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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:
August 2016 - March 2017
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)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): MIRAMER M140
- Physical state: Colorless liquid
- Analytical purity: 84.46 % ( CAS No.: 48145-04-6; EC No.: 256-360-6)
- Impurities (identity and concentrations): cas no. 61630-25-9; 8.6%; cas no. 122-99-6; 1.6 %
- Lot/batch No.:151014142
- Expiration date of the lot/batch: 31 December 2016
- Storage condition of test material: Under dry conditions, protected from light and at room temperature (20 ± 5 ºC)

Supplier: Miwon

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hannover
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rat, Wistar Hannover from Charles River Laboratories France
- Number: 107 mated females (nulliparous and non-pregnant); 29 males
- Age at study initiation: 11 weeks (Males/Females)
- Weight at study initiation: Males: xxx g Females: 196-310 grams (day 0 post coitum)
- Fasting period before study: No information
- Housing: Cages with standard, granulated, softwood Lignocel S8/15 bedding (supplied by Harlan Laboratories Models, S.L.).
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet ad libitum, batches: 121211MA, 122911MB, 042612MA expiry dates: 7 September 2012, 18 September 2012 and 21 January 2013 (supplied by Harlan Laboratories Models, S.L.). Pelleted standard Teklad 2018C rat/mouse maintenance diet batches: 122311MA and 041112MA expiry dates: 24 September 2012 and 6 January 2013 (supplied by Harlan Laboratories Models, S.L.) ad libitum, for lactating females and pups (until day 4postpartum).
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 5 days between arrival and pretest

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air-conditioned with ranges for room temperature 20 -23 °C
- Humidity (%): relative humidity 35-70%
- Air changes (per hr): 15-20 air changes per hour
- Photoperiod (hrs dark / hrs light): a 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To: 24 August 2016 to 04 October 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
REPARATION OF DOSING SOLUTIONS:

Method: Oral, by gastric gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Corn Oil (Sigma-Aldrich, cat.no. C8267), test item soluble in this vehicle and non-toxic in the concentration used.
- Concentration in vehicle:
Group 1: 0 mg/kg,
Group 2: 65 mg/kg
Group 3: 200 mg/kg
Group 4: 600 mg/kg

- Amount of vehicle (if gavage): 4 mL/kg /once daily
- Lot/batch no. (if required): MKBV2080V (expiry Date 05 Sep. 2018) and MKBW9504V (expiry Date 09 September 2018 / 06 October 2021)

- Purity: not specified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations were considered homogeneous when the coefficient of variation (CV) of
the results is ≤10%. The acceptance limits are 85-115% of the nominal content.
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 2:1
- Length of cohabitation: After acclimatization, females were housed with sexually mature males in a cage for a period approximately of 16 hours
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
The females received the test item from days 6 to 19 post coitum
Frequency of treatment:
Once per day
Duration of test:
At the scheduled necropsy on day 20 post coitum, females were sacrificed by CO2 inhalation and the fetuses removed by caesarean section.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
65 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
-Group 1: 22 (0 mg/kg),
-Group 2: 29 animals, (65 mg/kg),
-Group 3: 29 animals, (200 mg/kg),
-Group 4: 27 animals, (600 mg/kg).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a dose-range finding toxicity study in rats (Harlan Laboratories Study S55881Dose Range Finder for Prenatal Developmental Toxicity in Rats), using dose levels of 0, 100, 300 and 800 mg/kg/ bw/day.
Due to severe irritation of the gastric mucosa observed as thick whitish and rugous non-glandularmucosa in one female at 300 and in two females (one not pregnant) at 800 mg/kg/ bw/day, the highest dose level was considered too high for the main study. Furthermore, two mated females from the highest dose group showed abnormal gait, pilo erection,and hunched posture mainly on days 11-13 of pregnancy.
Based on these findings 600 mg/kg bw/day was chosen as the highest dose level for the main study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage signs on remaining days (together with the viability / mortality
Observation twice daily).

