2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined repeated dose oral toxicity and toxicity to reproduction study was conducted with di-pentaerythritol, according to OECD method 422 (draft version; 22 March 1990) (Powell et al, 1992). Sexually mature rats were exposed to di-pentaerythritol via gavage at 0, 500 and 1000 mg/kg bw/day. There were no findings at either dose level that would indicate an immediate requirement for more detailed studies. No effects of treatment were observed on food intake, bodyweight change, haematology, biochemistry, organ weights, post mortem findings and histopathology. There did not appear to be any effects on mating or on pup viability. The NOAEL for reproductive effects was considered to be 1000 mg/kg bw/day

 

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study planned

Study period:

Depending on finalisation of ECHA decision

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Di-Pentaerythritol
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: no previous GLP extended one generation guideline studies are available on Di-Pentaerythritol
- Available non-GLP studies: no relevant data available
- Historical human/control data: no data available
- (Q)SAR: no relevant data available; no profiler alerts in the OECD QSAR toolbox (for oestrogen binding; DART scheme; retinoic acid receptor binding)
- In vitro methods: no relevant data available
- Weight of evidence: no sufficient data available
- Grouping and read-across: no data available
- Substance-tailored exposure driven testing: Not applicable
- Approaches in addition to above: Not applicable
- Other reasons: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- The performance of the extended one-generation reproductive toxicity study - basic test design (Cohorts 1A, and 1B without extension) is a standard information requirement for substances with a tonnage > 1000 t/a.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:

- Details on study design / methodology proposed: An Extended One-Generation Reproductive Toxicity Study in rats, with basic test design (cohorts 1A and 1B without extension), by the oral route is proposed using the registered substance.

- There was no indication of particular concerns on (developmental) neurotoxicity or (developmental) immunotoxicity from current available data of the substance. No significant adverse maternal or developmental effects were observed in the pre-natal developmental toxicity studies in rats and rabbits up to 1000 mg/kg b.w. Cohorts 2A/2B or 3 is therefore not proposed.

- Additional IHC staining of pituitary in the OECD 408 study is still ongoing at the lab with the aim to understand any potential Prolactin changes and it’s correlation with the pituitary weight change and histopathology changes in the mammary gland in the OECD 408 study. Potential serum prolactin hormone analysis of the F0 and F1 animals are under discussion in the OECD 443 study design as well. This will be confirmed after the OECD 408 additional testing been completed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

The study design will be specified when results from the additional testing in the OECD 408 studies are available.

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.10.1991 to 26.05.1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)

Version / remarks:

draft 22 March 1990

GLP compliance:

no

Remarks:

The report states that the study was conducted according to the principles of GLP, but the work was not certified and was not subject to Quality Assurance review .

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): dipentaerythritol
- Substance type: white powder
- Physical state: solid
- Analytical purity: 96.2%
- Purity test date: received 2 September 1991
- Lot/batch No.: 9404
- Expiration date of the lot/batch: 2 November 1992
- Storage condition of test material: within the Formulation Department at room temperature in darkness

Species:

rat

Strain:

other: Crl:CD (SD) BR VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd.
- Age at study initiation: (P) Males 12 wks ±1 day, Females (sexually mature, virgin) 7 weeks ±1day;
- Weight at study initiation: (P) Males: 440-446 g; Females: 224-228 g
- Fasting period before study: overnight
- Housing: pre-mating; suspended stainless steel cages equipped with solid sides and wire grid front, back, floor, and top. During mating male and female pairs were housed in plastic breeding cages (North Kent Plastics, RM2 type).
- Acclimatisation period: 1 week
- Diet : Biosure Laboratory Animal Diet No. 1 ad libitum
- Water : tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Five males and five females were supplied for health check purposed. They were killed within 24 hours of arrival and subjected to routine macroscopic examination.

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose

Details on exposure:

The test material was ground in a mortar with a small volume of 1% methylcellulose until a smooth paste was formed. The formulation was then gradually made up to volume and mixed using a high speed homogeniser. A series of suspensions were then made to give the required concentrations. Formulations were prepared daily and dosed on the day of preparation.

Details on mating procedure:

Rats were paired 1 male to 1 female during the mating period (14 days). Mating was confirmed (by the presence of a sperm plug and vaginal smears). After the mating period, the males and females were returned to standard single housing.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Analytical verification of doses was not carried out.

