Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August to 10 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant with no deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Di-Penaerythritol
- Physical state: solid
- Analytical purity: 94.6%
- Lot/batch No.: 150300133
- Expiration date of the lot/batch: 03 Mar 2017
- Stability under test conditions: confirmed by the sponsor
- Storage condition of test material: ambient

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD) Sprague-Dawley rats (female)
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 9-10 weeks old
- Weight at study initiation: 201 - 305 g
- Housing: Up to 2 per cage in suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Sterilised white wood shavings were provided as bedding with a device for hiding in and an object for chewing as environmental enrichment.
- Diet (e.g. ad libitum): SDS RM1 diet, ad libitum
- Water (e.g. ad libitum): Water from the public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 34-64%
- Air changes (per hr): Ten or greater with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Doses of 0 (corn oil only), 25, 75 or 250 mg/mL were administered by oral gavage, at a constant dosing volume of 4 mL/kg bw. Dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations were also stirred continuously during dosing. Any residual volumes were discarded.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by Gas Chromatography with Flame Ionisation Detection (using a validated procedure). Dose formulaton samples were collected for analysis from all groups on Day 1 and Week 2 for determination of concentration, and from all test material groups for determination of homogeneity. Duplicate sets of samples (top, middle, and bottom samples (duplicate middle only for control)) for each sampling time point were sent to the analytical laboratory; the remaining samples were retained at the Test Facility as backup samples.
Stability analyses performed previously in conjunction with Charles River Study No. 434327 demonstrated that the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data were retained in the study records for Study No. 434327.
Details on mating procedure:
Time-mated females were delivered to the test facility.
Duration of treatment / exposure:
Days 6-19 of gestation
Frequency of treatment:
Once daily
Duration of test:
Day 6 to Day 20 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in agreement with the Sponsor following review of all available toxicological data, where dose levels of up to 1000 mg/kg bw/d were well tolerated with only minimal effects of toxicity.
- Rationale for animal assignment (if not random): random allocation on arrival

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes (general health, mortality and moribundity)
- Time schedule: twice daily; postdose observations for reaction to treatment were also made prior to daily and regularly throughout the day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from the start of dosing,

BODY WEIGHT: Yes
- Time schedule for examinations: once during pre-treatment (Day 5 of gestation) and daily (Day 6-20 of gestation) during dosing

FOOD CONSUMPTION: Yes
- Food consumption was measured quantitatively daily from Day 5 of gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20 of gestation
- Organs examined: All adult animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

OTHER:
Ovaries and uterine content:
The reproductive tract was dissected from the abdominal cavity. The gravid uterus was weighed. The uterus was opened and the contents were examined. The fetuses were removed from the uterus. The ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, placentae (size, colour or shape) any abnormalities were recorded, live and dead fetuses and early and late embryonic deaths.
Fetal examinations:
EXTERNAL ABNORMALITIES
Fetuses were examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that shows no sign of maceration), or a late embryonic death (macerated tissue identifiable as an embryo) fetus, with recognisable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may be of varying size).

BODY WEIGHT
The body weight of each fetus was recorded. Fetuses were individually identified within litters.

VISCERAL EXAMINATION AND SEX
Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol, examined internally for sex and eviscerated following initial fixation, the viscera were not examined from fetuses prior to disposal. The remaining half of the viable fetuses from each uterus were fixed in Bouin's fluid. The fetuses fixed in Bouin's fluid were examined for soft tissue abnormalities and sex using a freehand sectioning technique derived from that of Wilson.

SKELETAL EXAMINATION
The eviscerated carcasses were then macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, and then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.
Statistics:
Means and standard deviations were calculated for body weight, food consumption and pregnancy data. To assist interpretations, tests were applied to determine the statistical significance of observed differences between the control group and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Body weight and food consumption data were analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Indices:
Not applicable
Historical control data:
Records maintained at the test facility

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no mortalities, and no clinical signs of toxicity. Food consumption, group mean body weights and body weight gains were unaffected by treatment. There were no abnormalities detected at necropsy.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no effect of treatment on pregnancy frequency, gravid uterine weight, mean fetal weight, numbers of corpora lutea, number of implants or embryofetal survival including pre-implantation loss. The type and distribution of all fetal abnormalities and variations, including those indicating the extent of skeletal ossification were similar in all groups and did not indicate any association with treatment.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dose formulation analyses: All study samples analyzed had mean concentrations within or equal to the acceptance criteria of

± 10% of their theoretical concentrations. Day 1 Group 1 control samples showed test item peaks and Day 1 Group 2 samples were not

reported due to an unexpectedly high injection, therefore, as part of the investigation, the group 1 backup samples were analyzed in triplicate and the group 2 samples were rerun, and the results were within the acceptance criteria. Group 1 backup samples and group 2 re-run samples are reported. For homogeneity, the RSD of concentrations for all samples in each group was within the acceptance criteria of ≤ 10%, except for Week 2, Group 3 (12.1%) and group 4 (13.8%); therefore as part of the investigation, group 3 and 4 samples were rerun and the results (2.1% and 1.4%) were within the acceptance criteria. It is thought that the reason the week 2 group 3 and 4 results were outside acceptance criteria initially were due to a clogged syringe on the gas chromatograph. Back-up samples for Week 2 (Groups 3-4) were also analysed in triplicate, and the results were within the acceptance criteria. Back up sample results will not be reported since rerun samples were within the acceptance criteria. The dose formulations were within specification. Homogeneity testing showed that the formulation technique used produced homogeneous preparations.

Mortality (Table 1)

There were no premature deaths during the course of this study.

 

Clinical Observations (Table 1)

At dose levels up to and including 1000 mg/kg/day there were no clinical observations that were considered to be related to Di-Pentaerythritol administration.

 

Body Weights and Body Weight Changes (Table 2)

At dose levels up to and including 1000 mg/kg/day the group mean body weights and body weight gains were similar to the control group for the duration of the study.

  

Food Consumption (Table 3)

The group mean food consumption was similar across all groups.

 

Gross Pathology (Table 1)

At all dose levels, the type and distribution of gross necropsy findings did not indicate any association with test item administration.

 

Pregnancy Performance, Gravid Uterine and Fetal Weights (Table 4)

At dose levels up to and including 1000 mg/kg/day there was no effect on pregnancy frequency, gravid uterine weight, mean fetal weight, numbers of corpora lutea, number of implants or embryofetal survival including pre-implantation loss.

 

Fetal Abnormalities and Variants (Table 5, Table 6 and Table 7)

The type and distribution of all fetal abnormalities and variations, including those indicating the extent of skeletal ossification were similar in all groups and did not indicate any association with test item administration.

 

Applicant's summary and conclusion

Conclusions:
The NOEL for maternal and developmental toxicity was 1000 mg/kg bw/d.
Executive summary:

The potential for Di-Penta (Di-Pentaerythritol) to cause developmental toxicity in the rat was investigated in an OECD 414 compliant study. Four groups of 24 female Sprague-Dawley rats were dosed orally by gavage on Days 6-19 of gestation (where Day 0 was the day of detection of mating) at dose levels of 0 (corn oil only), 100, 300 or 1000 mg/kg bw/d. The following parameters were evaluated: clinical signs, body weights, food consumption, gross necropsy findings, gravid uterine weight, and examination of pregnancies and fetal examinations (external abnormalities, fetal weights, visceral and skeletal evaluations). Animals were killed on Day 20 of gestation. There were no effects of treatment on any of the measured parameters at any dose level; there was no maternal toxicity and no evidence for developmental toxicity. Therefore, under the conditions of the study, the maternal and embryofetal NOELs were considered to be 1000 mg/kg bw/d.