Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
from 2009-05-07 to 2009-12-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study has been performed in compliance with the: Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). There were no circumstances that may have affected the quality or integrity of the data.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
TYPES OF QA INSPECTIONS: study plan; study based: test item, test system, RD, dose preparation, sample taking, treatment, BW, FOB, necropsy; process based: clinical laboratory investigations, histotechnique, analytical work up; analytical appendix; report
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity of the substance tested in the experimental study:
Internal code: LCE08129
Description: solid
Batch Number: T82925
Purity: > 99% (dry extract)
Expiry Date (Retest Date): 15-Jul-2010
Storage Conditions: At room temperature (20 ± 5 °C)
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories Ltd., Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
Number of Animals: 40 males (10 per group) and 40 females (10 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males (286 to 327 g) and females (178 to 221 g)
Identification: Cage card and individual animal number (ear tattoo). Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH&CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 76/08).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
DOSE FORMULATIONS

The dose formulations were prepared daily using the test item as supplied by the Sponsor.

LCE08129 had to be crushed in order to get small portions for weighing. It was weighed into a glass beaker on a tared precision balance, heated in a water bath (max. 70 °C) and the vehicle was added (w/v) under agitation. Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

TREATMENT

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group); Group 2: 100 mg/kg/day; Group 3: 300 mg/kg/day; Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C23966, using dose levels of 100, 300 and 1000 mg/kg/day, resulting in a NOEL of 1000 mg/kg/day.
Dose Volume: 5 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males (28 days); females (approximately 7 weeks)
Details on mating procedure:
MATING, GESTATION AND LACTATION

During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day of mating was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONS

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs at room temperature). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

The application formulations investigated during the study were found to comprise LCE08129 in the range of 74.0% to 108.6%. Eleven out of 12 samples met the required content limit of ±20% with reference to the nominal concentration. The homogeneous distribution of LCE08129 in the preparations was approved because single results found did not deviate more than 3.3% (<15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept 4 hours at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.
Duration of treatment / exposure:
Males: 28 days
Females: Approximately 7 weeks
Frequency of treatment:
Daily
Details on study schedule:
Acclimatization: 7 days minimum (males and females)
First Test Item Administration: Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 14 days maximum (males and females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day 4 post partum (females); on day before sacrifice (males)
Necropsy: On day 5 post partum (females); on day 4 post partum (pups); after treatment of at least 28 days, when no longer needed for assessment of repro-ductive effects (males)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males (weekly during pre-pairing and after pairing periods); females (pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum). No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.


DETAILED CLINICAL OBSERVATIONS
Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.


FUNCTIONAL OBSERVATION BATERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:

- Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal.

- Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.

- Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.

- Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.

- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.


CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The assay was performed at Clinical Diagnostics Department (Harlan Laboratories Ltd., Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.


HEMATOLOGY
The following hematology parameters were determined:

Complete Blood Cell Count:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count
Platelet count

Coagulation:
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time


CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio


Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
TERMINATION OF THE STUDY

Males were sacrificed after treatment of 28 days. Dams were sacrificed on day 5 post partum.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

NECROPSY

All parent animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

All parent animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

The testes and epididymides of all parental males were weighed as pairs.

In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Liver
Thymus
Spleen

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate
Seminal vesicles with coagulating gland
Testes (in Bouin’s fixative)
Epididymides (in Bouin’s fixative)

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Ovaries

In addition, from the five males and females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions
Brain
Spinal chord
Small and large intestines (incl. Peyer’s patches)
Stomach
Liver
Kidneys
Adrenals
Spleen
Heart
Thymus
Thyroids, and parathyroids if possible
Trachea and lungs (preserved by inflation with fixative and then immersion)
Uterus (with vagina)
Urinary bladder
Lymph nodes (mesenterial, mandibular)
Peripheral nerve (sciatic)
Bone marrow

HISTOTECHNIQUE

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.



Postmortem examinations (offspring):
TERMINATION OF THE STUDY

Pups were sacrificed on day 4 post partum.

NECROPSY

All pups sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

All pups were sacrificed by an injection of sodium pentobarbital.