Duration of treatment / exposure:

Males: 7 weeks; Females: 9 weeks.

Frequency of treatment:

Daily

Details on study schedule:

Rats underwent 2 weeks of treatment prior to blood collection and commencement of the mating phase. The mating phase lasted for 2 weeks. Repeat blood collections were taken from female rats prior to parturition, at week 5. Males were sacrificed after 7 weeks of treatment. Females were sacrificed after 9 weeks of treatment of post partum day 4 (non-pregnant females were sacrificed after 9 weeks treatment).

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

20 per dose, 10 males, 10 females

Control animals:

yes

Details on study design:

A range finding study was conducted prior to the main study, the limit dose of 1000 mg/kg/day was established as a suitable high dosage level. Controls were concurrent but it is not stated whether they were unexposed, or administered the vehicle alone.

Positive control:

A positive control was not examined.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: recorded at allocation to groups, commencement of treatment and thereafter weekly. Females also weighed daily from the commencement of the mating period until parturition and Days 0 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, measured during the pre-treatment and pre-mate period for males and females, during the post mate period for males and post partum for females. Water intake was only measured by visual observation, and could therefore not be subjected to statistical analysis.


HAEMATOLOGY: Yes, assessed after two weeks of treatment, and again after 5 weeks treatment in females
- Anaesthetic used for blood collection: Light ether anaethesia
- Animals fasted: Yes, overnight
- Parameters examined: PCV, haemoglobin, RBC, MCHC, MCV, total and differential WBC, platelets, examination for abnormal cell morphology, thrombotest, reticulocyte count.


CLINICAL CHEMISTRY: Yes, assessed after two weeks of treatment, and again after 5 weeks treatment in females.
- Animals fasted: Yes, overnight
- Parameters examined: Glucose, GPT, GOT, CPK, gammaGT, total protein albumin, globulin, urea nitrogen, AP, total bilirubin, creatine, sodium, potassium, calcium, inorganic phosphorus, chloride, cholesterol.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily starting from 1 week prior to mating and throughout the mating phase.

Sperm parameters (parental animals):

Sperm parameters were not examined.

Litter observations:

For each litter the pups were counted, sexed, weighed and examined for gross abnormalities as soon as possible after birth. Litters were examined daily for dead/missing (i.e. cannibalised) pups. Pups were weighed again on Day 4 post partum.

Litter weight and mean pup weight were calculated from individual pup weight. Sex ratios were calculated at birth and at day 4.
Pup loss on completion of parturition was calculated according the formula: ((total no. young at birth - no. live young) / total no. young at birth) x100
Cumulative pup loss was calculated according to the formula: ((total no. young at birth - no. live young at Day 4) / total no. young at birth) x100.

Postmortem examinations (parental animals):

All rats were subject to gross necropsy at study termination, and examined for macroscopic abnormalities. Organ weights: adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, prostrate, testes, thymus, spleen. The following tissues were examined histologically (* denotes tissues examined from control and high dose groups only): adrenals*, brain*, epididymides*, heart*, kidneys*, liver*, lungs, mammary gland, macroscopically abnormal tissues, ovaries*, pituitary*, prostate*, seminal vesicles*, spleen*, testes*, thymus, thyroids, uterus* and vagina*.
Implantation loss was calculated according to the formula: ((no. implantation sites - total young born) / no. implantation sites) x 100.

Postmortem examinations (offspring):

Litters were examined for abnormalities and sex confirmed.

Statistics:

ANOVA followed by Williams' test, or Kruskal-Wallis 'H' statistic followed by Shirley's test where appropriate.

Reproductive indices:

Fertility index = (no. pregnant females / no. females showing evidence of copulation) x100.
Gestation index = (no. pregnant females with live young / no. pregnant females) x 100.

Offspring viability indices:

Viability index (day 4) = (no. live young at day 4 / no. live young at birth) x100.

Clinical signs:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (P0) " section below.

Mortality:

no mortality observed

Description (incidence):

For more details, see the " details on results (P0) " section below.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (P0) " section below.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

For more details, see the " details on results (P0) " section below.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (P0) " section below.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (P0) " section below.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (P0) " section below.

