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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 May 2012 - 4 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 2-methylpropanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2-{[(2-methylpropanoyl)oxy]methyl}-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propyl 3,5,5-trimethylhexanoate; 3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate
EC Number:
813-120-0
Cas Number:
1262967-45-2
Molecular formula:
C21H36O8 C26H46O8 C31H56O8 C36H66O8 C41H76O8
IUPAC Name:
3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 2-methylpropanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2-{[(2-methylpropanoyl)oxy]methyl}-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propyl 3,5,5-trimethylhexanoate; 3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: Jan 2013
- Purity test date: 95.8 %



RADIOLABELLING INFORMATION (if applicable): N/A



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material:bRoom temperature (ca 20 °C), in the dark under nitrogen
- Stability under test conditions: Stable

- Solubility and stability of the test substance in the solvent/vehicle: stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8C). 

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A



TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: The test material was used as supplied. The test item was prepared for administration as a series of graded concentrations in the vehicle - corn oil.
- Preliminary purification step (if any): Dose range finder was conducted prior to the main experiment ignorer to determine suitable dose levels.

- Final dilution of a dissolved solid, stock liquid or gel: 5 mL/kg
- Final preparation of a solid: N/A



FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Remarks:
Crl:CD(SD) rats
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: [yes/no]: Yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Weight range 228 to 283 g.
- Fasting period before study: Not Stated
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.



DETAILS OF FOOD AND WATER QUALITY:

- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet.
- Water (e.g. ad libitum): Polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.

- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES: From: To: 20 June 2012 - 19 July 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating.
Animals received the test material or vehicle control formulations orally at a volume-dose of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.
All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week. The volume administered to each animal was calculated from the most recently recorded scheduled bodyweight.
A daily record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Approximately 50 % of the final volume of vehicle was added to the test item and magnetically stirred until the test material had dispersed and homogenous. Specimen formulations were prepared at concentrations of 2 mg/mL and 200 mg/mL .

The stability was assessed following storage at ambient temperature (nominally 21 °C) for 0, 2 and 4 hours and 1 day, and refrigeration (nominally 2 to 8 ºC) for 1 day, 8 days and 15 days.

Single samples were taken for assay from the top, middle and bottom of the magnetically stirred formulation and homogeneity was determined by analysis of these samples. Stability was determined from the mean concentration of the test item in the vehicle at each sampling point.

The results of the analysis confirmed that the test item produced an homogenous suspension and was stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8 ºC).
Details on mating procedure:
The ainimals were paired on a 1:1 basis with stock males from the same source. Daily checks were made after pairing for evidence of mating, including ejected copulation plugs in cage trays and the presence of sperm in a vaginal smear.
Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Only females with a sperm positive vaginal smear or at least two copulation plugs were selected. Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily
Duration of test:
Day 6 to Day 19 after mating
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 Control (Corn oil)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 (test item)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 (test item)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 (test item)
No. of animals per sex per dose:
20
Control animals:
yes
Details on study design:
The oral gavage route of administration was chosen to simulate a condition of potential human exposure. The doses used (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor following a review of the results of a OECD 421 reproductive screening study using this compound performed in these laboratories (Huntingdon Life Sciences Report Number: OWH0020). It was expected that 100 mg/kg/day is the NOEL (no-observed-effect-level), and that there was the possibility of effects upon fetal weight at 300 mg/kg/day, and reduced fetal weight at 1000 mg/kg/day. The test substance was administered randomly assinged animals from Day 6 to 19 after mating.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes 

- Time schedule: Twice daily


BODY WEIGHT: Yes 

- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.



FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes 

- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive.

 

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

 


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20

- Organs examined: yes
 


OTHER:
In addition, a more detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.
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
The gravid uterus was weighed; this weight included the weight of the cervix and ovaries.
Fetal examinations:
All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded. The sex of each fetus was recorded.
Approximately half of the fetuses in each litter were eviscerated and their skeletons were fixed in Industrial Methylated Spirit prior to processing and staining with Alizarin Red. The remaining fetuses were fixed whole in Bouin’s fluid.
Statistics:
The following sequence of statistical tests was used for bodyweight, food consumption, organ weights and litter data: Bartlett's test, Dunnett's test, Shirley's test and Williams’ test
For gravid uterine weight, litter size and survival indices and fetal, placental and litter weight data, if 75 % of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

Pre/post implantation loss and sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991). Each treated group was compared to control using a Wald chi-square test. For pre-implantation loss, the numerator was Number of corpora lutea - Number of implantations, the denominator was Number of corpora lutea. For post-implantation loss, the numerator was Number of implantations - Number of live fetuses, the denominator was Number of implantations. For sex ratio, the numerator was Number of males, the denominator was Number of live fetuses.
For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test (Wilcoxon 1945).

