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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:
From 4 June 2019 to 15 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

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)
Version / remarks:
29 July 2016
Qualifier:
according to
Guideline:
other: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
The test solution contains NaOH due to the production method.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Han:Wistar of Wistar origin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90 Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 83-92 days; Females: 75-83 days
- Weight at study initiation: Males: 348-421 g; Females: 210-259 g
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex /cage.
Mating: 1 male and 1 female /cage.
Mated females: individually.
Males after mating: 2 animals /cage.
- Diet: Commercial ssniff® SM R/M-Z+H complete diet for rats and mice - breeding and maintenance and ssniff® SSNIFF rat/Souris Elevage E, 10 mm autoclavable complete feed for rats, ad libitum
- Water: Ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70 %
- Air changes (per hr): Above 10 air-exchanges/hour by a central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From 26 June 2019 to 21 August 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
VEHICLE:
- Concentration in vehicle: The test substance was formulated in the vehicle in concentrations of 30, 90 and 200 mg/mL.
- Amount of vehicle (if gavage): The dose volume was 5 mL/kg bw.
Details on mating procedure:
M/F ratio per cage: 1:1
- Length of cohabitation: 1-4 days
- Proof of pregnancy: Vaginal smears were prepared daily and examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422)
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed three times during the study. Five aliquots of 5 mL of each formulation and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at 2 - 8°C until analysis. Concentration of the test item in the dosing formulations varied between the range of 97.3 % and 105 % in comparison to the nominal values.
Duration of treatment / exposure:
Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy.
Male animals were dosed for 44 days (14 days pre-mating and 1-4 days mating, plus 26-29 days of post-mating period); and were sacrificed on Day 44.
Females were dosed for 14 days pre-mating, through 1-4 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice on Days 51 or 56.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: See supporting study: 14-Day Oral Gavage Dose Range Finding Study with EP1-Na (Danafloat 123) in Rats, Toxi-Coop Zrt., 2019
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: General clinical observations were made on parental animals once a day, after the administration.
Detailed examinations were made weekly, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter. 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. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week. General physical condition and behavior of animals were tested.

BODY WEIGHT:
- Time schedule for examinations:
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight was measured on the day of necropsy for female animals subjected to organ weighing (selected for further examinations).

FOOD CONSUMPTION:
The food consumption was determined weekly during the treatment period except mating phase (pre-mating days 0, 7, 13 and post-mating days 20, 27, 34 and 41 for male animals, pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

CLINICAL PATHOLOGY:
Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection.

HEMATOLOGY:
Parameters checked in Table 1 were examined.

BLOOD COAGULATION:
Parameters examined: Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT)

CLINICAL CHEMISTRY:
Parameters checked in Table 2 were examined.

DETERMINATION OF SERUM THYROID HORMONE:
Blood samples for determination of serum levels of thyroid hormones (FT4 and TSH) were collected from animals as follows:
- from 2-8 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter)
- from all dams and from 6-7 pups per litter on post-partum/post-natal day 13;
- from all parent male animals at termination on Day 44.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears daily before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each female animal.
Sperm parameters (parental animals):
Parameters examined in all male animals:
Weight of testis, epidymides and prostate and seminal vesicles with coagulating glands weight as a whole.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
Each litter was adjusted to 4 pups/sex/litter if feasible.

PARAMETERS EXAMINED
The following parameters were examined: Number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities. Litters were weighed on day 0, 4 and 13 post-natal. Any abnormal behavior of the offspring was recorded. The anogenital distance of each pup was determined on post-natal day 4. The number of nipples/areolae in male pups was counted on post-natal day 13.

GROSS EXAMINATION OF DEAD PUPS:
Dead or stillborn offspring were subjected to necropsy by macroscopic examination on the day when they were found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All male animals were sacrificed on Day 44
- Maternal animals: Dams selected for toxicology examination were sacrificed on Day 51; dams remained on Day 56 and non-pregnant female animal on Day 44.

