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:
2014-04-16 until 2014-05-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline compliant study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 1995
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines OPPTS 870.3550 Reproduction / Developmental Toxicity Screening Test, July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: pale yellow liquid

Test animals

Species:
rat
Strain:
other: Hsd.Brl.Han: of Wistar origin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male and female animals: 79 - 83 days old
- Weight at study initiation:
Male animals: 292 - 3622 g
Female animals: 189 - 220 g
- Housing: Type III polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: 2 animals / cage
Bedding: Lignocel Hygienic Animal Bedding, supplier J. Rettenmaier & Söhne GmbH+Co.KG (D-
73494 Rosenberg, Germany).
- Diet:
Animals received ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water:
Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
6 days

ENVIRONMENTAL CONDITIONS
- Temperature:
22 ± 3 °C
- Humidity:
30-70 %
- Air changes (per hr):
10 - 15
- Photoperiod:
12 hours daily, from 6.00 a.m. to 6

IN-LIFE DATES:
From: 2014-04-16
To: 2014-05-30

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Polyethylene glycol 400 (PEG 400)
Details on exposure:
REPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 200, 60 and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for 24 hours before use.
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study

VEHICLE
- Justification for use and choice of vehicle:
The test item is not soluble in water therefore PEG 400 was used for preparing formulations appropriate for oral administration. PEG 400 was a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle:
A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.
- Lot/batch no.: 12J110516
Details on mating procedure:
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females were cohabited with the same male until copulation occurred. Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
Recovery of Incozol EH from PEG-400 formulations at 20 and 200 mg/mL was 96 and 103 %.
Test item proved to be stable in PEG-400 formulations at 20 and 200 mg/mL concentration levels at least for 6 hours at room temperature and for 1 days in refrigerator.
Concentration of the test item in the dosing formulations varied in the range of 91 and 106 % of the nominal values at both analytical occasions.
Duration of treatment / exposure:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology. Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 41 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 or 4 (altogether for 41 – 44 days depending on day of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum.
Non-pregnant and not delivered female animals were treated up to and including the day before necropsy (altogether for 41 days).
Control animals were handled in an identical manner to the test groups receiving 5 mL vehicle/kg bw.
Frequency of treatment:
Once a day
Details on study schedule:
Male animals were dosed for 41 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 or 4 (altogether for 41 – 44 days depending on day of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum.
Non-pregnant and not delivered female animals were treated up to and including the day before necropsy (altogether for 41 days).
Control animals were handled in an identical manner to the test groups receiving 5 mL vehicle/kg bw.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 animals/sex in the control and dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting based on findings obtained in a previous repeated dose oral gavage toxicity study. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
Clinical Examination
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment at approximately the same time. More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling.

Weight Assessment
All parent animals were weighed with accuracy of 1 g. Parent male animals were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy. Parent females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partal day 0 (within 24 hours after parturition) and post-partal day 4. Female animals were weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

Food Consumption
The food consumption was determined weekly by reweighing the nonconsumed diet with an accuracy of 1 g during the treatment period except mating days (pre-mating and post mating for male animals and nonpregnant females, during pre-mating, gestation days 0, 7, 14 and 21, lactation days 0 and 4 for dams).

Examination of Placental Sign
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on day 13 of gestation. If the test was negative on day 13 the examination was repeated on day 14 of gestation.

Observation of the Delivery Process
Females were allowed to litter and rear their offspring. Delivery process was monitored whilst keeping possible interferences at a minimum. Observations were reported individually for each animal. The duration of gestation was recorded and was calculated from day 0 of pregnancy. Dams were observed whether they made a nest from the bedding material and cover their newborns or not. The sucking success was monitored by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
Oestrous cyclicity (parental animals):
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females were cohabited with the same male until copulation occurred. One pair was changed within the control group and within the high dose group after 14 day unsuccessful pairing.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete) and on day 4 postpartum with an accuracy of 0.1 g. In addition to the observations on parent animals, any abnormal behavior of the offspring was monitored. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead were subjected to necropsy by a macroscopic examination. On day of birth, a lung flotation test was performed on all pups found dead to separate stillborn from those that died after delivery (dead pups). The lung flotation test is negative for stillborn (pups that died intrauterine) but positive for pups that died after delivery.
Postmortem examinations (parental animals):
Pathology
Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isofluran CP® and then were exsanguinated. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.

