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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2014 -- 10 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age/weight: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 376 g (range: 330 g to 407 g) and the females were approximately 9 weeks old had a mean body weight of 230 g (range: 206 g to 261 g).
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 18 March 2014 to 10 May 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn 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.
The test item dose formulations were prepared for up to 10 days, stored at room temperature "prior-to-use" and delivered to the study room at room temperature.

VEHICLE
- Justification for use and choice of vehicle: vehicle already used in a previous 2-week preliminary toxicity study
- Concentration in vehicle: 6, 60, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): each female was placed with the same male until mating occurred. The pre-coital time was calculated for each female.
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as Day 0 post-coitum
- After successful mating each pregnant female was caged housed individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: remained within an acceptable range of variations (-4.4% to +5.0%) compared to nominal values (+/- 15% of the nominal concentrations).
Homogeneity: dose formulations prepared at 2 and 200 mg/mL in corn oil were found to be homogenous suspensions (homogeneity and stability study).
Stability: dose formulations prepared at 2 and 200 mg/mL in corn oil were found to be stable after 10 days at room temperature (homogeneity and stability study).
Duration of treatment / exposure:
In the males:
- 2 weeks before pairing,
- during the pairing period,
- until sacrifice (at least 5 weeks in total).

In the females:
- 2 weeks before pairing,
- during the pairing period,
- during gestation,
- during lactation until Day 5or 6 p.p. inclusive.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: on the first day of treatment, the males were approximately 10 weeks old and the females were approximately 9 weeks old. The males and the females were sexually mature and were not siblings. The females were virgin.
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 300, and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
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 a previous 2-week preliminary toxicity study performed in the same species and strain (a previous 2-week preliminary toxicity study). In this study, three groups of three males and three females received the test item by gavage at 100, 300 and 1000 mg/kg/day as a suspension in corn oil. Another group of three males and three females received the vehicle only. There were no mortality and no toxicological significant clinical signs in test item-treated groups and no toxicological significant effects on the mean body weight and mean food consumption. No obvious test item-related changes were observed in the mean organ weights or at the macroscopic post-mortem examination.

- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)

Examinations

Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mated, on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: Males: once a week, from the first day of treatment until the start of the pairing period. Females: on the first day of treatment, then once a week until mating, on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females were mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups)
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD PUPS:
- external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after the end of the pairing period (at least 5 weeks of treatment in total),
- Female animals: all surviving animals: on Day 6 p.p. or Day 7 p.p. (for females which delivered on 02 May 2014)

GROSS NECROPSY
Macroscopic post-mortem examination of the principal thoracic and abdominal organs .

HISTOPATHOLOGY
- all tissues listed in the Tissue Procedure Table from all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period [at the end of the mating period for males or on Day 6 p.p. or Day 7 p.p. (for females which delivered on 02 May 2014) for females],
- all macroscopic lesions of all groups.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

ORGAN WEIGHTS: epididymides, kidneys, liver, spleen, and testes.
Postmortem examinations (offspring):
SACRIFICE: on Day 5 post-partum

GROSS NECROPSY: on all found dead pups.

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
Data (body weight, food consumption and reproductive data) were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).

PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantation sites
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on Day 4 p.p. = 100 * (Number of surviving pups on Day 4 p.p. / Number of live born pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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 examined
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY:
There were no unscheduled deaths during the study.

CLINICAL SIGNS:
There were no clinical signs ascribed to the test item treatment, except ptyalism which was recorded occasionally in 1/10 lactating females at 30 mg/kg/day and in 1/10 males together with piloerection at 1000 mg/kg/day. Since these findings were isolated and poorly dose-related, they were considered to be of no toxicological relevance.

Other clinical signs noted in test item-treated animals were reflux at dosing and signs which are commonly observed in rat of this strain when housed in laboratory conditions (areas of hair loss, scabs and cutaneous lesions). The incidences were low and/or not dose-related.

BODY WEIGHT (GAIN):
There were no effects on mean body weight and mean body weight change in both sexes.

The slight lower mean body weight gain observed at 30 and 300 mg/kg/day (+35 g vs. +40 g in control data, p<0.05) during the gestation period (Day 7 to Day 14 p.c.) did not have any toxicologically significant impact on the mean body weight.

FOOD CONSUMPTION:
There were no effects on mean food consumption (g/animal/day) during the study.

REPRODUCTIVE PERFORMANCE:
Estrous cycle
As all females mated within less than 5 days, there were no toxicologically significant effects on estrus cycle.

Mating and fertility data
There were no effects on mating and fertility data.

Delivery data
There were no effects on mean duration of gestation and delivery data.