All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:
Following statistical methods were used to analyze locomotor activity, food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables coulc be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses and mean litter size.
Offspring viability indices:
From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratios and postnatal loss (up to day 4 post partum).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the dose levels of 300 and 1000 mg/kg bw/day all males and females pushed their head through the bedding material and showed salivation after application. These findings were considered to be signs of discomfort but not a toxic effect.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 1000 mg/kg bw/day males mean food consumption and mean body weight gain were slightly reduced after treatment start . This transient reduction was considered to be test item-related but not adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 1000 mg/kg bw/day males mean food consumption and mean body weight gain were slightly reduced after treatment start . This transient reduction was considered to be test item-related but not adverse.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
As examined by testis and epididymis weight.
Reproductive performance:
no effects observed

Details on results (P0)

1 IN-LIVE DATA - PARENTAL ANIMALS
1.1 DETAILED CLINICAL OBSERVATIONS (DAILY)
(See Summary Tables on pp. 22-23)

In group 3 and 4, all animals pushed their head through the bedding material and showed salivation after application during the whole treatment period. These findings were considered to be a sign of discomfort but not a toxic effect.

In group 3 and 4, one and two males, respectively, had rales on single days during treatment. Furthermore in group 4, one male which showed rales had also ruffled fur. These findings were considered to be not test item-related.

1.2 DETAILED CLINICAL OBSERVATIONS (WEEKLY)
No test item-related findings were noted during the detailed weekly clinical observations of males and females in any group.

1.3 FUNCTIONAL OBSERVATIONAL BATTERY
(See Summary Tables on pp. 24-27; A description of the test parameters and scoring used on pp. 28-32)

None of the parameters under investigation during the functional observational battery (appearance, behavior in the open field, grip strength, landing food splay, body temperature) gave an indication of a test item-related effect.

In group 4, male no. 35 in group 4 was aggressive, had unkempt and ruffled fur and vocalization over 10 seconds. Due to the isolated occurrence, this behavior was considered to be incidental. Furthermore, for three males the number of rearings was decreased compared to the control group. Since the locomotor measurements did not show any reduced activity, this finding was considered to be incidental. For two females, a wet chin was noted due to the observed salivations.

Also in group 1, 2 and 3, single animals were noted, which showed an aggressive behavior, a reduced number of rearings or a spontaneous vocalization.

1.4 LOCOMOTOR ACTIVITY
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor.

No indication of a test item-related effect was noted in any group.

1.5 FOOD CONSUMPTION - MALES
(See Figures on pp. 2-3, Summary Tables on pp. 33-34 and 38-39)

Pre-pairing and After Pairing Periods

In group 4, mean food consumption was transiently statistically significantly reduced after treatment start ( 15.3% compared to the control group from day 1 - 8 of the pre-pairing period). Thereafter, mean food consumption was not affected by treatment with the test item. This transient reduction was considered to be test item-related but not adverse.

In group 3, mean food consumption was lower in the second week of the pre-pairing period ( 9.1% compared to the control group). Due to the absence of a dose dependency, this finding was considered to be incidental.

In group 2, mean food consumption was similar to the control group.

1.6 FOOD CONSUMPTION - FEMALES
(See Figures on pp. 4-6, Summary Tables on pp. 35-37 and 40-42)

Pre-pairing, Gestation and Lactation Periods

Mean food consumption was similar in all groups and in all periods and no test item-related effects were observed. The overall mean food consumption per day e.g. in the pre-pairing period was 14.3 g, 13.5 g, 13.8 g and 13.8 g in order of ascending dose levels.

1.7 BODY WEIGHTS - MALES
(See Figures on pp. 7-9 and 14-16, Summary Tables on pp. 43-45, 51-53 and 59-61)

Pre-pairing, Pairing and After Pairing Periods

In group 4, mean body weight gain was slightly reduced after treatment start (+3.5% compared to +6.2% in the control group from day 1 - 8 of the pre-pairing period) and remained reduced until the end of the period. This reduction, which was on single days statistical significant, was considered to be test item-related but not adverse. Mean body weights were similar to the control group during the whole treatment period.

In group 3, mean body weight gain was slightly lower during the second week of the pre-pairing period (+2.5% compared to the control group from day 8 - 14 of the pre-pairing period). As a consequence, mean body weights were minor lower during the following periods. These effects were considered not to be test item-related but a result of biological variability.

In group 2, mean body weights and mean body weight gain were similar to the control group.