- Cage side observations checked in table [yes] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs for signs of reaction to treatment and/or
symptoms of ill health during days 6-20 of pregnancy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, daily from 6 to 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20 post coitum, females were sacrificed by CO2 inhalation
and the fetuses removed by caesarean section.
- Organs examined: all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea.

OTHER:
- Food consumption Periods: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post coitum.
- Body weight Days 0, 3, daily from 6 to 20 post coitum.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: When no implantation sites were evident, the uterus was placed in an aqueous solution of
ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The following comparisons are performed:
Group 1 vs Groups 2, 3 and 4 to analyze food consumption, body weights, clinical signs and reproduction data:
Means and standard deviations of various data were calculated and included in the report.
For continuous data, a parametric analysis was performed when Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
A non-parametric analysis was performed when Bartlett's test is still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test, unless there is evidence against a monotonic dose-response when Steel's test was performed instead.
Indices:
NA
Historical control data:
NA

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were recorded. The salivation recorded at 600 mg/kg/day (before, during and after administration) could be attributed to the palatability of the test item.

Some findings as coat hair loss (dorsal or ventral surface, head, forelimbs and hind limbs), encrustation (ear and head) and wound were observed sporadically.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Description (incidence and severity):
No test-item-related findings were recorded in the macroscopic examination at hysterectomy
in the rats treated with the test item.

Some findings were recorded among all groups: dilated uterus horns or containing reddish
fluid/hemorrhagic contents, reddish kidney medulla and alopecia in different body areas.
These findings were considered to be within the range of normal background lesions that may
be seen in rats of this strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no indication of a food consumption effect.
There was no effect on body weights.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant differences observed at 600 mg/kg/day in pre-implantation losses were devoid of any toxicological relevance due to the fact that the females received the test item from days 6 to 19 post coitum, once the implantation had already occurred. They were considered a consequence of the biological variability and not test-item related. A slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d compared to control group (8.3%). However, the available historical control data (2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%.
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:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly reduced uterus weight and litter weight was observed at 600 mg/kg/day when compared to control group.
The reduction was related to 2 litters (No 94 and 100) with observed values outside the range of control group.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effect found at the highest dose (600mg/kg bw/day)

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
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 specified
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings which could be attributed to the treatment were observed.

0 mg/kg/day
Litter no. 12 and 13: One fetus (no. 9 in both litters) presented protrusion of intestine.

600 mg/kg/day
Litter no. 113: One fetus (no. 2) had thin skin in right side of head.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day there was a slightly increased incidence of incompletely ossified cranial centers, sternebrae and sacrocaudal vertebrae compared to concurrent Control. All except cranial centers are just outside of historical control data from previous S.A.U study.

At 200 mg/kg/day there was a slightly increased incidence of incompletely ossified thoracic vertebrae compared to concurrent Control which was outside of historical control data.

At 65 mg/kg/day there was a slightly increased incidence of fused/partially fused maxilla/jugal (precocious ossification) compared to concurrent Control which was outside of historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some isolated major abnormalities were recorded in a total of four fetuses: Control group
(litters no. 12 and 13, fetuses no. 9), doses 200 mg/kg/day (fetus no. 6 and 12 from litter 68)
and 600 mg/kg/day (no. 5 from litter 103 and fetus no 1 form litter 94). As no dose-effect relationship was observed, it
cannot be considered attributable to the test item.

Some minor visceral abnormalities were observed in all the groups, including Control.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect found at the highest dose (600mg/kg bw/day)

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Summary of Performance of Mated Females

Group
Dose (mg/kg/day)

1
0

2
65

3
200

4
600

Number of mated females

22

29

29

27

Number of pregnant females

20

21

21

23

The following females were not pregnant:

Group 1: 4 and 7

Group 2: 31, 33, 34, 39, 40, 42, 48 and 58

Group 3: 61, 62, 64, 74, 78, 79, 87 and 89

Group 4: 92, 97, 98 and 109.