There were no deaths during the study. There were no clinical signs associated with treatment. The bodyweight gain of treated males was similar to that of controls throughout the study. Females given 1000 mg/kg/day showed reduced gain during pregnancy but recovered post partum.
No effects of treatment on food intake were apparent over the time periods of measurement.
There were no changes in the haematological and biochemical parameters measured that were clearly indicative of a reaction to treatment.
There were no differences in organ weights that indicated any treatment-related effects.
There were no differences or changes noted at histopathological examination that indicated any treatment-related effects.
The oestrus cycle was unaffected by treatment and the pregnancy rate was good in all groups. Time to mating and duration of pregnancy were within normal limits. Two females were not pregnant, and one dam exhibited total resorption of her litter.

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 effects were seen

Clinical signs:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (F1) " section below.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

For more details, see the " details on results (F1) " section below.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (F1) " section below.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

For more details, see the " details on results (F1) " section below.

Histopathological findings:

not examined

There were no obvious effects of treatment on litter size or sex ratio at birth, or on survival at post partum day 4. Litter and mean pup weight also appeared unaffected. There were no findings at necropsy, and no clinical signs were reported. A small number of pups died at birth, or by post partum day 4, none of these deaths were attributed to treatment. Litter data are shown in Table 1.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no obvious effects of treatment on litter size or sex ratio at birth, or on survival at post partum day 4. Litter and mean pup weight also appeared unaffected. There were no findings at necropsy, and no clinical signs were reported.

Critical effects observed:

no

Reproductive effects observed:

no

Table 1: Litter data

Group	No. of Animals		Implant sites	Implant loss %	At birth								At day 4
					Litter size					Pup loss %	Litter Wt (g)	Mean pup wt. (g)	Litter size				Cumulative loss%	Litter wt (g)	Mean pup wt.(g)
	Mated	Pregnant			male	female	Total	%males	live				male	female	Total	%males
Control	10	10	17.5	4.6	9	7.7	16.7	54.9	16.3	2.3	102.9	6.3	8.7	7.3	16	55.3	4.2	161.6	10.2
500 mg/kg/day	10	8	18.4	4.1	9.4	8.3	17.6	53.4	17.5	0.7	107.8	6.2	9.3	8	17.3	53.8	2.1	165.2	9.6
1000 mg/kg/day	10	9	17.1	3.7	7.7	8.8	16.4	46.2	16.4	0	101.4	6.2	7.3	8.6	15.9	45.8	3.4	158.2	10

Conclusions:

The study author concluded that there were no findings at either of the dosage levels employed in this study to indicate an immediate requirement for more detailed studies.

Executive summary:

The study was conducted to assess the repeated dose oral toxicity and toxicity to reproduction of dipentaerythritol, in accordance with OECD guideline 422 (draft version; 22 March 1990).

Sexually mature rats were exposed to dipentaerythritol via oral gavage at dose levels 0, 500, and 1000 mg/kg bw/d. The rats were exposed throughout pregnancy and the effects on both parents and the F1 generation were assessed.

Male rats were exposed for 7 weeks prior to sacrifice and necropsy. Females were exposed for 9 weeks until post partum day 4, when they along with their pups were sacrificed and subjected to gross necropsy.

There were no findings at either of the dosage levels in this study to indicate an immediate requirement for more detailed studies. No effects of treatment were observed on food intake, bodyweight change, haematology, biochemistry, organ weights, post mortem findings and histopathology. There did not appear to be any effects on mating or on pup viability.

The NOAEL for reproductive and developmental effects can therefore be considered to be 1000 mg/kg bw/d.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

An OECD screening study is available for the submission substance. Sufficient to address requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study in the rat was performed using dose levels of 0, 100, 300 and 1000 mg/kg bw/d. No maternal toxicity was observed up to and including the highest dose level. There were no effects of treatment on pregnancy performance parameters, embryofetal survival or fetal weights, fetal abnormalities and variants. Under the conditions of the study, the maternal and embryofetal NOAELs were considered to be 1000 mg/kg bw/day.

A rabbit study was conducted to assess the maternal and developmental toxicity of Dipentaerythritol, in accordance with OECD guideline 414. Female rabbits were administered doses of 0, 100, 300 or 1000 mg/kg bw/day daily via oral gavage from GD 6 to 28 (inclusive). No adverse effects were observed on maternal or developmental toxicity. It was therefore concluded that 1000 mg/kg/day was the maternal no observed effect level (NOEL) and the fetal no observed adverse effect level (NOAEL).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.08.2015 to 29.02.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- 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

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system 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:

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

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:

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

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

For more details, see the Table 4 attached below.

