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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June 2016 to 28 August 2016
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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(2Z)-3-({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl}({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl)prop-2-enoic acid; (2Z)-3-[bis({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl})carbamoyl]prop-2-enoic acid; (2Z)-3-[bis({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl]prop-2-enoic acid
EC Number:
800-430-6
Cas Number:
1419212-76-2
Molecular formula:
ca. C44H75N3O5 - C48H81N305
IUPAC Name:
(2Z)-3-({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl}({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl)prop-2-enoic acid; (2Z)-3-[bis({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl})carbamoyl]prop-2-enoic acid; (2Z)-3-[bis({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl]prop-2-enoic acid
Details on test material:
Identification Reaction products of tall oil fatty acids with diethylenetriamine and maleic anhydride
Appearance Dark brown viscous liquid/paste
Batch Development sample 15-11-1
Purity/Composition UVCB
Test item storage At room temperature
Stable under storage conditions until 19 November 2017 (expiry date)

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain: Crl:WI(Han)
- Age at study initiation: 10-14 weeks
- Sex: Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
- Weight at study initiation: weighed on day 2: 172 to 240 g
- Fasting period before study: none
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm), Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material
- Feed: Free access to pelleted rodent diet ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
prepared daily within 6 hours prior to dosing. Formulations were heated to a maximum of 63.8°C for a maximum duration of 34 minutes to obtain visual homogeneity. Formulations were allowed to cool down below 40°C before dosing. Adjustment was made for specific gravity of the substance and vehicle. No correction was made for the purity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on lab trial and sponsor information
- Dose volume 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples were taken of all concentrations on one occasion during treatment and assessed for accuracy and homogeneity. Samples were diluted with THF in order to obtain solutions of 0.1% formic acid in 50/50 (v/v) THF/water and concentrations within the calibration range. During the development of the analytical method stock solutions were stable for 12 hours when stored at room temperature.
HPLC with MS dectection
Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector: Xevo TQ-S mass spectrometer (Waters)
Column: Acquity UPLC BEH C8, 50 mm × 2.1 mm i.d., dp = 1.7 µm (Waters)
Column temperature: 40°C ± 1°C
Injection volume: 5 µL
Mobile phase: 0.1% formic acid in 90/10 (v/v) acetonitrile/water
Flow 0.6 mL/min
MS detection
Ionisation source ESI+
Cone voltage 20 V
Collision energy 30eV
Quantitation sum of m/z 628.5 --> m/z 306 and m/z 630.4 --> m/z 308

Calibration range: 5 - 100 µg/L --> quadratic (r>99%)
QC samples: 1mg/g 109% ; 200 mg/g 99-104%
Accuracy: 93-106%
Homogeneity: CV 2.7-2.8%
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
day 6-20 post-coitum
Frequency of treatment:
daily
Duration of test:
day 2-21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose-range finding study
Four groups of 6 females were exposed to 0, 100, 300 and 1000 mg/kg for Days 6 to 20 post-coitum inclusive by oral gavage. These dose levels were based on a 14-day pilot study in which no toxicity was observed with treatment up to 1000 mg/kg. In a 90-day repeated dose study no effects on clinical signs, body weight or food consumption were observed by treatment up to 1000 mg/kg in the first weeks after dosing.
In this study no effects were found related to maternal or developmental toxicity up to 1000 mg/kg bw.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (mortality/viability twice daily) form day 2 post-coitum onwards

BODY WEIGHT: Yes
- Time schedule for examinations: on day 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: No quantative examination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post-coitum
- Organs examined: external, thoracic and abdominal examination, with special attention being paid to the reproductive organs

OTHER: ovary and uterine horn of all animals
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
•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 (early and late resorptions).
Fetal examinations:
- Weight of each fetus.
- Sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- External examinations: Yes: [all per litter, including late resorptions]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Dunnett-test , Steel-test, Fisher Exact-test, Mann Whitney test, Kruskal-Wallis nonparametric ANOVA test, Dunn’s test
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance
Indices:
Pre-implantation loss (%), Post-implantation loss (%), Viable fetuses affected/litter (%)

Historical control data:
yes included in an appendix to the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental alopecia in one animal in group 1, 3 and 4
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not quantitative assessed. No effects reported
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
uterus weight: no treatment related effects (average 68.7, 73.1, 69.0 and 65.7 g at 0, 100, 300 and 1000 mg/kg bw)
Gross pathological findings:
no effects observed
Description (incidence and severity):
Alopecia observed in 1 animal in control, at 300 and 1000 mg/kg bw was considered not toxicologically relevant
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
none observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related effects
pre-implantation loss: 34, 4, 5 and 14 at 0, 100, 300 and 1000 mg/kg bw
postimplantation loss: 13, 19, 21 and 27 at 0, 100, 300 and 1000 mg/kg bw
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
No treatment related effects
early resorptions: 11, 18, 21 and 26 at 0, 100, 300 and 1000 mg/kg bw
late resorptions:2, 1, 0 and 1 at 0, 100, 300 and 1000 mg/kg bw
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses in any of the dose group
The number of viable fetuses at 1000 mg/kg bw was reduced compared to controls (88% versus 93% in controls), but this decrease was only slightly outside the historical control values and therefore considered not toxicologically relevant.
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:
not examined
Description (incidence and severity):
1 female at 1000 mg/kg bw was not pregnant
Details on maternal toxic effects:
none observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
mean fetal body weight: 5.0, 5.2, 5.1 and 5.2 at at 0, 100, 300 and 1000 mg/kg bw

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
Description (incidence and severity):
mean number of fetuses per litter: 10, 10.5, 10.2 and 9.3 per litter at 0, 100, 300 and 1000 mg/kg bw

The decrease at 1000 mg/kg bw did not reach statistical significance and was only slightly outside the range of the available historical data. Therefore this effects was considered toxicologically irrelevant
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
one control fetus: omphalocele
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Not treatment related
100 mg/kg bw: smal mandible (1), bent limb bone(1), vertebral centra anomaly (1) and sternoschisis (1)
300 mg/kg bw: bent limb bone(4)
1000 mg/kg bw: malpositioned metatarsals (1) and bent limb bone(1)
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
one fetus at 100 mg/kg bw: one kidney missing
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal variations : 84.8%, 78.0%, 84.2% and 79.7% per litter at 0, 100, 300 and 1000 mg/kg bw
Details on embryotoxic / teratogenic effects:
no treatment related effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related effects

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no
Dose response relationship:
no

Applicant's summary and conclusion

Conclusions:
The substance did not induce maternal and developmental toxicity up to 1000 mg/kg bw.
Executive summary:

Twenty-two female rats/group were exposed to the substance during day 6 to 20 post-coitum at 0, 100, 300 and 1000 mg/kg bw by gavage. No effects on maternal mortality, bodyweight (gain), clinical signs, food consumption and macroscopy were found. No effects were reported on maternal develomental parameters like abortions, implantation loss, resorptions and fetal deaths. The litter size as well as the sex ratio, number and weight of the fetuses did not differ between dose groups. No treatment related external, visceral and/or skeletal malformations were observed. The NOAEL for maternal toxicity is 1000 mg/kg bw. The NOAEL for developmental toxicity is 1000 mg/kg bw.

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