The testes, epididymides and brain of all male adult animals were weighed.
The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were fixed in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

Histopathology
Detailed histological examination was performed on the ovaries, testes and epididymides of the animals in the control and high dose groups and in non-pregnant females and males cohabited with in the 100 mg/kg bw/day groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):
Pups were carefully examined for gross (external) abnormalities and euthanized on postnatal day 4.
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)
Statistics:
The statistical evaluation of appropriate data (marked with † above) were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of intergroup differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value.
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables".
Offspring viability indices:
The offspring viability indices were calculated: survivla index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Results and discussion

Results: P0 (first parental animals)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity: No adverse effects observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive performance: No adverse effects observed.

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

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity in offspring: No adverse effects observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The reproductive performance and developmental toxicity in F1 offspring of Incocol EH were assessed in a study performed according to OECD Guideline 421 in male and female Hsd.Brl.Han: Wistar rats. The No Observed Adverse Effect Levels (NOAEL) were determined as follows:  

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day
Executive summary:

The purpose of this reproduction/developmental toxicity screening test was to obtain initial information concerning the effect of the test item Incozol EH on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post partum associated with oral administration to rats at repeated doses.

Four groups of Hsd.Brl.Han: Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 1000, 300 and 100 mg/kg bw/day corresponding to concentrations of 0, 200, 60 and 20 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, Polyethylene glycol 400 (PEG 400). The suitability of the vehicle for the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. Incozol EH concentrations in the dosing formulations varied in the acceptable range between 91% and 106% of the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 41 days). Females were additionally exposed through the gestation period and up to lactation days 3 or 4, i.e. up to the day before necropsy (altogether for 41 – 44 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. The dams were allowed to litter, and rear their offspring up to day 4 postpartum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 4. All parental animals were subjected to gross pathology one day after the last treatment. Histopathology examination was performed on reproductive organs (testes, epididymides and ovaries) in the control and high dose groups. The reproductive organs of cohabited males and non-pregnant females in the low dose group were also processed and evaluated histopathologically.

Results

Mortality

There was no mortality at any dose level (1000, 300 and 100 mg/kg bw/day).

Clinical observation

Adverse signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. At the same intervals, the behavior and physical condition of the animals was not impaired at each dose level (1000, 300 or 100 mg/kg bw/day) during the entire treatment period.

Body weight and body weight gain

The mean body weight gain of male animals at 1000 mg/kg bw/day was in a varying degree slightly reduced during the treatment period.

However, this slight change was not associated with relevant or significant changes in the mean body weight. Therefore, this observation was judged to be no toxicologically relevant adverse effect.

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at 1000, 300 and 100 mg/kg bw/day during the entire

study (pre-mating, post-mating, gestation and lactation periods).

Reproduction

There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals.

Necropsy

Specific macroscopic alterations related to the test item were not found at necropsy.

Organ weight

There were no test item related changes in brain, testes and epididymides weights of male animals at any dose level.

Histopathology

Histopathological examinations of male and female genital organs (ovaries, testes and epididymides) did not reveal any test item related changes at any dose level.

Offspring

No adverse findings on offspring development (mortality, clinical signs, body weight) or at necropsy were detected in the offspring terminated as scheduled.

Conclusion

Under the conditions of the present study, Incozol EH administered at 1000, 300 or 100 mg/kg bw/day did not led to signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats. The development of the F1 offspring from conception to day 4 postpartum after repeated oral administration was not impaired at any dose level. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day