Pentaerythritol, reaction product with fatty acids, C8 to 18 (even numbered) and/or branched and/or unsaturated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2013-Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD/EU test guideline and in compliance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): „Fatty acids, C8-10 and C14-18 and C16-18- unsaturated, ester with pentaerythritol and 2 ethylhexanoic acid”; “Glycerides, C16-18 and C18-unsaturated mono- and di-“
- Physical state: Yellow to amber liquid with a characteristic sweet odour.
- Storage condition of test material: in a well-ventilated place, stored at ambient temperature, protected from direct sunlight.

Test animals

Species:

rat

Strain:

other: Crl: CD (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 175.4 - 238.5 g (day 0 pregnancy)
- Fasting period before study: no
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus); Granulated textured wood released for animal bedding was used as bedding.
material in the cages.
- Diet: Commercial ssniff R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ad libitum
- Water: Tap water ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15 (exceeded up to a maximum of 91% on 3 days caused by servicing; this did not affect the integrity of the study)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 June 2013 To: July 04 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item mixtures were freshly prepared each day. The test item was suspended in the vehicle corn oil to the appropriate concentrations of 0, 75, 225 and 500 mg/mL and was administered orally at a constant
volume of 2 mL/kg b.w. to the animals once daily from the 6th to the 19th day of pregnancy. The homogeneity of the suspension was maintained by using a magnetic stirrer throughout the daily administration. The dose of the test item was adjusted to the animal's body weight daily.

Details on analytical verification of doses or concentrations:

For the analysis of the test item formulations, samples of approximately 10 mL were taken at the following times and stored at -20°C until analysis: Analysis of concentration was performed at start of the administration period and at study termination, Analysis of homogeneity was done at the start of the administration period.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: until pregnancy
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 to 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

Until day 20 of gestation

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 450, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25 females treated (20 litters/group evaluated)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the present study the same concentrations of the test article were used as in the 28-day toxicity study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from gestation day 0

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: daily monitoring by visual appraisal of the drinking water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: ovaries and uteri

OTHER: In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals would have been found dead. In case of macroscopical findings, the affected maternal
tissues were preserved in 7% buffered formalin for possible future histopathological examinations.

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 resorptions: Yes, early resorptions <2 mm, late resorptions >2 mm

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

The following statistical methods were used:
For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test
(p ≤ 0.05 and p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p ≤ 0.05 and p ≤ 0.01.

For the comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the FISHER's exact test (n < 100)
or chi2-test with YATES' correction for continuity (n ≥ 100)
(p ≤ 0.05 and p ≤ 0.01)
was employed.

Indices:

Corpora lutea: number per dam, absolute number per group, mean per group

Implantations: number per dam, distribution in the uterine horns, absolute number per group, mean per group

Resorptions: number per dam, distribution in the uterine horns, absolute number per group, mean per group, mean % per group

Resorption rate [%] = (resorptions/implantations) x 100

Weight of placentae: individual data per fetus, mean per litter, mean per group, litter mean per group, litter mean per sex and group

Weight of fetuses: individual data per fetus, mean per litter, mean per sex and litter, litter mean per group, litter mean per sex and group

Fetuses: number per dam (alive and dead), number of fetuses per sex and dam, distribution in the uterine horns, absolute number of fetuses alive per group, mean number of fetuses alive per group, mean % of fetuses alive per group, mean % per sex and group

Dead fetuses: number per dam, mean per group

Runts: number per dam, mean per group

Malformed fetuses: individual data per fetus, mean % per group and type of malformation

Malformation rate [%] =( malformed fetuses/fetuses) x 100

Fetuses with variations: individual data, mean % per group and type of variation

Variation rate [%] = (fetuses with variations/fetuses) x 100

Fetuses with retardations: individual data per fetus, mean % per group and type of retardation

Retardation rate [%] = (fetuses with retardations/fetuses) x 100

Pre-implantation loss [%] = (corpora lutea - implantations/corpora lutea) x 100

Post-implantation loss [%] = ((implantations – living fetuses)/implantations) x 100

