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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
Study period:
08 Jun 2018 - 25 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate
EC Number:
256-032-2
EC Name:
(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate
Cas Number:
42978-66-5
Molecular formula:
C15 H24 O6
IUPAC Name:
(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 15-21 weeks old
- Weight at study initiation: between 3043 g and 4268 g
- Housing: housed individually in cages with perforated floors (Ebeco, Germany)
- Diet: ad libitum, Pelleted diet for rabbits (Global Diet 2030 from Harlan)
- Water: ad libitum, Municipal tap water
- Acclimation period: least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 38 to 97
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension; protected from light. If not used on the same day of preparation, the aliquots were stored in the refrigerator set to maintain 4°C for a maximum of 8 days. On the day of use, they were removed from the refrigerator and allowed to warm to room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during dosing.m Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: 1% Aqueous carboxymethyl cellulose with 0.5% Tween 80
Analytical verification of doses or concentrations:
yes
Remarks:
Acquity UPLC system
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
from Day 6 to Day 28 post-coitum
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the tolerability study and dose range finder and in an attempt to produce graded responses to the test item. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity including mortality was observed at 750 mg/kg. Therefore, the dose levels selected in the current main teratology study were 0, 50, 150 and 450 mg/kg bw/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
All animals (including female no. 75 euthanized before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. In addition, the gastro-intestinal tract was examined for signs of irritation/corrosion; the oesophagus was examined internally. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed. Each ovary and uterine horn of all animals was dissected and examined as quickly as possible.
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: [all per litter]

- Skeletal examinations: Yes: [all per litter]
Subsequently, the skeletal examination was done on all fetuses from all groups from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

- Head examinations: Yes: [half per litter]
Statistics:
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). 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 were compared using the Mann Whitney test. 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.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. 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:
see table 1

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicological relevant findings up to 450 mg/kg.
Reduced faeces production (up to a moderate degree) was observed in 14, 18, 19 and 18 females in the control, 50, 150 and 450 mg/kg groups, respectively, on several days during treatment. As all groups were affected (including the vehicle control group) and in the absence of a dose-related trend, no toxicological significance was attached to this observation. Incidental findings of note were red fluid on the manure tray in one high dose female, and lean appearance and/or piloerection in another high dose female. These findings were considered not to be toxicological relevant as they were observed in the vehicle control group at the same incidence (red fluid on the manure tray, lean appearance), were transient (lean appearance and piloerection) and all gravid females in this study had a normal pregnancy.
One female in Group 3 was noted with missing toes (two missing toes on the right foreleg and 1 missing toe on the left foreleg; taken from the study daybook). This finding was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Any 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, and/or were observed in control females only. They were therefore of no toxicologically relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatmentwith the test item.
One female from the high dose group had to be euthanized after 4 days of treatment. Immediately after dosing on post-coitum Day 9, she was observed with respiratory problems (gasping) and moribund. At necropsy, red foamy content of the trachea, several reddish foci in left caudal lobe of the lungs and grey-white discolouration of the left caudal lobe of the lungs were noted. Taken together, these findings were indicative of complications during the oral gavage procedure. Therefore, the preterm sacrifice of this female was considered unrelated to the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight, body weight gain before and after correction for (gravid) uterus weight of treated Group 2, 3 and 4 females were unaffected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 450 mg/kg. In the high and mid dose group each, a trend towards slightly lower absolute and relative food consumption compared to the concurrent control group was noted from Days 6-15 and Days 12-15 post-coitum, respectively. As changes were only slight (reaching no statistical significance) and transient (complete recovery was seen from Days 15-18 post-coitum onwards), it was not considered as adverse.
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
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Moreover, the examination of the gastro-intestinal tract and the internal examination of the oesophagus did not reveal any signs of irritation and/or corrosion. The observation of missing toes in one Group 3 female was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Remaining incidental findings among control and treated animals included watery-clear cysts of the oviducts, alopecia, scabs or an isolated wound. These findings are occasionally seen among rabbits used in these types of study, and in the absence of a dose relationship they were considered unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.
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
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Overall, the number of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and treatment groups were considered to be unaffected by treatment. Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 450 mg/kg.
Mean combined (male and female) fetal body weights were 37.8, 38.2, 39.1 and 38.0 gram for the control, 50, 150 and 450 mg/kg groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 450 mg/kg.
Mean sex ratios (males:females) were 46:54, 50:50, 44:56 and 56:44 for the control, 50, 150 and 450 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.4, 9.8, 10.1 and 9.3 fetuses/litter for the control, 50, 150 and 450 mg/kg groups, respectively. As all values remained within the available historical control range the slightly lower litter size in the high dose group was considered unrelated to treatment.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 450 mg/kg/day.
Two external malformations were observed in this study. Two fetus of Groups 2 and 4, respectively, both had an open eye with relating findings of the eye/lens noted at soft tissue cephalic examination (i.e. eyelids not joined, lens attached to cornea and/or lens misshapen). Due to the single occurrence of this malformation in a low and high dose fetus, it was considered to be chance finding. A flexure of the carpal and/or tarsal was the other malformation and this occurred in the control group in one fetus (both tarsals, without apparent skeletal origin) and in late resorptions in two fetuses (one or both carpals, not skeletally examined). Because carpal and/or tarsal flexures were observed at the control level only, this was considered spontaneous in origin. External variations were not observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment at 450 mg/kg/day.
The only fetus with a malformation was one fetus of the high dose, which had a vertebral anomaly with associated rib anomaly. A vertebral anomaly is the most common skeletal malformation among historical control fetuses and at the single occurrence noted in this study, it is considered spontaneous in origin. A statistical significant increase in the litter incidence of fetuses with caudal shift of pelvic girdle was observed in the high dose group. This variation occurred at an incidence of 21.5%
versus 8.7% per litter in the concurrent control group. Because the litter incidence of caudal shift of pelvic girdle was well within the historical control data range (mean 16.5% per litter; P5 – P95: 2.8 - 34.5% per litter), the higher incidence of this finding in Group 4 was considered to have occurred by chance and not to be toxicologically relevant. All other variations noted were not considered treatment related as they occurred infrequently, at frequencies that were within the range of available historical control data, or were observed in control fetuses only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 450 mg/kg/day. Visceral malformations occurred in 0 (0), 1 (1), 4 (3) and 3 (2) fetuses (litters) in the control, 50, 150 and 450 mg/kg groups, respectively.
In Group 4, the three malformed foetuses (out of two litters) had a malpositioned testis. This malformation was also noted in Group 3 fetus, but not in fetuses at the control and low dose levels. Nevertheless, because a malpositioned testis is one of the most common visceral malformations in historical control fetuses, this was considered not to be treatment related. The other malformed Group 3 fetuses all had a multiple cardiovascular malformation. As no cases occurred in Group 4 and none of
the individual malformations was uncommon among historical control fetuses these were considered not toxicologically relevant. The affected fetus in Group 2 had a diverticulum of the intestine, which at the single occurrence at the low dose was considered spontaneous in origin.
All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The objectives of this study OECD 414 study (ReachCentrum, 2019) were to determine the potential of the read-across substance TGPDA (CAS 42978-66-5) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive.No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg bw/d groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established
Executive summary:

The objectives of this study OECD 414 study (ReachCentrum, 2019) were to determine the potential of the read-across substance TGPDA (CAS 42978-66-5) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg bw/day, based on the results of the dose range finder and tolerability study. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity was observed at 750 mg/kg. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents, and maternal pregnancy data. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in 1% aqueous carboxymethyl cellulose with 0.5% Tween 80 were prepared accurately and homogenously.

No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg bw/d groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established. Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg bw/d.