1.8 BODY WEIGHTS - FEMALES
(See Figures on pp. 10-13 and 17-20, Summary Tables on pp.46-50, 54-58 and 62-64)

Pre-pairing, Pairing, Gestation and Lactation Periods

Mean body weights and mean body weight gain were similar in all groups and in all periods and no test item related effects were observed. The overall mean body weight gain e.g. in the pre-pairing period was +9.0%, +5.9%, +10.0% and +9.4% in order of ascending dose levels.


2 CLINICAL LABORATORY INVESTIGATIONS
2.1 HEMATOLOGY
(See Summary Tables on pp. 65-66; Historical Data on pp. 67-68)

No test item-related effects were noted for males and females. The occasionally statistically significantly altered values were either in the range of the historical control data or no dose dependent manner was noted.

2.2 CLINICAL BIOCHEMISTRY
(See Summary Tables on pp. 69-70; Historical Data on pp. 71-72)

No test item-related effects were noted for males and females. The occasionally statistically significantly altered values were either in the range of the historical control data or no dose dependent manner was noted.

Exceptionally in group 4 males, the level of triglycerides was statistically significantly increased and not covered by the attached historical control data (0.91 nmol/l versus 0.50 nmol/l in the control group). Since no other test item-related altered parameters were noted in these group and the common 95% tolerance limit for males in this age was between 0.20 - 1.14 nmol/l, this finding was considered to be incidental.


3 REPRODUCTION AND BREEDING DATA
3.1 MATING PERFORMANCE AND FERTILITY
All females were mated within the first pairing period.

The median and mean precoital times were not affected by treatment with the test item. Mean precoital times were 2.7, 3.1, 2.7 and 2.9 days in order of ascending dose levels. The median precoital time was 2, 4, 3 and 3 days in order of ascending dose level.

One female each in group 3 and 4 was incidentally not pregnant. Therefore, the fertility index was 90% in these groups and 100% in group 1 and 2.

3.2 DURATION OF GESTATION
The mean duration of gestation was not affected by treatment with the test item. Mean duration of gestation was 21.5, 21.3, 21.7 and 21.7 days in order of ascending dose level.

3.3 CORPORA LUTEA COUNT
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (13.0, 13.9, 14.8 and 14.8 in order of ascending dose level) and gave no indication of a test item-related effect.

3.4 IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The mean number of implantations per dam and the mean implantation loss was similar in all groups and not affected by treatment with the test item. The mean implantation loss per dam was 0.6, 0.4, 1.6 and 0.9 in order of ascending dose levels.

3.5 LITTER SIZE AT FIRST LITTER CHECK
The mean number of living pups at first litter check was similar in all groups (11.2, 12.8, 12.6 and 13.0 pups in order of ascending dose levels). No dead pups were found at first litter check in any group.

3.6 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
The mean postnatal loss was not affected by treatment with the test item. In group 3, one male pup was incidentally found dead on day 2 post partum. As a result, the mean postnatal loss between days 0 - 4 post partum was 0 in group 1, 2 and 4 and 0.1 in group 3.


4 TERMINAL FINDINGS - PARENTAL ANIMALS
4.1 ORGAN WEIGHTS
(See Summary Tables on pp. 73-78; Historical Data on pp. 79-102)

In males and females, no test item-related findings were noted on organ weights or organ weight ratios in any group.

In group 4 females, the body weight ratio of the liver was statistically significantly higher (3.33% compared to 3.00% in the control group). Since this value was in the range of the historical control data (3.11 - 3.63%) and no histopathological correlate was noted, this finding was considered to be incidental.

4.2 MACROSCOPICAL FINDINGS
(See Summary Tables on pp. 103-104)

In males and females, no test item-related macroscopical findings were noted in any group.

Due to the isolated occurrence and in the absence of a histopathological correlate following findings were considered to be incidental:

Group 1: Male nos. 4 and 5 had a dark red discolored thymus and female no. 44 had a left uterus horn reduced in size.

Group 2: Male no. 11 had yellowish discoloration on the mucosa of the pylorus region in the stomach and the right kidney was adherent to the liver and showed a pelvic dilation, male no. 12 had dark red discolorations on the meninges of the brain, male no. 13 had an isolated foci on the thymus, male no. 16 showed a left testis reduced in size and female no. 54 had reddish discoloration on the mandibular lymph node.

Group 3: Male no. 25 had yellowish discoloration on the mucosa of the pylorus region in the stomach.

