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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:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
By error, the brains of control fetuses A004-6 and A005-12 were not assessed by mid-coronal slice. Evaluation: Sufficient control data was available for a proper evaluation. The study integrity was not adversely affected by the deviations.
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear colourless liquid
Details on test material:
stored at room temperature

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Female albino rabbits, New Zealand White (NZW) strain (SPF-Quality), from a non-inbred laboratory colony. Nulliparous, non-pregnant and untreated females were used at initiation of the study.
Stock male NZW rabbits were used for mating with the females. These males were adult and proven fertile. After mating these males were placed back in their stock and might be used for future studies.

Source: Charles River Deutschland, Sulzfeld, Germany.
Age at delivery: Females were approximately 17 weeks.
Acclimatization: At least 5 days prior to pairing.
Health inspection: upon receipt of the animals.


Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
The temperature range in the animal room of this Test Facility (18-24°C) was slightly higher than the range proposed in the OECD 414 guideline (18±3°C). Laboratory historical control data did not indicate any effect of this slightly higher range on the animals condition.
Accommodation: Females were individually housed in labelled cages with perforated floors and shelters.
Diet: Free access to pelleted diet for rabbits. In addition, pressed hay and wooden sticks were provided during the study period.
Water: Free access to tap-water.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Oral gavage, using a plastic catheter attached to a plastic disposable syringe. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Accuracy and homogeneity of formulations were confirmed by chemical analyses.

Prior to start of the main study, stability of the test item in the vehicle was demonstrated as part of the method development and validation study.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Days 6 to 28 post-coitum, inclusive
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
7 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose levels
In a dose range finding study in which dose levels of 7, 20 and 60 mg/kg were tested, slight toxicity was noted at 60 mg/kg (reduced maternal body weight gain and food consumption, reduced faeces production, and reduced fetal body weight). In the preceding tolerability study mortality occurred at 120 mg/kg. Based on these findings dose levels of 7, 20 and 60 mg/kg were selected for the main study.

Examinations

Maternal examinations:
Mortality / Viability: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7). The circumstance of any death was recorded in detail.

Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights: Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

Food consumption: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

Water consumption: Subjective appraisal was maintained during the study.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Animals found dead or sacrificed before planned necropsy were subjected to relevant examinations of the ovaries and uterine horns.
Fetal examinations:
External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (=60mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from all groups.

The specimens were archived in glycerin with bronopol as preservative.

Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss, post-implantation loss, viable fetuses affected/litter
Historical control data:
Yes, general pregnancy performance, fetal malformations and variations (attached)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Reduced faeces production was noted in all groups, including the control group, but a dose related increase in incidence and severity was seen in all groups treated with the test item.
Additional treatment-related findings at the highest dose level (60 mg/kg) consisted of piloerection in 3/22 animals and a lean appearance in 5/22 animals. At the lower dose levels (7 and 20 mg/kg) no additional toxicologically relevant clinical signs were noted.

In the low dose group, one female was noted with red staining of the vagina on Days 16 and 17 post-coitum. She and another females from this group had also red fluid on the manure tray on Days 25 and 28 post-coitum. As these findings were limited to the low dose group, and since these 2 females had normal litters, no toxicological significance was attached to this observation. Both findings are seen more often for rabbits in this type of study.

Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study, did not show any apparent dose-related trend, were limited to isolated cases and/or were also observed during the pre-treatment period. Therefore, these signs were considered to be of no toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 60 mg/kg four females were killed in extremis on Days 16 or 17 post-coitum. All females had a lean appearance and slightly to severely reduced production of faeces (which was pale in one animal). Additionally, two animals showed piloerection, one was lethargic and another one had a pale skin. All females showed severely reduced to no food intake, generally from postcoitum Day 6 onwards, resulting in significant body weight loss. Animals were affected with a body weight loss of 9-13% on Day 15 post-coitum.

At 20 mg/kg, one female died spontaneously on Day 25 post-coitum. This female showed no clinical signs of toxicity and her body weight development and food consumption were normal. Macroscopic findings in this female included lung lesions (many dark red foci, perforation(s) in the right caudal lobe) and dark red fluid in the thoracic cavity, both indicative for an oral gavage accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain at 60 mg/kg was severely reduced from the start of treatment onwards, resulting in statistically significantly reduced mean body weights from post-coitum Day 12 onwards. Corrected body weight gain (for uterus) was also statistically significantly reduced
at 60 mg/kg (-10.5%, versus -3.3% in the control group). Body weight loss occurred in more than half of the animals, most severely in the females that were killed in extremis or delivered early. Body weight gain of the 60 mg/kg females that did not lose weight was generally at the lower end or slightly below the concurrent control range.

