.

Food consumption:
- The group treated at 200 mg/kg/day had statistically significantly lower mean food consumption from day 6 to day 12 post-coitum and lower, not statistically significant, food consumption until day 15 post-coitum.
- There were no effects on mean food consumption at 20 or 60 mg/kg/day.

MATERNAL TERMINAL EXAMINATIONS
Gravid uterus weight:
- There were no effects on mean gravid uterus weight at the dose-levels of 60 and 200 mg/kg/day, although the group treated at 20 mg/kg/day did have a lower mean value than the controls.
- The group treated at 200 mg/kg/day had a statistically significantly lower net weight change (mean body weight change taking into account the gravid uterus weight) than the controls.

Macroscopic post-mortem examination:
- One female treated at 200 mg/kg/day had a reddish-colored focus on the lung.
- One female treated at 60 mg/kg/day had urinary bladder and left kidney dilatation along with urinary bladder stones. These were considered to be unrelated to treatment with the test item.
- Brownish-colored contents in uterine horns were observed in one control female and one female treated at 20 mg/kg/day both of which had the highest post-implantation loss in their group.

Hysterectomy data:
- Generally, the mean number of corpora lutea and implantations were higher in the groups treated with test item than in the controls but the mean pre-implantation loss was also higher, although no dose-relationship was observed.
- The lack of a noticeable increase in the number of resorptions or the mean post-implantation loss indicates that this increase in the mean pre-implantation loss was unlikely to be related to treatment with the test item. The mean number of fetuses per female remained comparable with the controls at all dose-levels.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
20
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Remarks:
(preliminary)
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Body weight:
- There were no effects of treatment with the test item on the mean fetal body weight at any dose-level.
- The mean percentage of male fetuses was slightly lower in the group treated at 20 mg/kg/day, but no dose-relationship was observed so this could be related to the low number of females per group.


External examination:
- Malformations: one fetus from each of the groups treated at 0, 20 or 60 mg/kg/day had gastroschisis and another control fetus had exencephaly. Given the similar incidence in the control and test item-treated groups, no relationship to test item is indicated.
- Variations: no variations were observed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The dose-level of 200 mg/kg/day elicited severe clinical signs of ventral decubitus, half-closed eyes and reddish vaginal discharge and there were two unscheduled deaths (one female found dead and another prematurely sacrificed). Mean food consumption was statistically significantly lower for the first 6 days of treatment and there was an initial mean body weight loss.
Treatment with the dose-levels of 20 and 60 mg/kg/day resulted in initially lower mean body weight gains and clinical signs at 60 mg/kg/day including vaginal discharge. All effects showed a dose-relationship in incidence and/or severity.
All groups showed hypersalivation, and loud breathing was also observed at 60 and 200 mg/kg/day. These signs are likely to be related to the oily nature of the vehicle, but a higher incidence at higher dose-levels indicates that the test item elicits an additional effect.
There were no effects on pregnancy parameters in all dose groups, and development of offspring was not affected by treatement with the test item.

Under the experimental conditions of this preliminary study, a NOAEL for systemic toxicity of 20 mg/kg bw/d can be derived, because the only finding observed at this dose level was a slight initial lower of body weight. No treatment-related effects on fetal development and no fetal external abnormalities were observed in this preliminary study.