GROSS NECROPSY
- Gross necropsy was performed on all parental animals and consisted of external and internal examinations including the cranial, thoracic and abdominal cavities. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, and all organs showing macroscopic lesions of all adult animals were preserved. Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter. Tissues and organs (see Table 3) were collected from 5 male and five female animals randomly selected from each group.

Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups – including non-pregnant female and its mating partner – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1000 mg/kg bw/day) groups and in dead female animals at 450 mg/kg bw/day.
Additionally, organs and tissues were processed and evaluated histologically on the basis of necropsy observations.
Postmortem examinations (offspring):
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).
Offspring were sacrificed on Day 13 or shortly thereafter and carefully examined for gross abnormalities. All organs showing macroscopic lesions were preserved and evaluated histologically.
Statistics:
The statistical evaluation was 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 inter-group 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.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Reproductive indices:
Male and female mating and fertility index, female gestation index
Offspring viability indices:
Survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item related mortality at any dose level.
One dam at 450 mg/kg bw/day (1/12), was found dead on post-partum (lactation) day 5. There were no preceding clinical signs for this animal. Based on findings at necropsy and histological evaluation, the cause of death was judged to be individual disease and not related to the treatment as no death occurred at the higher (1000 mg/kg bw/day) dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly reduced mean body weight gain in male and female animals at 1000 mg/kg bw/day resulted in minor changes in the mean body weight (≥ - 7 % relative to control) therefore was considered to have little or no toxicological relevance.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Squamous cell hyperplasia in the non-grandular part of the stomach was observed in some female animals at 450 and 1000 mg/kg bw/day (4/4 and 6/6 respectively)

Reproductive function / performance (P0)

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

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
30 other: mg/mL (administered to animals of 150 mg/kg bw/day group)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Stomach irritation
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The thyroid hormone (free T4 and TSH) levels were not affected by the test item in offspring sampled on post-natal day 13.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: General health parameters

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, EP1-Na administered at 150, 450 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or gave any indication to cause endocrine effects in parental male and female Han:WIST rats as far as investigated in this study.
There were no signs of systemic toxicity in male or female animals at 150, 450 or 1000 mg/kg bw/day. In the female animals at 450 and 1000 mg/kg bw/day, squamous cell hyperplasia in the non-glandular part of the stomach referred to the local effect of the test item. Besides of this local tissue irritation no other histological findings were noted.
The development of the F1 offspring was not impaired from birth to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 150, 450 or 1000 mg/kg bw/day.

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
NOAEC (stomach irritation, female rats): 30 mg/mL (administered to animals of 150 mg/kg bw/day group)
NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day

Remark: The test substance as produced and marketed contains NaOH due to the production method.
Executive summary:

The toxicity of the substance was assessed in a Combined Dose Toxicity Study with the Reproductive/Developmental Screening Test according to OECD 422 (adopted 29 July 2016). Doses of 0 (control), 150, 450 and 1000 mg/kg bw/day was administered by gavage to 12 Wistar rats/sex/dose. Male rats were dosed daily for 44 days starting 14 days before mating up to the day before necroscopy. Female rats were dosed daily for 51 or 56 days starting 14 days before mating up to lactation day 13 or 17, the day before necroscopy.


 


The test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or gave any indication to cause endocrine effects in parental male and female Han:WIST rats as far as investigated in this study.
There were no signs of systemic toxicity in male or female animals at 150, 450 or 1000 mg/kg bw/day. In the female animals at 450 and 1000 mg/kg bw/day, squamous cell hyperplasia in the non-glandular part of the stomach referred to the local effect of the test item. Besides of this local tissue irritation no other histological findings were noted.
The development of the F1 offspring was not impaired from birth to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 150, 450 or 1000 mg/kg bw/day.


 


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
NOAEC (stomach irritation, female rats): 30 mg/mL (administered to animals of 150 mg/kg bw/day group)
NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day


 


Remark: The test substance as produced and marketed contains NaOH due to the production method.