Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)-study in progress
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Justification for Read Across is given in Section 13 of IUCLID

Data source

Reference
Reference Type:
other: details from study plan
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25th June 2018
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Council Regulation (EC) No. 440/2008, Published in O.J.L. 142, 2008
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Similar Substance 02_EL
IUPAC Name:
Similar Substance 02_EL

Test animals

Species:
rat
Strain:
Wistar
Remarks:
CRL (SPF quality)
Details on test animals or test system and environmental conditions:
Supplier: SPF breeding, VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
Sex: sexually adult females and males for DRFE and main study
Acclimatization: at least 5 days
Housing conditions:DRFE: monitored conditions, microbiologically defined background
Main study: SPF – specified pathogens free animal room,
Animals per cage: before mating – 2 rats of the same sex in one cage in mating period – one male and two females in one cage, pregnant females – individually
Food: pelleted standard diet for experimental animals ad libitum
Water: drinking water ad libitum
Light cycle: 12 hour light/12 hour dark
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Bedding: sterilized shavings of soft wood or Lignocel
Selection of females: pregnant females will be randomly selected to the experimental groups
Identification of animals: the animals will be identified by the colour marks on their fur, each cage will be marked with the number of animals, sex, number of cage, name and dose level of the test item

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Preparation of test formulation and Administration: The formulation of the test item will be administered to the stomach by gavage. The test item concentration at single dose level will be adjusted so that the administered volume will be constant at all dose levels – 1 ml/100 g body weight. The administration form of the test item will be prepared daily before administration and during administration it will be mixed by the magnetic stirrer.
Vehicle: Aqua pro iniectione
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and Homogeneity of test formulation: Before the start of the study stability and homogeneity of test formulation will be determined at the test facility by HPLC.
Details on mating procedure:
After acclimatization females will be mated with males of established fertility (1 male and 2 females). Control of fertilization will be made by the help of the vaginal smears. Vaginal smears will be carried out after 24 hours of the first removing to male and then daily at the same time and the presence of sperms will be examined. Day 0 of pregnancy will be the day on which sperms in vaginal smear will be found. Pregnant females will be randomly selected to the experimental groups – about 20 pregnant females at each group

Examination of vaginal smears
Each morning during the mating period vaginal smears will be prepared from all the mated females. These smears will be examined for presence of spermatozoa. Vaginal smears will be prepared and examined
Duration of treatment / exposure:
from implantation (the 5th day after fertilisation) to one day prior to the day of scheduled euthanasia (the 19th day after fertilisation).
Frequency of treatment:
7 days per week
No. of animals per sex per dose:
DRF: 6 females and 3 males per dose level and control group
Main study: 20 pregnant females for each dose level (total number of animals before mating: 100 females and 25 males).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for DRFE OECD 414 were chosen according to the results of DRFE OECD 408
- Selection of animal species: laboratory rat has been chosen because the testing laboratory has long experience with this species and therefore the historical data on key parameters are available. As per the OECD TG 414, the preferred rodent species is the rat.

DOSE RANGE FINDING STUDY
After acclimatization females will be mated with males of established fertility (1 male and 2 females). Control of fertilization will be made by the help of the vaginal smears. Vaginal smears will be carried out after 24 hours of the first removing of male from the cage. Then daily at the same time the presence of sperms will be examined. Day 0 of pregnancy will be the day on which sperms in vaginal smear will be found out.
The test item will be administered in graduated dose levels to pregnant females from implantation (the 5th day after fertilisation) to one day prior to the day of scheduled euthanasia (the 19th day after fertilisation). The application form (test item in aqua pro iniectione ) will be administered to the stomach by gavage. The vehicle control group will be administered by aqua pro iniectione in the same volume. The test item concentration at single dose level will be adjusted so that the administered volume will be constant at all dose levels – 1 ml/100 g body weight. The animals will be treated 7 days per week at the same time (8.00 – 10.00 am).
On the 20th day of pregnancy, blood will be collected from the females under the light ether narcosis for haematological examination and then the females will be necropsied, macroscopic abnormalities will be recorded and the examination of reproductive parameters and mascroscopic examination of all foetuses will be performed. In the dose-range finding experiment the following haematological parameters will be determined: total erythrocyte count, total leucocyte count, total platelet count, haematocrit, haemoglobin concentration and average volume of erythrocyte.
The revision of the external surface of the body will be performed. During macroscopy,all orifices, the cranial, thoracic and abdominal cavities will be examined and uterus (incl. the cervix) will be removed and weighed. Ingravid uteri, the number of viable foetuses, number of dead foetuses, number of early resorption (implantation without recognizable embryo/foetus) and number of late resorption (dead embryo or foetus with external degenerative changes) will be recorded. The numbers of corpora lutea of both ovaries will be recorded. Each foetus will be examined for external and soft tissue alterations.
In DRFE the male rats will serve only for mating (they will not be administered the test item).
Time course of examinations in DRFE
mortality/viability: twice daily
body weight: 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
health condition control: daily
general clinical observations: daily - during the administration period (after application)
laboratory examinations:
- haematological examination: 20th day of pregnancy
- pathological examination of females: 20th day of pregnancy
- pathological examination of foetuses: 20th day of pregnancy