Dead fetuses:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

For more details, see the Table 4 attached below.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For more details, see the "embryotoxic / teratogenic effects" section below.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For more details, see the "embryotoxic / teratogenic effects" section below.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For more details, see the "embryotoxic / teratogenic effects" section below.

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.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: 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 a

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

 

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 April 2021 (animal arrival) to 04-07 May 2021 (necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: • Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

yes

Remarks:

As noted for OECD TG 414.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

As noted for OECD TG 414.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Humidity was slightly lower than the minimum limit of 45% during 5 days of the study. Temperature was slightly higher than the recommended 21˚C during 2 days of the study. These deviations were not considered to compromise the study.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL INFORMATION:
- Test item provided by the study sponsor
- Material IUPAC name: 2,2’-[oxybis(methylene)bis[2- (hydroxymethyl)propane- I ,3-diol]
- Batch number: 200806770
- Purity 96.28%
- White crystalline powder (odourless)
- Stored at ambient temperature (15 to 25°C)
- Expiry date: 11 August 2022

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available for the laboratory.

TEST ANIMALS
- Source: Envigo RMS (UK)
- Age at study initiation: 18 to 22 weeks
- Weight at study initiation: 2.68 to 4.31 kg
- Fasting period before study: n/a
- Housing: Individually housed in suspended cages fitted with perforated floor panels and mounted in batteries.
- Diet (e.g. ad libitum): Teklad 2930 Diet restricted to 200 g/animal/day
- Water (e.g. ad libitum): Potable water provided ad libitum.
- Acclimation period: Five days before commencement of treatment (GD1 to
GD5).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 to 21°C
- Humidity (%): 45 to 70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 14 hours light: 10 hours dark.

IN-LIFE DATES: From 06-09 April 2021 (animal arrival), to 04-07 May 2021 (necropsy).

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose (aq).

Details on exposure:

The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer. A series of formulations at the required concentrations were prepared by dilution of individual weighing of the test item.
The volume of the dose administered to animals was 5 ml/kg. The formulated concentration for the doses of 100, 300 and 1000 mg/kg bw/day was 20, 60 and 200 mg/mL, respectively.
The formulations were prepared weekly and stored refrigerated at 2-8°C.

Analytical verification of doses or concentrations:

yes

Remarks:

Samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test item.

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test item.

Details on mating procedure:

Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females were not closely related. After mating each female injected intravenously with 25 i.u. luteinizing hormone. The day of mating was considered to be Gestation day 0.

Duration of treatment / exposure:

Gestation day 6 to 28 (inclusive)

Frequency of treatment:

Once daily for the duration of the treatment.

Duration of test:

06 April 2021 (animal arrival) to 04-07 May 2021 (necropsy).

Dose / conc.:

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

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were selected based on the results of a preliminary embryo-fetal study conducted in the New Zealand White rabbit at dose levels of 0, 500, 750 and 1000 mg/kg bw/day. Such doses were well tolerated and there were no findings that would preclude the use of 1000 mg/kg bw/day as the high dose on the main study. A slightly higher mean placental weight was observed at 1000 mg/kg bw/day was observed but there were no adverse effects on fetal weight or macropathology. Therefore, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

Maternal examinations:

Mortality:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss. A complete necropsy was performed in all cases.

Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing:
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
- Pre-dose observation
- One to two hours after completion of dosing
- As late as possible in the working day.

Clinical Signs:
A detailed physical examination was performed on each animal on Days 1, 6, 12, 18, 23 and 29 after mating to monitor general health.

Body Weight:
The weight of each adult was recorded on arrival (Day 1), on Day 3 and then on Days 6 to 29 after mating.

Food Consumption:
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 after mating.

Ovaries and uterine content:

For females surviving to term, the gravid uterine weight (including cervix and ovaries) were recorded.

The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn, the number of corpora lutea, implantation sites, resorption sites (classified as early or late), fetuses (live and dead) were recorded.
Apparently non-pregnant animals and for apparently empty uterine horns, the absence or number of uterine implantation sites was confirmed.
For females exhibiting pregnancy loss, the expelled uterine contents were identified and examined, as appropriate.

Blood sampling:

Not performed.

