Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2017 - August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
waxy/ yellowish

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Time-mated Wistar rats (Crl:WI[Han]) were supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, at an age of about 10-12 weeks. Only animals free from clinical signs of disease were used for the investigations.

During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²). Individual housing is preferred over group housing as the close individual monitoring of food and water consumption as well as of clinical signs of toxicity in pregnant animals is crucial during this period of increased sensitivity. In addition, the control for signs of abortion or fetal loss can only be done in a reliable fashion with single-housed animals.
Dust-free wooden bedding was used in this study. For enrichment, wooden gnawing blocks were offered (Typ Lignocel® block large, supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany). The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a range of temperature of 20-24°C and a range of relative humidity of 30-70%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study. The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h). The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles)
were available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (corn oil) in the same way. The volume administered each day was 4 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of the analysis of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory.
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.
Frequency of treatment:
once a day
Duration of test:
On GD 20, the females were sacrificed in a randomized order and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Dose / conc.:
7 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE RATIONALE:
In a reproduction/developmental toxicity screening test (OECD 422), Diisopropanol-p-toluidin was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 2.5 mg/kg bw/d (test group 1), 10 mg/kg bw/d (test group 2) and 40 (males)/ 20 (females) mg/kg bw/d (test group 3). Almost all (9 out of 10) females of test group 3 (40 mg/kg bw/d) showed severe tremors on premating day 1 and three females (40 mg/kg bw/d) were found dead on premating day 1. After the death of these animals receiving 40 mg/kg bw/d, the dose level was reduced to 20 mg/kg bw/d (only in females) from study day 2 onwards. No further findings were observed.

Based on the lethal / severe findings in the OECD 422 and at the request of the sponsor, the following dose levels were chosen for the present prenatal developmental toxicity study in Wistar rats:
- 2.5 mg/kg body weight/day: as low-dose level
- 7 mg/kg body weight/day: as mid-dose level
- 20 mg/kg body weight/day: as high-dose level

The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:
Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

Clinical symptoms
A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

Food consumption
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

Body weight data
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results

Corrected (net) body weight gain
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).
Ovaries and uterine content:
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in
randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology.
The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI
from uteri from apparently non-pregnant animals and the empty uterus horn in the
case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the
uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams
and the gestational parameters (except of gross pathology including organ weights) were conducted
by technicians unaware of treatment group in order to minimize bias. For this purpose
animal numbers were encoded.

These data were used to calculate conception rate and pre- and postimplantation losses.
Fetal examinations:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus
and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded.
Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
Statistics:
see attached background material
Historical control data:
see attached background material

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 2.5, 7 or 20 mg/kg bw/d during the entire study period.
For one dam of the low-dose group (No. 36 - 2.5 mg/kg bw/d) vaginal hemorrhage before and after treatment (>2h<5h) was recorded on GD 19. Since it was only one affected animal and there was no relation to dose, it was not assessed as treatment-related.

Please see also attached summary document.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 2.5, 7 or 20 mg/kg bw/d).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (2.5, 7 and 20 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
The statistically significantly increased body weight gain value in test group 3 during GD 19-20 was assessed as incidental.

Please see also attached summary document.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the high-, mid- and low-dose dams (20, 7 and 2.5 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.
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
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):
Uterus weight
The mean gravid uterus weights of the animals of test groups 1-3 (2.5, 7 and 20 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Weight of the placentae
The mean placental weights of test groups 1-3 were comparable to the concurrent control group.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Two spontaneous findings were noted in two individual females of test group 3 (20 mg/kg bw/d), i.e. dilated renal pelvis (No. 77) and discolored kidneys (No. 78). No necropsy findings which could be attributed to the test substance were seen in any dam.

Please see also attached summary document.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Reproduction data
The conception rate was 96% in the control and the mid-dose groups (0 and 7 mg/kg bw/d) and 100% in the low- and high-dose groups (2.5 and 20 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

For historical control data see also attached background material.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment-related, advers effects were observed
Remarks on result:
other: highest tested dose

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (2.5, 7 and 20 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External malformations were detected in test groups 1 (kinked tail) and 2 (multiple external malformations) (2.5 and 7 mg/kg bw/d). In one case, these external malformations were associated with skeletal malformations. None of these malformations were assessed as treatment-related since they were not related to dose and the finding multiple external malformations can be found in the historical control data at comparable incidences.
The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

For historical control data see also attached background material.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were noted in one fetus, each, in test groups 1 (cleft sternum) and 2 (multiple skeletal malformations) (2.5 and 7 mg/kg bw/d). One male fetus of the mid-dose group had multiple skeletal findings concerning skull, forelimbs, sternum and vertebral column. The incidences of the total skeletal malformations were not dose-dependent. The malformations were not assessed as treatment-related since they occurred in single fetuses without relation to dose and were within the range of the respective historical control data.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

The increased incidences of skeletal variations were not related to the dose and/or they were inside the historical control range (see attached background material). Therefore, they are not considered as treatment-related.

For historical control data see also attached background material.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment-related, advers effects observed
Remarks on result:
other: highest tested dose

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study, the test substance Diisopropanol-p-toluidin was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.
Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle. Neither clinical examinations nor determination of food consumption and body weights revealed any relevant difference between animals receiving 2.5, 7 or 20 mg/kg bw/d Diisopropanol-p-toluidin and the control. No differences of toxicological relevance between the control and the treated groups were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.
Executive summary:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Diisopropanol-p-toluidin to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 20 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 20 mg/kg bw/d.