At 7 and 20 mg/kg, mean body weight gain was slightly (not statistically significantly) reduced from post-coitum Day 15 onwards. Changes as compared to the concurrent control group were relatively slight, reaching statistical significance on post-coitum Days 18
(20 mg/kg) and 21 (7 and 20 mg/kg) only. A few females of these groups occasionally showed slight body weight loss (generally between 1 and 5%, up to 9% in one female at 7 mg/kg). Corrected body weight gain (for uterus) at 7 and 20 mg/kg was unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated at 60 mg/kg had statistically significantly reduced food consumption between Days 6-24 post-coitum (absolute values) and Days 6-21 post-coitum (relative to body weight). Changes were most pronounced in the second week of treatment (Days 12-15 post-coitum), when
mean food intake in the high dose group was approximately 60% lower as compared to the mean of the concurrent control group. Thereafter, food consumption at 60 mg/kg remained low but the mean differences from controls were smaller and no longer statistically significant. This could be explained by the lower food consumption of (a few) control females between Days 21-29 post-coitum (compared with their food consumption in the preceding periods), which resulted in lower control group mean values.

At 7 and 20 mg/kg, mean food consumption was slightly lower as compared to the concurrent control group, reaching statistical significance for the 20 mg/kg group from Day 12-15 postcoitum (absolute and relative) and the 7 mg/kg group from Day 18-21 post-coitum (absolute
only). As changes were relatively slight and in the absence of a clear dose-related trend, no toxicological relevance was attached to this finding.

In general, relatively large variations in food consumption occurred among individual animals of all groups (including controls). This is more often seen for rabbits of this age and strain which are housed and treated under the conditions in this study.
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:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The only test item -related gross finding at necropsy was the emaciated appearance noted in two high-dose females treated at 60 mg/kg (related to the test item-induced growth retardation).

One female in the low dose group had agenesis of her right uterus horn. This was a congenital abnormality and by no means related to treatment that started after the animal had reached adolence.

The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show any apparent dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
The numbers of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and test groups were similar and in the range of normal biological variation.

There were 1, 2, 3 and 0 non-pregnant females in the control, 7, 20 and 60 mg/kg groups, respectively. All remaining females were pregnant. As in the high dose group four females were euthanized preterm, and in the mid dose group one female died due to an oral gavage accident, the number of litters available for fetal evaluation was 21, 20, 18 and 18, respectively. This includes the high dose female that had an early delivery on Day 29 post-coitum. She delivered 9 dead fetuses.

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
One female at dose 60 mg/kg b.w. had an early delivery on Day 29 post-coitum (the morning of planned necropsy). The female had a body weight loss of 21% and 23% before and after correction for gravid uterus weight, respectively. She delivered 9 dead fetuses.
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:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): 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 (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weight was adversely affected at 60 mg/kg as indicated by the significantly lower weights of male and female fetuses (both sexes combined: 34.5 gram at 60 mg/kg versus 41.7 gram in controls).

At 7 and 20 mg/kg, mean fetal body weights (males and females separately and both sexes combined) were unaffected by treatment. All values remained within the available historical range.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Fetal body weight was adversely affected at 60 mg/kg as indicated by the significantly lower weights of male and female fetuses (both sexes combined: 34.5 gram at 60 mg/kg versus 41.7 gram in controls).

At 7 and 20 mg/kg, mean fetal body weights (males and females separately and both sexes combined) were unaffected by treatment. All values remained within the available historical range.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on external morphology following treatment up to 60 mg/kg.

The only external malformation observed in this study was noted in fetus at the high dose level (60 mg/kg). This fetus had a carpal flexure for which no skeletal cause was found.
Due to its single occurrence, this malformation was considered a chance finding.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related skeletal malformations were noted following treatment up to 60 mg/kg.

Skeletal malformations were observed in 2 (2), 4 (4), 1 (1) and 2 (2) fetuses (litters) in Groups 1, 2, 3 and 4, respectively. Both fetuses in Group 4 (60 mg/kg) had fused sternebrae, but as this sternal malformation was also noted in two control fetuses and two Group 2 (7 mg/kg) fetuses it was considered not to be treatment-related.