Fetal examinations:

Examination of all viable fetuses and placenta were dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Fetuses examined externally with abnormalities were recorded. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fetal Pathology Examination:
Bouin’s fixed heads: Serial sections were examined for soft tissue abnormalities.
Alizarin Red stained fetuses and torsos: Assessed for skeletal development and abnormalities.

Statistics:

The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights:

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. For all other analyses the H1 approximate test, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Historical control data:

Historical control data was used to compare potential adverse events.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs at routine physical examination that could be attributed to treatment and no signs were observed in association with dose administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female receiving 1000 mg/kg bw/day (no. 73) was killed for welfare reasons on GD21 after exhibiting signs of abortion (red discharge vaginal area with fetus evident in under tray). Macroscopic examination of the female revealed dark renal pelvis in both kidneys and the left kidney was an abnormal shape with an irregular surface. Uterine examination revealed eight implantation sites, three of which were early resorptions and the remaining five sites were empty following abortion. This was an isolated incidence and is considered to be unrelated to administration of Di-Pentaerythritol.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain and maternal weight gain following adjustment for the gravid uterine weight was unaffected by treatment at dose levels up to and including 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by administration of Di-Pentaerythritol at all dose levels.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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):

Macroscopic examination of females on GD29 did not reveal any findings that could be related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

No adverse effects were observed on maternal animals on mortality, clinical observations, body weight, gravid uterine weight, food consumption or macropathology.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female receiving 1000 mg/kg bw/day (no. 73) was killed for welfare reasons on GD21 after exhibiting signs of abortion (red discharge vaginal area with fetus evident in under tray). This was an isolated incidence and is considered to be unrelated to administration of Di-Pentaerythritol.

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

Details on maternal toxic effects:

No adverse effects were noted on litter data as assessed by the number of implantations, resorptions, live young and the extent of pre- and post-implantation loss. Whilst a spontaneous absorption was observed, it was considered an isolated incident and not treatment related.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

other: No adverse effects observed

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

endocrine findings

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

In the absence of any effect on embryo-fetal survival or any structural abnormalities in the fetal male and female reproductive organs at 1000 mg/kg/day, the statistically significant higher mean percentage of male fetuses at this dose level was concluded to be fortuitous.

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):

Skeletal examination revealed a slight increase in incidence of full supernumerary 13th rib and 20 thoracolumbar vertebrae at 100 and 1000 mg/kg/day when compared with concurrent Control but the incidences were within the litter historical control data (HCD)range. At 100 mg/kg/day there was a slight increase in incidence of caudal shift of pelvic girdle compared with concurrent Control, but within the HCD range. As these abnormalities are variants, they are not considered adverse.

Visceral malformations:

not specified

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

No adverse effects were observed on mean placental, litter and fetal weights. Whilst some abnormalities were observed on on fetal pathology they were not considered to be significant or treatment related.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: No adverse effects observed.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

No adverse effects were observed on maternal or developmental toxicity. It was therefore concluded that 1000 mg/kg/day was the maternal no observed effect level (NOEL) and the fetal no observed adverse effect level (NOAEL).

Executive summary:

This study was conducted to assess the maternal and developmental toxicity of Dipentaerythritol, in accordance with OECD guideline 414. Female rabbits were administered doses of 0, 100, 300 or 1000 mg/kg bw/day daily via oral gavage from GD 6 to 28 (inclusive).

No adverse effects were observed on maternal condition, body weight, food consumption, maternal macropathology or embryo-fetal survival/development.

Fetal skeletal examination revealed a slight increase in incidence of the minor findings full supernumerary 13th rib and 20 thoracolumbar vertebrae at 100 and 1000 mg/kg bw/day and a slight increase in incidence of caudal shift of pelvic girdle at 100 mg/kg bw/day, but both were within the historical control range; as these abnormalities are variants, they were not considered adverse.

It was therefore concluded that 1000 mg/kg bw/day was the maternal no observed effect level (NOEL) and the fetal no observed adverse effect level (NOAEL).

 

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A recent GLP and guideline-compliant study is available. Sufficient to address requirements.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No evidence of reproductive or developmental toxicity potential was seen in a combined screening assay performed with di-pentaerythritol at dose levels of up to and including the limit dose of 1000 mg/kg bw/day. No effects were observed in  developmental toxicity studies in rats and rabbits performed with di-pentaerythritol at dose levels of up to 1000 mg/kg bw/day. Therefore, no classification is therefore required for reproductive toxicity according to Regulation (EC) No 1272/2008.