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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-02-04 to 2020-03-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one
EC Number:
403-830-5
EC Name:
6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one
Cas Number:
89331-94-2
Molecular formula:
C35H36N2O3
IUPAC Name:
6'-(dibutylamino)-3'-methyl-2'-(phenylamino)-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 189-236 g
- Housing:
Before mating: 1-2 females per cage
1-2 males per cage
During mating: 1 male and 1-2 females / cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum, ssniff® SM R/M " breeding and maintenance autoclavable complete feed (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum, tap water from municipal supply
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 – 24.1
- Humidity (%): 30 - 52
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared in the formulation laboratory of the Test Facility not longer than 3 days before the administration. Formulations were stored in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore 1% Methylcellulose was used.
- Concentration in vehicle: 6.25, 25, 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: 1908-4485
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five samples from different places were taken from each concentration. Similarly, five samples were taken from the vehicle (Control group) and analyzed. The suitability of the chosen vehicle for the test item at the intended concentrations was verified up front. WinCon-2 recovery was 104 and 101 % of nominal concentrations at 5 and 100 mg/mL in 1% Methylcellulose, respectively. WinCon-2 proved to be stable at room temperature for 24 hours (mean recovery was 102 % of starting concentration at 5 mg/mL and 103 % at 100 mg/mL) and at 5 ± 3°C for 3 days (recovery was 102 % of starting concentration at 5 mg/mL and 99 % at 100 mg/mL). A separate analytical report provided these results.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: 2 - 4 hours
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from gestational day 5 to 19
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25 to 26
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting was based on the results obtained in a study with the same test item 6' -(Dibutylamino)-3' -methyl-2' -phenylaminospiro (isobenzofuran-l(3H),9'-(9H)-xanthene)-3-one) “ODB-2 a study in the rat of reproductive function on one generation by oral administration” performed at Huntingdon Research Centre Ltd. 1993 (Guideline for Testing of Chemicals, No. 415, adopted May 1983.) where the dosages of 62.5, 250 or 1000 mg/kg/day did not lead to any effect on the growth and reproductive capacity of male and female rats or development of their offspring. The dosage of the test item at which no signs of toxicity was recorded was therefore considered to be 1000 mg/kg/day which is the limit dose according to the OECD 414 test guideline.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (signs of morbidity and mortality were checked twice daily)
- Cage side observations included check of behavior and general condition, duration and severity of the clinical signs and observations for signs of morbidity and mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Uterus with cervix and ovaries, organ weight of thyroid glands together with the parathyroid glands

OTHER:
Examination of placental signs: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.

Blood Collection and Determination of Serum Levels of TSH, FT3 and FT4: Blood samples collected for TSH and Thyroid Hormones (FT3 and FT4) measurements were drawn from all sperm positive females from the
sublingual vein in the morning on the day of necropsy within a short timeframe (not exceeding two hours).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually. The statistical evaluation of data was performed with the 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 is detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If the result of the Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Dams or litters were excluded from the data evaluation in cases of:
- Non pregnant females (no implantation, no corpora lutea), total exclusion
- Disease or death of the dam unrelated to the treatment (total exclusion)
Although these animals were excluded from the data evaluation the Study Report contains all data of these animals, too. A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses.
Historical control data:
The results were compared to the laboratory's historical control data (refer to "Historical control data" in "Overall remarks, attachments").

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs observed in the females of all dose groups.
1000 mg/kg bw/day: Clinical signs were only observed in 1 moribund female which was euthanized on GD 16 (1/26; unrelated to the test item).
Refer to Appendix 2.1 ("Experimental summary tables" in "Overall remarks, attachments").
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
62.5 mg/kg bw/day: 1/25 sperm positive females died due to a technical reason (mis-gavage).
1000 mg/kg bw/day: 1/26 females was moribund and therefore euthanized on GD 16 (unrelated to the test item).
Refer to Appendix 1 ("Experimental summary tables" in "Overall remarks, attachments").
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight, body weight gain in the different periods as well as corrected body weight of the females was similar in all groups.
Refer to Appendix 3.1 ("Experimental summary tables" in "Overall remarks, attachments").
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no significant differences in the food consumption of the dams in the dose groups.
Refer to Appendix 5.1 ("Experimental summary tables" in "Overall remarks, attachments").
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
The free thyroid hormone (FT3 and FT4) and TSH levels were similar in all dose groups.
Refer to Appendix 6.1 ("Experimental summary tables" in "Overall remarks, attachments").

