Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2016 - May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B102 06.11.2015
- Purity: 98.8 corr. area%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 143.0 - 190.9 g
- Housing: the rats were housed individually in Polycarbonate cages type III (floor area about 800 cm²)
- Diet (e.g. ad libitum): Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): potable tap water in water bottles
- Acclimation period: The animals were acclimated to the laboratory conditions between start of the study and first administration (GD 6).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance solutions were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in deionized water at room temperature over a period of 7 days had been verified prior to the start of the study
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. The following day was designated as “GD 1”.
Duration of treatment / exposure:
From implantation to one day prior to the expected day of parturition (GD 6-19).
Frequency of treatment:
Once a day
Duration of test:
ON GD20 all surviving dams were sacrified and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. During the administration period (GD 6-19) all animals were checked daily for any abnormal clinically signs before administration as well as within 2 hours and within 5 hours after administration.


DETAILED CLINICAL OBSERVATIONS: Yes
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. A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

BODY WEIGHT: Yes
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.
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).


FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
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.

POST-MORTEM EXAMINATIONS: Yes / No / No data
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
Ovaries and uterine content:
Weight of the unopened uterus, Number of corpora lutea, Number and distribution of implantation sites classified as: Live fetuses
Dead implantations:
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)
Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
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.
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:
Means and standard deviations were calculated. In addition the following statistical analysis will be carried out:
Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus,
number of corpora lutea, number ofimplantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight. DUNNETT-Test
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings. FISHERS EXACT-Test
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter.WILCOXON-Test
Indices:
Conception rate, preimplantation loss, postimplantation loss.
Historical control data:
Historical control data were available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of the low-, mid- and high-dose dams (5, 20 or 60 mg/kg bw/d) were in general comparable to the concurrent
control group throughout the entire study period. Consistent to the reduced food consumption value from GD 6 to 8, the average body
weightgain of the dams in test group 3 (60 mg/kg bw/d) was statistically significantly decreased onGD 6-8. During GD 6-8 10 out of 25 animals showed a body weight loss (up to -15.3 g) which recovered during GD 8-10 and thereafter. If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the mean body weight gain of the high-dose damswas comparable to the
concurrent control group. The decreased body weight gain of the highdosedams during GD 6-8 was considered to be treatment-related and adverse.
The average body weight gain of the low- and mid-dose dams (5 or 20 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was distinctly and statistically significantly lower in test group 3 (60 mg/kg bw/d - about 24% below the concurrent control value).
Furthermore, the carcass weight of the high-dose dams was also reduced (without attaining statistical significance, about 3% below controls). The decrease in corrected body weight gain was considered to be treatment-related and adverse.
The corrected body weight gain of test groups 1 and 2 (5 and 20 mg/kg bw/d) revealed no difference of any biological relevance to
the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test group 3 (60 mg/kg bw/d) was statistically significantly decreased on GD 6-8 (about 17% below control), but recovered afterwards and was comparable to the concurrent control group throughout the remaining study period (GD 8- 20). The decrease showed a high standard deviation value which might be caused by two individuals with very low food consumption values. However, if calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the mean food consumption of the high-dose dams was generally comparable to the concurrent control. Therefore, the reduced food consumption value during GD 6-8 was considered to be spontaneous and not treatment-related.
The mean food consumption of the dams in test groups 1 and 2 (5 and 20 mg/kg bw/d) was quite similar to the concurrent control throughout the entire study period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20 in dams of test group 3 (60 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin, hematocrit and mean
corpuscular hemoglobin concentration (MCHC) values were significantly decreased and absolute reticulocyte counts were increased.
Absolute reticulocyte counts were already statistically significantly higher in dams of test group 2 (20 mg/kg bw/d), but this was an isolated alteration of the red blood cell parameters and the mean was within the historical control range (absolute reticulocytes 77.4 – 187.0 giga/L). Therefore, increased absolute reticulocyte counts in dams of test group 2 were regarded as incidental and not
treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At gestation day 20, in dams of test group 3 (60 mg/kg bw/d) glucose and total bilirubin values were significantly increased and
creatinine values were significantly decreased. However, all means were within historical control ranges (glucose 4.76-5.81 mmol/L;
total bilirubin 0.67-1.66 μmol/L; creatinine 22.8-31.5 μmol/L). Therefore, all mentioned alterations were regarded as incidental and not
treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of all test groups (1-3) showed significantly increased relative liver weights, in animals of test groups 2 (20 mg/kg bw/d) and
3 (60 mg/kg bw/d) absolute liver weights were increased in addition. The increased absolute (+111%) and relative (+114%) liver weights of test group 3 animals and increased absolute (+8%) and relative (+9%) liver weights of test group 2 animals were assumed as
treatment related. A relation to treatment of the very slightly increase relative liver weights (+4%) in test group 1 animals is rather
unlikely, since only relative weight parameters were significantly changed and since the weight increase was very slight.
The increased absolute and relative weight of the spleen in test group 3 animals were assumed as treatment related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The duodenal wall was thickened in 13/25 animals of test group 3 (60 mg/kg bw/d) and 2/25 animals of test group 2 (20 mg/kg bw/d).
The thickening of duodenal wall was assumed as treatment related.
All other 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.

