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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
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:
liquid

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean values of the low-, mid- and high-dose samples (1, 4 and 12 mg/kg bw/d) were below the expected range of 90% to 110% of the nominal concentrations. For the low-, mid- and high-dose, three samples each were measured. One out of
three analytical value of the high-dose samples was within the expected range (sample no. 10: 91.2%). The mean values of 54, 65 and 75% of the nominal concentrations correspond to an actual low-, mid- and high dose of 0.5, 2.6 and 9 mg/kg bw/d, respectively. This did not affect the validity of the study. However, in the robust study summary, the nominal doses were used.
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 mg/kg bw/day (nominal)
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
Dose rational
In a maternal toxicity study, 7 pregnant Wistar rats per group received doses of 0, 8 and 25 mg/kg bw/d 2-propyn-1-ol orally by gavage over 14 days from gestation day (GD) 6 through GD 19. The study aimed to select the dose levels for the subsequent OECD 414 study.
Clinical examinations including body weight, food consumption, uterus and carcass weight and clinical pathology (clinical chemistry and hematology) were assessed and showed no relevant changes besides lower total protein levels (albumins and globulins) at 25 mg/kg bw/d. At 25 mg/kg bw/d, a statistically significant increase in absolute (+29%) and relative (+30%) liver weights was observed. Histopathological evaluation of these animals showed hypertrophy (n=6), nuclear inclusions (n=2), single cell necrosis (n=2) and multifocal necrosis (n=1) in liver. At 8 mg/kg bw/d, organ weights showed a non-statistically significant increase in absolute (107%) and a statistically significant increase in relative (110%) liver weights. A histopathological examination of the livers of that group showed no effects.

Due to the clear adverse findings in the livers at 25 mg/kg bw/d, half of the dose level was selected for the OECD 414 as high dose: 12 mg/kg bw/d. This dose was assumed to be appropriate since, it was higher than 8 mg/kg bw/d with slight findings only, but below the dosage of 25 mg/kg bw/d resulting in pronounced maternal toxicity.

Based on the available data the following dose levels were chosen for the present prenatal developmental toxicity study in Wistar rats:
1 mg/kg body weight/day: as low-dose level
4 mg/kg body weight/day: as mid-dose level
12 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:
CLINICAL EXAMINATIONS OF THE DAMS
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).

TERMINAL EXAMINATIONS OF THE DAMS
Cesarean section
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, special attention being given to the reproductive organs. 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 were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.

Pathology
Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Liver
The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Liver

No further examinations or procedures were performed in the study.
Ovaries and uterine content:
see maternal examinations
Fetal examinations:
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
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 1, 4 or 12 mg/kg bw/d during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 1, 4 or 12 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (1, 4 and 12 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1, 2 and 3 (1, 4 and 12 mg/kg bw/d) was 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:
effects observed, treatment-related
Description (incidence and severity):
When compared to control group 0 (set to 100%), the mean absolute weights of the livers were significantly increased in test groups 2 and 3. When compared to control group 0 (set to 100%), the mean relative weights of the livers were significantly increased in test groups 2 and 3.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data. The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d):
• female No. 8 – not pregnant

Test group 1 (1 mg/kg bw/d):
• females Nos. 26, 42 and 43 – not pregnant

Test group 2 (4 mg/kg bw/d):
• female No. 68 – not pregnant

Test group 3 (12 mg/kg bw/d):
• females Nos. 76 and 90 – not pregnant
Other effects:
no effects observed
Description (incidence and severity):
Weight of the placentae
The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
12 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
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 (1, 4 and 12 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:
effects observed, non-treatment-related
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 control group. The marginally but statistically significantly higher male fetal weight in test group 3 (3.8 g) is within the historical control range (HCD: 3.7 g, [2.8-4.5]) and, therefore, not considered to be of any biological relevance.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus each in test groups 1, 2 and 3 (1, 4 and 12 mg/kg bw/d) had external malformations, In two cases, these external malformations were seen in combination with either soft tissue or skeletal malformations. None of these malformations were considered to be related to the treatment since they showed no relation to dose and were scattered observations in individual fetuses with no specific ontogenetic pattern. The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was comparable to the historical control data.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were noted in three fetuses of the low-dose group (1 mg/kg bw/d) and one fetus of the mid-dose group (4 mg/kg bw/d). One low-dose fetus had associated external malformations. Although the overall incidence of skeletal malformations was statistically significantly increased in test group 1, the individual malformations were without relation to dose and the overall incidences were clearly within the historical control range (mean value: 0.9 %, range: 0.0 - 3.1%). Thus, the findings were not considered to be treatment-related.