There were no effects of treatment with the test item in the control, 62.5 and 1000 mg/kg bw/d dose groups. The mean thyroid weight was statistically significantly lower (p<0.05) in the 250 mg/kg bw/day group, however without a dose response. Hence, it was not attributed to any effect of the test item. Thyroid weight was considered to be not effected by treatment.
Refer to Appendix 7.1 ("Experimental summary tables" in "Overall remarks, attachments").

The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Cyst forming was recorded for the thyroid gland of one control female.
Refer to Appendix 8.1 ("Experimental summary tables" in "Overall remarks, attachments").
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item in the control, 62.5 and 1000 mg/kg bw/d dose groups. The mean thyroid weight was statistically significantly lower (p<0.05) in the 250 mg/kg bw/day group, however without a dose response. Hence, it was not attributed to any effect of the test item.
Thyroid weight was considered to be not effected by treatment.
There were no test item related differences in gravid uterine weight among the dosing groups.
Refer to Appendix 7.1 and 4.1 ("Experimental summary tables" in "Overall remarks, attachments").
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A dilated renal pelvis was found in two females each in the 62.5, 250 and 1000 mg/kg bw/day dose groups and one female in the control group. This finding was not attributed to the treatment considering the lack of dose response. Diaphragmatic hernia as a developmental disorder (hence not related to the treatment) was observed in one female in the 1000 mg/kg bw/day group. The uterine horn was filled with fluid to distention in one female in the 1000 mg/kg bw/day group. Considering the low incidence, this was not attributed to an effect of the test item. Blood was found in the uterine horn of one dam in the 62.5 and one in the 250 mg/kg bw/day dose group.
Refer to Appendix 2.1 ("Experimental summary tables" in "Overall remarks, attachments").
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Cyst forming was recorded for the thyroid gland of one control female.
Refer to Appendix 8.1 ("Experimental summary tables" in "Overall remarks, attachments").
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no increase indicated in the mean percent of pre- and post-implantation loss (early embryonic, late- and fetal death) at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no test item related effect at any dose level.
Refer to Appendix 1 ("Experimental summary tables" in "Overall remarks, attachments").
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment related differences in the body weight of the fetuses.
Refer to Appendix 10.1 ("Experimental summary tables" in "Overall remarks, attachments").
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No effects observed. Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex distribution of the fetuses were not influenced by the treatment.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The number of evaluated litters/fetuses was 25/270, 22/235, 23/242 and 22/227 in the control, 62.5, 250 and 1000 mg/kg bw/day groups respectively. There was no dose related increase in the incidence of growth retardation.
Refer to Appendix 9.1, 9.2 and 10.1 ("Experimental summary tables" in "Overall remarks, attachments").
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no treatment related differences in the absolute or normalized ano-genital distance at any dose level.
Refer to Appendix 10.1 ("Experimental summary tables" in "Overall remarks, attachments").
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was 1/22 litters with malformed fetuses in the 1000 mg/kg bw/day groups (edema and multiply malformed; 0733152/1). There were no statistically significant differences in the incidence of malformations over all dosing groups.
Refer to Appendix 11.1 and 12.1 ("Experimental summary tables" in "Overall remarks, attachments").
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 1/25, 1/22, 1/23 and 1/22 litters with malformed fetuses (in the control, 62.5, 250 and 1000 mg/kg bw/day groups).
Control group: Scapula bent; radius bent, humerus slightly bent, thicker, femur short, bent (0617194/1)
62.5 mg/kg bw/day: humerus slightly shorter, radius slightly bent (0831119/5)
250 mg/kg bw/d: Thoracic centrum asymmetric, bipartite, cartilage dumb-bell shaped (0513160/7)
1000 mg/kg bw/day: Scapula bent, Mis developed mandible (agnathia) (0733152/1)
There were no statistically significant differences in the incidence of malformations over all dosing groups.