Group 4: Male no. 33 had an urinary bladder with a milky-cloudy fluid content, male no. 35 had yellowish discoloration on the mucosa of the pylorus region in the stomach and male no. 36 showed seminal vesicles reduced in size.

4.3 HISTOPATHOLOGY FINDINGS
The test item LCE08129 produced no histological evidence of toxicological properties in the organs and tissues examined.

During sperm staging no differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No test item-related effects were noted in reproduction and development up to and including 1000 mg/kg body weight/day. NOEL = highest dose tested
Remarks on result:
other: Generation: reproduction and development (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

1 LITTER DATA - F1 PUPS
1.1 EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No abnormal findings were noted at first litter check or during the first 4 days post partum.

In group 3, each for one male pup and one female pup from the same litter a bloody wound on nose or cervical region was noted.

1.2 SEX RATIOS
Sex ratios at first litter check and on day 4 post partum were not affected by treatment with the test item. There was a statistical significant higher proportion of males in group 3 which was considered to be incidental due to the lack of dose-dependency.

1.3 BODY WEIGHTS TO DAY 4 POST PARTUM
Mean pup weights on day 1 post partum were not affected by treatment with the test item. On day 1 post partum mean pup weights were 6.3, 6.0, 6.2 and 6.4 g in order of ascending dose level.

Mean pup weight development until day 4 post partum was slightly lower in group 2 and 4 (+49.2%, +41.7%, +48.4% and +39.1% in order of ascending dose levels), but due to the lack of a dose-dependency and no statistical significant differences, this finding was considered to be a result of biological variability.

1.4 MACROSCOPICAL FINDINGS
No abnormal findings were noted at macroscopic examination of the pups in any group.


Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

SUMMARY OF PERFORMANCE - P ANIMALS BREEDING FOR F1 LITTERS

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

41-50

51-60

61-70

71-80

Number of females paired

10

10

10

10

Number of females mated

10

10

10

10

Number of females,which were not pregnant (A)

0

0

1

1

Number of females which reared their pups until day 4 post partum

10

10

9

9

(A)  Female nos. 67 and 73 were not pregnant

Applicant's summary and conclusion

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item LCE08129 to rats. LCE08129 was administered in olive oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. LCE08129 was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Based on the results of this study a general NOAEL (No Observed Adverse Effect Level) was established at 1000 mg/kg body weight/day. The NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 1000 mg/kg body weight/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of LCE08129 on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

LCE08129 was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

 

Group 1:              0 mg/kg body weight/day (control group)

Group 2:          100 mg/kg body weight/day

Group 3:          300 mg/kg body weight/day

Group 4:        1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (olive oil).

 

The following results were obtained:

 

 

PARENTAL ANIMALS

Mortality and General Tolerability

All animals survived until the scheduled necropsy.

 

In group 3 and 4, all animals pushed their head through the bedding material and showed salivation after application during the whole treatment period. These findings were considered to be a sign of discomfort but not a toxic effect.

 

Food Consumption and Body Weights

In group 4 males, mean food consumption and mean body weight gain were slightly reduced after treatment start. Mean body weights were similar to the control group. This transient reduction was considered to be test item-related but not adverse.

 

In group 2 and 3 males, mean food consumption, mean body weight gain and mean body weights were not affected by treatment with the test item.

 

In females, mean food consumption, mean body weight gain and mean body weights were not affected by treatment with the test item.

 

Functional ObservationalBatteryand Locomotor Activity

None of the parameters under investigation during the functional observational battery (appearance, behavior in the open field, grip strength, landing food splay, body temperature) and the locomotor activity measurements gave an indication of a test item-related effect.

 

Clinical Laboratory Investigations

No test item-related effects were noted from the clinical laboratory investigations.

 

Reproduction and Breeding Data

All pairs mated. No effects were noted in the reproduction and breeding data.

 

Organ Weights

No test item-related findings were noted on organ weights or organ weight ratios in any group.

 

Macroscopical Findings and Histopathological Examinations

No test item-related macroscopical or histopathological findings were observed.

 

 

LITTER DATA - F1 PUPS

Findings at First Litter Check and during Lactation

The mean number of pups at first litter check was not affected by the treatment with the test item. No abnormal pup was noted at any dose level during external examinations.

 

Pup Weights to Day 4 Post Partum

Pup weights were not affected by treatment with the test item.

 

Macroscopical Findings

No abnormal findings were noted during macroscopical examination of the pups.