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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-06-23 to 2021-XX-XX
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 25, 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : 10 weeks
- Basis for dose level selection: DRF (on the basis of OECD 421) was performed (rf. to study entry for study number 552-421-5133 and 552-421-5133R)
- Inclusion/exclusion of extension of Cohort 1B: not yet possible
- Termination time for F2 : not yet possible
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : not yet possible
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : not yet possible
- Route of administration : gavage; A 14 d palatability pre-test has been performed with APS in diet at doses of 250 and 500 mg/kg bw/d (please refer to IUCLID section 7.5.1). The test substance proved to be instable in diet (analytical study not reported in the dossier).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) not older than 9 wks; (F1) not older than 9 wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Housing:
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
F1 offspring (after weaning): 2 or three animals of the same sex/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item will be formulated in the vehicle. Formulations will be prepared in the formulation laboratory of Test Facility and used within 3 days.

VEHICLE
- Concentration in vehicle: 10, 20, 40 mg/mL
- Treatment Volume: A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility if less than 20 pregnant females per group have been achieved.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations will be performed in the Analytical Laboratory of Test Facility. Five samples will be taken from different places from each concentration (Groups 2, 3 and 4) and at least three replicates are measured on 5 occasions. Similarly, five samples will be taken from the control solution (Group 1) from different places and analyzed.
Duration of treatment / exposure:
Parental male and female animals: 18 weeks
F1 (cohort 1A): 12 weeks
F1 (cohort 1B): 13 weeks
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 19 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day
Remarks:
10 mg/mL
Dose / conc.:
100 mg/kg bw/day
Remarks:
20 mg/mL
Dose / conc.:
200 mg/kg bw/day
Remarks:
40 mg/mL (dose was reduced back to 180 mg/kg bw/d due to moribund condition of male and female animals)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained from previous studies with Diammonium Peroxodisulphate in rats (Pre-Tests for OECD 443 Study with Diammonium Peroxodisulphate in the Rat, study nos. 552-421-5133 and 552-421-5133R) and in agreement with the Sponsor. 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. In case of severe signs of toxicity, the high dose will be reduced in the course of the study.
- Fasting period before blood sampling for clinical biochemistry: yes, 16 hours

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: same as weighing

BODY WEIGHT: Yes
- Time schedule for examinations:
Parental males will be weighed on the first day of dosing (day 0) and weekly thereafter.
Parental females will be weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 2, 4, 7, 9, 11, 14, 16, 18, 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21. Body weight of the pregnant female animals will only be evaluated statistically on gestation days 0, 7, 14 and 21. Body weight data will be reported individually for adult animals.

F1 animals selected for follow-up examinations will be weighed on post-natal day 22, then twice a week during the two weeks following weaning, and once weekly thereafter.
For selected F1 offspring, the body weight will be recorded on the day when they attain puberty (completion of balano-preputial separation or vaginal patency).

Fasted body weight will be measured on the day of necropsy for all animals (P and F1).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual inspection
Oestrous cyclicity (parental animals):
Estrous cycle will be monitored by examining vaginal smears from each parental female animal daily for two weeks before the mating starts. Vaginal smears will also be prepared and estrous cycle will be monitored daily during the mating period until evidence of copulation. Vaginal smear will also be prepared on the day of the necropsy of parental animals. Vaginal smears will be examined for all F1 Cohort 1A females selected for follow-up examinations after the onset of vaginal patency until the first cornified smear is recorded thus determining the time interval between these events.
Estrous cycle of F1 adult female animals will be examined for a period of two weeks commencing on PND89 and PND96 in Cohort 1A and Cohort 1B, respectively, including necropsy days. Vaginal smears will be stained with 1 % aqueous methylene blue solution. After drying, the smears will be examined with a light microscope.
Sperm parameters (parental animals):
Parameters examined in all control and high dose P/F1 (cohort 1A) male parental generations:
testis weight, epididymis weight, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead, if it can be identified.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [after the optionally extended post-mating period]
- Maternal animals: All surviving animals [on post-partum day 22 or shortly after]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination (only in control and high dose animals) and weighed, respectively:
All parental animals and all adult F1 animals of Cohort 1A:
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one unit (with their fluids)
- brain,
- liver,
- kidneys,
- spleen,
- thymus,
- pituitary,
- thyroid glands (post-fixation)
- adrenal glands

In animals of Cohort 1B, the weight of following organs will be determined:
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one unit (with their fluids)
- brain
- pituitary
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 22 days of age, and all F2 offspring on PND 4 or shortly thereafter.
- These animals were subjected to postmortem examinations macroscopic and microscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

IMMUNOTOXIC EXAMINATION

At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis will be performed in 10 male and 10 female Cohort 1A animals from each group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) as follows:
- weighing of the lymph nodes associated with and distant from the route of exposure (submandibular and popliteal lymph nodes);
- splenic lymphocyte subpopulation analysis (CD4+ and CD8+ Tlymphocytes, B lymphocytes and natural killer cells) by flow cytometry method using one half of the spleen
Statistics:
The statistical evaluation of appropriate data will be performed with the statistical program package SPSS PC+4.0.
Reproductive indices:
Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100

Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant):
Males: Number of males impregnating a females / Total number of males with confirmed mating x 100
Females: Number of pregnant females / Number of sperm positive females x 100

Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100
Offspring viability indices:
Post-implantation mortality: Number of implantations – Number of liveborns / Number of implantation x 100

Post-natal mortality: Number of liveborns – Number of live pups on PND 13 / Number of liveborns x 100

Survival Index: Number of live pups on PND 13 / Number of liveborns x 100

Sex ratio: Number of pups examined – Number of pups males (females) / Number of pups examined x 100

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks on result:
other: Will be completed when study has been finalised.

Results: F1 generation

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks on result:
other: Will be completed when study has been finalised.

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Applicant's summary and conclusion