Significant differences between Control and treated groups were expressed at the
5% (p<0.05) or 1% (p<0.01) level. The key to the annotation used on the tables that contain statistical results is given below.
Av Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.
Sh Treated groups compared to Control using Shirley’s test.
Wa Treated groups compared to Control using Wald’s test
Wc Treated groups compared to Control using Wilcoxon rank sum test Wi Treated groups compared to Control using Williams’ test.
* p<0.05 ** p<0.01
Indices:
Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occuring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.
Historical control data:
Included in the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Signs seen in association with dosing included occasional chin rubbing for some females in all groups throughout gestation and for most females receiving the test item at 1000 mg/kg/day towards the end of gestation. No other signs in association with dosing were recorded.
Dermal irritation (if dermal study):
not examined
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
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:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
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
Changes in number of pregnant:
no effects observed
Other effects:
not specified

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:
clinical signs

Maternal abnormalities

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:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day there was a slightly higher incidence of incompletely ossified/ unossified cranial centres, hyoid, and pelvic bones compared with concurrent controls. These are all within historical control data levels.

There was also a low incidence of folded retinas. In the absence of any related findings, this is also considered not to be treatment related.

At 300 mg/kg/day there was a slightly higher incidence of incompletely ossified/ unossified cranial centres compared with concurrent controls. This is within historical control data levels.
Visceral malformations:
no effects observed
Other effects:
not specified

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 adverse effect observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Tables containing raw data are attached in full study report.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test item to pregnant CD rats during the organogenesis and fetal growth phase of gestation resulted in slight maternal toxicity at 1000 mg/kg/day while embyro-fetal survival, growth and development were considered not affected. Fetal and litter weight of females which had received the test item at 1000 mg/kg/day were slightly, but statistically significantly lower than the weight of the concurrent control (approximately 95 % of control values). Offspring birth weight in females treated throughout pregnancy (HLS study OWH0020) were much more severely affected and were nearly 20 % below control values, suggesting that continued treatment at this level could be damaging to late fetal growth.

Based on the condition of the study, it was concluded that the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development was 1000 mg/kg/day.
Executive summary:

OECD 414 (2012) - In Embryo-Fetal Development al toxicity study (OECD 414), Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was administered to  20 female Crl:CD(SD) strain rats  during the organogenesis and fetal growth phases of pregnancy via oral gavage at dose of 100, 300 and 1000 mg/kg/dayfrom Day 6 to Day 19 after mating. In addition,  a group of 20 female control rats were administered corn oil only at the same dose volume throughout the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. A summary of adult responses to the test item are described below; Systemic toxicity (maternal)

 

Mortality- There were no deaths and no clinical signs seen considered related to treatment with the test item.

Clinical signs- occasional chin rubbing for some females in all groups throughout gestation and for most females receiving the test item at 1000 mg/kg/day towards the end of gestation. No other signs in association with dosing were recorded.  

Bodyweight - There were no maternal bodyweight or bodyweight gain.

Food consumption and efficiency - Food consumption for females receiving the test item at 100, 300 or 1000 mg/kg/day was generally similar to that of the Control throughout gestation but the food consumption of females receiving the test item at 1000 mg/kg/day was slightly lower than that of the Control between Days 18-19 of gestation.

  

Histopathological changes and Necropsy - There were no macroscopic findings at necropsy which were considered related to treatment with the test item from Day 6 to Day 19 after mating.

  

Organ weights - Values for the weight of the gravid uterus, adjusted bodyweight and adjusted bodyweight change at dose levels up to 1000 mg/kg/day were similar to control values and considered unaffected by treatment with the test item.

All females were found to be pregnant at necropsy. Litter data as assessed by the mean numbers of corpora lutea, implantations, embryo-fetal resorptions, live young, sex ratio and the extent of pre- and post-implantation loss was similar across all groups.

Developmental toxicity (Fetal)

Embyro-fetal survival, growth and development were considered not affected by treatment at dose levels up to 1000 mg/kg/day

Fetal and litter weight of females which had received the test item at 1000 mg/kg/day were marginally lower than the weight of the concurrent control (approximately 95 % of control values, statistically significant for overall fetal weight and male fetuses). Offspring birth weight in females treated throughout pregnancy (HLS study OWH0020) were much more severely affected and were nearly 20 % below control values, suggesting that continued treatment at this level could be damaging to late fetal growth.

There were incidence of major and minor abnormalities and skeletal variants without any relationship to treatment. This included, a slightly higher incidence of incompletely ossified/ unossified cranial centres, hyoid, and pelvic bones compared with concurrent controls at 1000 mg/kg/day. At this dose, there was also a low incidence of folded retinas, however, in the absence of any related findings, it was considered not to be treatment related.  At 300 mg/kg/day there was a slightly higher incidence of incompletely ossified/ unossified cranial centres compared with concurrent controls. This is within historical control data levels.

Based on the results of this study, it is concluded that the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development was 1000 mg/kg/day.

The study was acceptable and satisfies the guideline requirements for an OECD 414 in the rat.