Examinations

Maternal examinations:
Health condition control
Control of the health condition will be performed once a day during animal handling before the application of the test item.

Body weight of females
The body weight of animals will be recorded on automatic balances with group average computing module. First weighing will be performed on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy. Weight increment will be computed as an average per group (in grams).

Food consumption
Food consumption will be determined at three-day intervals; it will coincide with the terms of body weight recording.

Clinical observations of the dams
During the administration period clinical observation will be made in order to record possible clinical effects of the test item application and all changes in behaviour of females. Females will be observed in natural conditions in their cages after application - once a day at the similar time each day. The observation time will be determined according to peak period of incidence of clinical effects observed during the dose-range finding experiment. The condition of the animals will be recorded including mortality, moribundity, pertinent behavioural changes, and all signs of overt toxicity

Pathological examination of females
Females will be killed one day prior to the expected day of delivery and macroscopic examination for any structural abnormalities and pathological changes will be performed. Females with signs of abortion or premature delivery will be also euthanized and macroscopically examined.

Biometry and histopathological examination of thyroid gland
The thyroid glands will be preserved in fixation medium from all pregnant females. The thyroid weight will be determined after fixation. For histopathological processing the routine histological paraffin technique with synoptic haematoxylin- eosin staining will be used.
The histopathology will be performed in all groups

Determination of thyroid hormones
Blood samples from the pregnant females will be assessed for serum levels of thyroid hormones (T3, T4, TSH) by ELISA kit.
The blood sampling will be performed from all females, because the pregnancy status is determined during the necropsy. The blood samples for this examination will be taken from orbital plexus by glass micropipette under the light ether narcosis before necropsy within a short timeframe (e.g. two hours) in the morning. The blood serum samples will be prepared by spontaneous coagulation and they will be centrifuged 10 minutes in centrifuge. Serum samples will be frozen until the thyroid hormone analysis.

Experimental data collection
Body weight: 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
Food consumption: 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
Health condition control: daily
Clinical observations: daily during the whole administration period (after application)
Laboratory examinations: blood collection for thyroid hormones determination – 20th day of pregnancy
pathological examination of females – 20th day of pregnancy
histopathological examination of thyroid gland – after processing
Ovaries and uterine content:
As soon as possible after euthanasia gravid uterus (incl. the cervix) will be removed and weighed. In uterus number of viable foetuses, number of dead foetuses, number of early resorptions (implantations without recognisable embryo/foetus) and number of late resorptions (dead embryo or foetus with external degenerative changes) will be recorded. Number of corpora lutea on ovaries will be found out.
Uteri of non-pregnant females will be examined to confirm the non-pregnant status (by the help of ammonium sulphide staining)
Fetal examinations:
Pathological examination of foetuses and anogenital distance (AGD) measurement
Sex and individual body weight of foetuses will be recorded. Each foetus will be examined for external alterations. The AGD will be measured in all live foetuses. For measuring digital calliper will be used.

One half of each litter will be examined for soft tissue alterations using careful gross dissection. External foetal sex will be compared with internal (gonadal) sex in these foetuses. These examination will be performed on the day of necropsy.
Second half of each litter will be processed and microscopically examined for skeletal alterations after Alizarin red staining
pathological examination of foetuses – 20th day of pregnancy
Statistics:
Statistical treatment of main study results
The software Statgraphic ® Centurion (version XVII, USA) will be used for statistical analysis. The data from control group will be compared with data from treated groups. The results statistically significant on probability level 0.05 will be indicated

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks on result:
other: to be determined

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks on result:
other: to be determined

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

The test item is administered in graduated dose levels to pregnant females from implantation (5th day after fertilisation) to one day prior to the day of scheduled euthanasia (19th day after fertilisation). The females are killed on the 20th day of gestation, uterine contents of females are examined and the foetuses are be evaluated for externally visible anomalies and for soft tissue and skeletal changes.

Categories Display