ORGAN WEIGHTS:
There were no test item-related organ weight changes.
There were lower absolute and relative-to-body spleen weights in females treated at 30, 300 or 1000 mg/kg/day. These differences were poorly dose-related, did not reach statistical significance and were of low magnitude (up to -22% in relative-to-body weights at 1000 mg/kg/day). They were considered to be mostly the contribution of high weights in controls (mean absolute weight: 0.99 g; mean relative to body weight ratio: 0.33%) with higher standard deviation when compared to the other groups. In historical control data of comparable studies performed in 2009-2014 period, the mean absolute weight is 0.776 g and the mean relative-to-body weight is 0.26%. These results are similar to those noted in test item-treated groups. Consequently, it was concluded that the spleen weight differences were not related to test item administration.
The other organ weights were of low magnitude and thus were considered to be unrelated to test item administration.

GROSS PATHOLOGY:
There were no test item-related macroscopic post-mortem changes.
The few gross findings correlated microscopically with spontaneous lesions seen occasionally in the rats of these strain and age, with no dose-relationship.

HISTOPATHOLOGY:
There were no test item-related microscopic changes in testes/epididymides or in ovaries/oviducts submitted for microscopic examination.
The few gross findings examined microscopically were considered to be consistent with spontaneous lesions seen occasionally the rats of these strain and age.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

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:
not examined
Histopathological findings:
not examined

Details on results (F1)

MORTALITY/VIABILITY/CLINICAL SIGNS:
There were no effects on pup viability.

None of the clinical signs mentioned below were considered to be related to the treatment with the test item (comparable incidences in control pups or not dose-related): nodule on ombilical cord, dehydration, hematoma, generalized pallor, abdominal breathing and/or necrosed limb.

BODY WEIGHT (GAIN):
There were no effects on mean pup body weight and mean body weight change.

PUP SEX RATIO
There were no effects on the mean percentage of male pups at birth.

GROSS PATHOLOGY:
There were no observations at necropsy of dead pups which were indicative of a test item treatment related effect.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating and (for females) throughout gestation and until Day 5 or Day 6 post-partum, at dose-levels of 30, 300 or 1000 mg/kg/day.
In the absence of significant effect, the dose-level of 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for parental systemic toxicity and for reproductive performance (mating, fertility and gestation).
In the absence of significant effect on pups, the No Observed Effect Level (NOEL) for pups was considered to be 1000 mg/kg/day.

Executive summary:

The potential reprotoxic effects of EKKE was evaluated following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 5 or Day 6 post-partum (p.p.). This study provides initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition. This study was performed according to OECD guideline 421. Three groups of ten male and ten female Sprague-Dawley rats received EKKE daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5 or Day 6 p.p.. EKKE was administered as a suspension in the vehicle, corn oil, at dose-levels of 30, 300 or 1000 mg/kg/day. Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dose-volume of 5 mL/kg/day was used.  The concentrations of the dose formulations were checked in study Weeks 1, 3 and 6. The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment period. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post-coitum  (p.c.) and lactation on Days 1 and 5  p.p.. Estrous cycle was monitored on all females during the mating period. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 5 p.p..  The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1 and 5 p.p..  Males were sacrificed after at least 5 weeks of treatment and dams on Day 6 or Day 7p.p.. Final body weights and selected organs weights (kidneys, liver, epididymides, testes and spleen) were recorded and a complete macroscopic  post-mortem  examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on ovaries (with oviducts), epididymides and testes from all males sacrificed at the end of the treatment period and all females sacrificed on Day 6 or Day 7 p.p. in the control and high-dose groups and on all macroscopic lesions. Pups were sacrificed on Day 5  p.p.  and discarded without necropsy.

EKKE concentrations in the administered dose formulations analyzed in Weeks 1, 3 and 6 were within an acceptable range of variation when compared to the nominal values (±15%). In F0 animals, there were no unscheduled deaths. EKKE-related clinical signs were limited to ptyalism which was noted on rare occasions in isolated animals at 30 and 1000 mg/kg/day and was considered to be of minor toxicological relevance.  There were no toxicologically relevant effects on body weight gain, body weight, food consumption and on mating, fertility and delivery data.  There were no EKKE-related organ weight and macroscopic post-mortem changes. There were no EKKE-related microscopic changes in testes/epididymides or in ovaries/oviducts submitted for microscopic examination. In pups, there were no EKKE-related effects on pup data by Day 5 p.p. (viability, clinical signs, body weight, sex ratio, macroscopic findings).

EKKE was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating and (for females) throughout gestation and until Day 5 or Day 6 post-partum, at dose-levels of 30, 300 or 1000 mg/kg/day. In the absence of significant effect, the dose-level of 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for parental systemic toxicity and for reproductive performance (mating, fertility and gestation). In the absence of significant effect on pups, the No Observed Effect Level (NOEL) for pups was considered to be 1000 mg/kg/day.