Historical control data:

included in report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No test substance related effects were observed on mortality, clinical signs, consumption of food and water, body weight gain, uterus weight, number of resorptions and live fetuses or the values calculated for the postimplantation losses.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No test substance related effects were observed on mortality, sex distribution, weights of placenta and fetuses, number of fetuses with malformations or variations. Skeletal variations were related to the skull (distinct areas not ossified), the sternum (sternebra(e) misaligned to a slight degree or bipartite) and the rib(s) (accessory 14th rib(s), not ossified or wavy). The incidences of these skeletal variations were low and in the range of the laboratory backgound data. As these differences show no dose-response relationship and were in the range of the Laboratory background data, they are considered to be not test-substance related.
The skeletal retardations observed were related to the skull (incomplete ossification of frontal, parietal, interparietal and / or supraoccipital area), the hyoid (not ossified), the sternum (sternebra(e) not or incompletely ossified or reduced in size), the thoracic vertebral bodies (bipartite, dumbbell-shaped or not ossified), the pelvic vertebral bodies (less than 5 pelvic vertebral bodies ossified), the caudal vertebral bodies (no or only one body ossified), os pubis (not or incompletely ossified) or os ischii (not or incompletely ossified), metacarpalia or metatarsalia (absence of ossification in metacarpalia / metatarsalia 2 to 5) and the os fibula (not ossified). No test item-related influence was noted for the fetal incidences of skeletal retardations at 150, 450 or 1000 mg test article/kg b.w./day. The fetal incidences of nearly all findings were in the range of LPT background data, and in the absence of a dose response relationship. The finding of a not ossified os fibula is very rare and not included in the background data. However, this single occurrence, noted for one fetus of the low dose group, has to be considered as spontaneous and not test item-related.
Soft tissue variations were noted in the form of dilatations of the 3rd or 4th cerebral ventricle, dilatation of the renal pelvis (uni- or bilateral), misplaced kidney
and haemorrhagic foci in the liver. No test item-related influence was noted in the incidence of soft tissue variations at any tested dose level and in the range oflaboratory background data

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analysis of test item formulations:

Concentration: The measured concentrations of the test item in the test item vehicle mixtures, were between 103.1% and 105.2% of the nominal concentrations of the low, the intermediate and the high dose group. These values indicated correctly prepared test item vehicle mixtures.

Homogeneity: The measured concentrations of the test item in the test item vehicle mixtures in all samples were between 100.5% and 108.8% of the nominal concentrations for the low, the intermediate and the high dose group. These values indicated that there was no phase separation between the test item and the vehicle during the procedure of administration of the test item vehicle mixtures to the animals.

Applicant's summary and conclusion

Conclusions:

In this prenatal developmental toxicity study in rats, no test substance related maternal toxicity or fetal toxicty was observed up to an including 1000 mg/kg bw/day, the highest dose tested.

Executive summary:

Prenatal developmental toxicity of Fatty acids, C8-10 and C14-18 and C16-18- unsaturated, ester with pentaerythritol and 2 ethylhexanoic acid”; “Glycerides, C16-18 and C18-unsaturated mono- and di-“ was studied according to OECD 414 guideline and GLP principles. Female rats were exposed from gestation day 6 to 19 prior to expected parturition to 0, 150, 450 or 1000 mg/kg bw/day.

No maternal toxicity was observed up to and including 1000 mg/kg bw/day (highest dose tested). Fetal effects were restricted to slight increased incidences in skeletal variations (non-ossified areas in the skull), skeletal retardation (incomplete ossification). However, as these slight increased incidences in skeletal variations/retardation were generally in the range of the laboratory background data and were without dose-relationship, these effects are considered not substance related. Based on these results, a NOAEL for maternal toxicity, fetal toxicity and teratogenicity of at least 1000 mg/kg bw/day (the highest dose tested) is derived.