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
Details on maternal toxic effects:
For more details cf. "Attached background material"

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
haematology
gross pathology

Results (fetuses)

Fetal body weight changes:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Only one low-dose fetus with multiple external malformations was seen: female fetus No. 47- 10 (5 mg/kg bw/d) had an exophthalmus on its left eye, micrognathia and microstomia. These external findings were associated with multiple skeletal malformations concerning the skull.
The overall incidences of external malformations were comparable to those found in the historical control data and, therefore, assessed as incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Individual fetuses of test groups 1-3 (5, 20 or 60 mg/kg bw/d) had skeletal malformations. One low-dose fetus had associated external malformations. The finding ‘misshapentuberositas deltoidea’ was observed in one single fetus of test groups 2 and 3 each.
However, the finding can be found in the historical control data in a higher incidence and wasassessed as not treatment-related.
The number of these malformations adds up to a statisticallysignificantly higher value for the total skeletal malformation rate in the mid-dose group(mean of affected fetuses/litter: 2.3). However, the values are clearly inside the historical controlrange (HCD of affected fetuses/litter: 0.8%, andshowed no dose-dependency. Therefore, it was assessed as not treatment-related.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Three soft tissue variations were detected in all test groups including the control (0, 5, 20 or 60 mg/kg bw/d), i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences.

Fetal skeletal unclassified cartilage obvervations
Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups.. The observed unclassified cartilage findings were related to the skull, the ribs and
the sternum and, in most cases, did not show any relation to dosing. The incidence of ‘branched rib cartilage’ was statistically
significantly increased in test groups 1-3 (2.5%* / 3.7%** / 3.7%** affected fetuses per litter versus 0.0% in control) but was well within the historical control range (HCD of affected fetuses per litter: 2.1% [0.0 - 6.0]). Therefore, it was not assessed as treatment-related.
The overall incidences of skeletal unclassified cartilage observations in the substance- treated groups did not differ significantly from
the concurrent control group.
Details on embryotoxic / teratogenic effects:
External, soft tissue and skeletal malformations were noted in all test groups (0, 5, 20 and 60 mg/kg bw/d). Some fetuses were multiple malformed: one male control fetus had a situs inversus combined with an abnormal lung lobation. For one male fetus (5 mg/kg bw/d) severely malformed cervical vertebrae were recorded. The findings in one female fetus (5 mg/kg bw/d) consisted of multiple external malformations (exophthalmus, micrognathia, microstomia), comprising
severely malformed skull bones, while another male fetus (20 mg/kg bw/d) had a severely malformed vertebral column and/or ribs. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of the low-, mid- and high-dose groups. All of them are present in the historical control data of the rat strain. Thus, a relationship of these four cases of malformed fetuses to the treatment is not assumed. Three malformations, i.e. split scapula, malpositioned and bipartite sternebra and misshapen tuberositas deltoidea, were not related to dose and most of them can be found in the historical control data. An association of these findings to the treatment is also not assumed. The total number of the fetal skeletal malformations added up to a statistically significantly higher value in the mid-dose group (mean of affected fetuses/litter: 2.3) compared to control.
However, the values were clearly inside the historical control range (HCD of affected fetuses/litter: 0.8%, [0.0 - 3.0]; and showed no dose-dependency. Therefore,
it was assessed as not treatment-related and not adverse.
External variations did not occur in any of the fetuses of this study. Some soft tissue variations and a range of skeletal variations occurred in all test groups including the controls. None of the total incidences showed a relation to dosing. The majority of individual variations were 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.
One skeletal variation – wavy ribs - was dose-related increased without statistical significance and, in the high dose group, above the historical control range (mean of affected fetuses per litter: 11.0 %, HCD of affected fetuses per litter: 4.6% [0.8 - 8.7]). Wavy ribs were noted in 15 pups out of 11 litters (test group 3, 60 mg/kg bw/d). Wavy ribs represent a common finding in prenatal toxicity studies. Numerous studies have been conducted to study the reversibility of wavy ribs during postnatal development. They have shown that wavy ribs represent a reversible finding. The finding represents minor departures from normal morphology
or slight delays of ossification, which did not affect morphology. In the high-dose group they occur in the presence of maternal toxicity. Thus, the marginal increase of the finding above the background rate of the used rat strain is considered neither as adverse nor of toxicological relevance.
No unclassified external and no 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 all test groups (0, 5, 20 and 60 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.
The incidence of ‘branched rib cartilage’ was statistically significantly increased in test groups 1-3 (2.5%* / 3.7%** / 3.7%** affected fetuses per litter versus 0.0% in control) but was well within the historical control range (HCD: 2.1% [0.0 - 6.0]). Therefore, it was not assessed as a treatment-related, adverse event. The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.
Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to a dose of 60 mg/kg bw/d.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

For more details cf. "Attached background material"

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 3- Hexyne-2,5-diol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity in the mid- and high-dose group (20 and 60 mg/kg bw/d), such as decreased (corrected net) body weight gain, a normocytic, hypochromic, regenerative anemia based on altered hematology parameters (associated with increased spleen weights at 60 mg/kg bw/d), and thickening of duodenal wall in pathology. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 5 mg/kg bw/d.
Under the conditions of the present study the test substance is not teratogenic in rats. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/d, the highest dose tested.