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 in the majority of cases without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

The finding ‘incomplete ossification skull; unchanged cartilage’ was statistically significantly increased in affected fetuses/litter in test groups 2 and 3 and in litter incidence (39%) in test group 3. However, all values were clearly inside the historical control range (HCD of litter incidence: mean 28.5%, [4 - 56%]). Therefore, the finding was not assessed as treatment-related. The findings ‘dumbbell ossification of thoracic centrum’ and ‘misshapen sacral vertebra’ were not related to dose and the mean values were below or close to the mean of the historical control data. Therefore, both findings were not assessed as treatment-related. The finding ‘incomplete ossification of basisphenoid’ was statistically significantly increased in test group 1 (litter incidence: n=21**, 95%). The value was slightly outside of the range of the historical control data (mean 51.4%, [20.8 - 87.5%]). However, the increase was not dose-related and, therefore, it was not assessed as treatment-related.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dose and were not assessed as treatment-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus each of test groups 2 and 3 (4 and 12 mg/kg bw/d) had soft tissue malformations. For one affected fetus, these soft tissue findings were seen in combination with an external malformation. The finding right-sided aortic arch was not related to dosing and, therefore, not assessed as treatment-related. The malformations hydronephrosis and hydroureter were only observed in one individual fetus of one single litter. Since no other fetus or litter of the same or lower test groups were affected, the findings were, therefore, assessed as spontaneous and
not treatment-related. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.

Four soft tissue variations, i.e. malpositioned subclavian artery origin, short innominate, dilated renal pelvis and dilated ureter, were detected in all test groups including the controls. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups and, therefore, not considered biologically relevant.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 12 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

There were noted external, soft tissue and skeletal malformations in test groups 1, 2 and 3 (1, 4 and 12 mg/kg bw/d). However, the low-dose mean affected fetuses per litter (mean 1.2%) were statistically significantly different from concurrent control. All of these individual malformations (mandibular micrognathia, microstomia, severely malformed skull bones, misshapen tuberositas deltoidea) of test group 1 were scattered observations in individual fetuses of different litters. Some of them are present in the historical control data. Due to the absent relation to dose, an association of these findings to the treatment is not assumed. Three fetuses were multiple malformed in each test group. Male low-dose fetus No. 47-10 (1 mg/kg bw/d) had a mandibular micrognathia and microstomia, comprising severely malformed skull bones during skeletal examination. For male mid-dose fetus No. 57-03 (4 mg/kg bw/d) a gastroschisis and a right-sided aortic arch were recorded, while high-dose female fetus No. 98-02 (12 mg/kg bw/d) had a hydronephrosis and a hydroureter. No ontogenetic pattern is recognizable for these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. Most of them can be found in the historical control data of the rat strain. An association of these,findings to the treatment is not assumed. Further malformations, i.e. meningocele (in test group 3) and misshapen tuberositas deltoidea (in test group 2), were observed in individual fetuses, were not related to dose and can mostly be found in the historical control data. An association of these findings to the treatment is also not assumed.

External variations did not occur in any of the fetuses of the study. Some soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. In test groups 1-3, all statistically significantly increased fetal skeletal variations were within the historical control range. None of the incidences showed a relation to dose. The skeletal variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.

No unclassified external and unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0, 1, 4 and 12 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest dose tested (12 mg/kg bw/d).

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-propyn-1-ol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 12 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 12 mg/kg bw/d.
Executive summary:

In a prenatal developmental toxicity study, the test substance 2-propyn-1-ol 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. Generally, clinical observations including food consumption and body weight/body weight gain revealed no toxicologically relevant differences between the animals receiving 1, 4 and 12 mg/kg bw/d 2-propyn-1-ol and controls.

Regarding pathology, the target organ was the liver. Increased absolute and relative liver weights in test groups 2 and 3 were regarded as treatment-related but not adverse. They were above the historical control data (absolute: 10.81 – 11.45 g; relative: 4.55 – 4.73). Since the weight increase was only minimal, it was regarded as an adaptive change.

No differences of toxicological relevance between the control and the treated groups (1, 4 or 12 mg/kg bw/d) 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.