The other skeletal malformations in this study were a vertebral anomaly with or without associated rib anomaly in 2 Group 2 (7 mg/kg) fetuses and a rib anomaly in Group 3 (20 mg/kg) fetus. However, because of their incidence and group distribution these were considered chance findings.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on visceral morphology following treatment up to 60 mg/kg.

In Group 4 (60 mg/kg), four fetuses had a visceral malformation and in three of them the accessory lung lobe was missing. However, as this malformation also occurred in two control fetuses and was seen at a higher mean litter incidence in historical controls (1.7% versus 1.4% per litter), this was
considered not to be treatment related. The fourth fetus was delivered dead and appeared to have internal hydrocephaly at serial sectioning of the head, which was considered a chance finding because of its single occurrence.

Other viscerally malformed fetuses of treated dams were observed in Group 2 (7 mg/kg) and in Group 3 (20 mg/kg) Because of their single occurrence these malformations were considered chance findings. Apart from a control fetus with multiple malformations, there were no other visceral malformations observed in this study.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At 60 mg/kg, delayed fetal ossification (classified as skeletal variation) was noted. There were no test item-related skeletal variations following treatment at 7 and 20 mg/kg.

Among the skeletal variations, there were several ossification parameters indicating delayed fetal ossification in Group 4 (60 mg/kg). This was demonstrated by higher incidences compared to the controls for unossified metatarsals (14.2% versus 4.9% per litter), unossified hyoid bodies (7.1% versus 1.0% per litter), unossified tarsals (6.3% versus 0.4% per litter) and unossified pubis (2.0% versus 0.4% per litter). Only the increase of unossified hyoid body was statistically significant, but all the above mentioned incidences of Group 4 were higher than the historical control maximum values (10.1% for unossified metatarsals, 3.2% for unossified hyoid bodies, 2.1% for unossified tarsals and 0.8% for unossified pubis).
Therefore, it was considered that these signs of delayed fetal ossification in Group 4 were related to treatment. Moreover, the delayed fetal ossification in Group 4 was in line with the lower mean fetal body weights in that group (34.5 versus 41.7 grams in Group 1).

In Group 3 (20 mg/kg), it was noted that the mean litter incidences of unossified metatarsals (11.3% per litter), unossified tarsals (2.9% per litter) and unossified pubis (0.9% per litter) were above the respective historical control maximum values (10.1%, 2.1% and 0.8% per
litter) as well. However, in comparison with Group 4 (60 mg/kg), these values were only marginally higher than the maximum values and the Group 3 mean fetal body weights were about the same as those in the control group (39.6 versus 41.7 grams, respectively). Moreover, no statistical changes were found in comparison to the control group.
Therefore, these signs of delayed ossification in Group 3 were considered not to be toxicologically relevant.

Of the remaining skeletal variations, the finding of 13th full ribs showed a curious group distribution. Incidences were 30.9%, 56.1%, 44.9% and 53.8% in Groups 1, 2, 3 and 4, respectively, and statistical significance was reached for the higher values in Groups 2 and 4.
An explanation for this could not be given, but the distribution does not indicate a treatment relationship. Moreover, all incidences were within the historical control range (26.8 - 71.4% per litter) and a higher incidence for this finding is considered not have any detrimental effect. Therefore, the curious group distribution of 13th full ribs is considered to have occurred by chance.

The other skeletal variations that were noted occurred in the absence of a dose-related incidence trend, occurred infrequently or were observed in control fetuses only. Therefore, they were considered not to be related to treatment with the test item.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: skeletal variations at dose level of 60 mg/kg b.w.
Remarks on result:
other: effects related to maternal toxicity

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Applicant's summary and conclusion

Conclusions:
Treatment of pregnant female New Zealand White rabbits by daily oral gavage with 1,4-Butenediol (B2D) from Day 6 to 28 post-coitum at doses of 7, 20 and 60 mg/kg revealed significant maternal toxicity at 60 mg/kg (mortality, clinical signs, lower body weights and reduced food consumption). Secondary to the maternal toxicity at the highest dose level, some developmental toxicity (reduced fetal body weight and delayed ossification) was observed at 60 mg/kg.

No developmental toxicity was observed at the lower dose levels tested (7 and 20 mg/kg) which were maternally non-toxic.

Based on these results, a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity of 20 mg/kg was derived.