Applicant's summary and conclusion

Conclusions:
The toxicity and adverse effects of 2-phenoxyethyl acrylate on prenatal development toxicity were investigated in accordance with the requirements of OECD 414 test guideline at dose levels 0, 65, 200, and 600 mg/kg bw/day. It should be noted that a higher dose level than 600 mg/kg bw/d could not be used for this study due to the observations in the dose range finding study where severe local irritation in the stomach was observed at 800 mg/kg bw/d.

The administration with 2-Phenoxyethyl Acrylate by oral gavage, did not cause mortality, changes in body-weight and food consumption and caused no adverse clinical signs at any of the doses. Salivation recorded at 600 mg/kg/ bw/day was attributable to the palatability of the test item. A slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d compared to control group (8.3%). However, the available historical control data (2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%.

In conclusion, as the increase in postimplantation loss was not significant at the dose level of 600 mg/kg/ bw/d, a formal NOAEL for maternel toxicity as well as for fertility/developmental toxicity of 600 mg/kg bw/d can be concluded in this study when assessed on its own.
However, as postimplantation loss has also been seen in the dose-range finding study and in the OECD 422 study at a dose level of 800 mg/kg bw/d the post-implantation loss at 600 mg/kg bw/d in this OECD 414 study imay be considered to be related to the dosing and thus, a more precautious NOAEL of 200 mg/kg bw/d for increased postimplantation loss may be concluded.


Executive summary:

The toxicity and adverse effects of 2-phenoxyethyl acrylate on prenatal development toxicity were investigated in accordance with the requirements of OECD 414 test guideline.

2-phenoxyethyl acrylate was administered by oral gavage to four groups (0, 65, 200, and 600 mg/kg bw/day), each of 22-29 female rats. Treatment started at Day 6 post coitum and ended at Day 19 post coitum. At day 20 post coitum caesarean section and necropsy were performed.

 

The administration with 2-Phenoxyethyl Acrylate by oral gavage, did not cause mortality, changes in body-weight and food consumption and caused no adverse clinical signs at any of the doses. Salivation recorded at 600 mg/kg/ bw/day was attributable to the palatability of the test item. A slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d compared to control group (8.3%). However, the available historical control data (2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%.

There were no test-item related changes in the observations derived from hysterectomy. The skeletal examination of the fetuses showed a slightly increased incidence in incomplete ossification considered a transient stage in fetal development. As this finding is expected to be resolved in time, this finding is not considered adverse. The slightly increased incidence of fused/partially fused maxilla/jugal (precocious ossification) is not considered to be relevant, as it was only recorded at the low dose (65 mg/kg/ bw/day). The external, skeletal and visceral examination of the fetuses showed the presence of major abnormalities in two fetuses (no. 9 from the litters no. 12 and 13) from the Control group, two fetuses (no. 6 and 12 from litter 68) at 200 mg/kg/ bw/day and two fetuses (no. 5 from litter 103 and no. 1 from litter 94) at the dose of 600 mg/kg/ bw/day. Despite the fact that these abnormalities were also observed in some of the groups treated with the test item, the incidence and distribution recorded could be considered not dose-related.

It should be noted that a higher dose level than 600 mg/kg bw/d (with a higher potential for systemic uptake and systemic toxicity) could not be used for this study due to the observations in the dose range finding study where severe local irritation in the stomach was observed at 300 and 800 mg/kg bw/d.

In conclusion, as the increase in postimplantation loss was not significant at the dose level of 600 mg/kg/ bw/d, a formal NOAEL for maternel toxicity as well as for fertility/developmental toxicity of 600 mg/kg bw/d can be concluded in this study when assessed on its own.

However, as postimplantation loss has also been seen in the dose-range finding study and in the OECD 422 study at a dose level of 800 mg/kg bw/d the postimplantation loss at 600 mg/kg bw/d in this OECD 414  study may be considered related to the dosing and thus, a more precautious NOAEL of 200 mg/kg bw/d for increased postimplantation loss may be concluded.