Refer to Appendix 11.1 and 14.1 ("Experimental summary tables" in "Overall remarks, attachments").
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral malformations were reported.
Refer to Appendix 11.1 and 13.1 ("Experimental summary tables" in "Overall remarks, attachments").
Details on embryotoxic / teratogenic effects:
Fetal examinations

The number of litters with malformations was one in all groups. There was no increase of variations during fetal examinations indicated:

External examination: There was no increase indicated in the incidence of external variations and no effect of treatment with the test item was observed. Body weight retardation of the fetuses (below 2.85 g for males and 2.77 g for females) as well as hyperflexion of the forelimbs (not proved skeletally) were evaluated as external variations. There was no dose related increase in the incidence of growth retardation. The incidence was statistically significantly (p<0.05) lower in the 62,5 mg/kg bw/day group. Hyperflexion of the forelimbs (not proved at skeletal examination) was found in two control fetuses (besides the above discussed fetus No.: 0733152/1 in the high dose). One fetus was found as slightly pale in the 250 mg/kg bw/day group, which was not considered as test item related considering the low incidence. Considering the lack of dose response or the low incidence the above variations were not attributed to the treatment.

Visceral examination: There were no statistically significant increases in the incidence of visceral variations and no effect of treatment with the test item was indicated. The variations/abnormalities were statistically significantly lower (p<0.05) in the 250 mg/kg bw/day group.
Slightly dilated third brain ventricle and lateral ventricles were recorded in the control group (5 fetuses) and high dose (2 fetuses). Hydroureter were found in all groups without a dose response. Hydroureter accompanied with dilated renal pelvis was observed in one single fetus in the 1000 mg/kg bw/day group. A slightly malpositioned kidney was found in one control and one high dose fetus. Slightly smaller kidney was recorded for one control fetus. Considering the lack of dose response or the low incidence the above variations were not attributed to the treatment.

Skeletal examination: There was no statistically significant increase in the incidence of skeletal variations and no effect of treatment with the test item was indicated. Statistically significant decreases were seen sporadically in the dose groups (3 ossified sternebra in the 62.5, incompletely or not ossified pubic or ischia in the 62.5, 250 and 1000, less than 3/3.5 ossified metacarpal/metatarsal in the 62.5 and 1000 mg/kg bw/day groups, each significance p<0.05). It was noticed that the occurrence of skeletal variations in the control group was nearly twice as high as in the dosing groups (18.8 fetuses with abnormalities compared with 7.9 – 14.3 fetuses in the dosing groups). Skeletal variations were mainly observed in 2 litters (0210157, 0617194) with between 83 % and 100 % of the fetuses having at least one variation. Thus, the sporadic decrease in the dose groups is mainly based on the higher incidence of skeletal variations observed in the control group.

Refer to Appendix 11.1 and 14.1 ("Experimental summary tables" in "Overall remarks, attachments").

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with WinCon-2 at the dose levels of 62.5, 250 and 1000 mg/kg bw/day did not cause any maternal effects. Fetal examinations did not reveal any adverse effects on the fetuses.
Executive summary:

WinCon-2 was examined for its possible prenatal developmental toxicity. Groups of 25, 26 and 26 sperm-positive female Han: of Wistar origin rats were treated with WinCon-2 by oral administration daily at three dose levels of 62.5, 250 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% methylcellulose. The treatment volume was 10 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. WinCon-2 in 1% methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 oC) at the concentrations of 5 and 100 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 102 and 108 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.


 


Results


Evaluated litters


In total, on gestation day 20 there were 25, 22, 23 and 22 evaluated litters in the control, 62.5, 250 and 1000 mg/kg bw/day group, respectively.


 


Mortality, clinical signs, necropsy findings


None of the females died before scheduled necropsy due to the test item and there were no test item related clinical signs recorded in the dose groups. No treatment related necropsy findings were observed.


 


Food consumption, body weight


Food consumption and body weight development were not affected by the test item.


 


FT3, FT4 and TSH level


The test item did not influence the thyroid hormone levels in any of the treated animals.


 


Thyroid weight


There were no test item related differences in thyroid weight among the dosing groups.


 


Histopathology of thyroid glands


The treatment did not result in histological changes of the thyroid gland in any of the dose groups.


 


Intrauterine mortality


Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment.


 


Fetal examinations


There were no test item related adverse effects on the fetal- and placental weight, ano-genital distance, external and visceral development of fetuses. The number of litters with malformations was one in all groups. There was no increase of variations during fetal examinations indicated.


 


Conclusion


Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with WinCon-2 at the dose levels of 62.5, 250 and 1000 mg/kg bw/day did not cause any maternal effects. Fetal examinations did not reveal any adverse effects on the fetuses.


Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:


NOAEL (maternal toxicity): 1000 mg/kg bw/day


NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day