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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 January 2021 to Present

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Performed according to guideline study OECD 408 and under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Remarks:

Statement to be provided on finalisation of the study report.

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

204-794-1

EC Name:

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

Cas Number:

126-58-9

Molecular formula:

C10H22O7

IUPAC Name:

2,2'-[oxybis(methylene)]bis[2-(hydroxymethyl)propane-1,3-diol]

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in
man and the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain
was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited
- Age at study initiation: 6 to 8 weeks.
- Weight at study initiation: 208 to 293 g (males) / 161 to 212 g (females)
- Housing: Three to four of the same sex
-Cages: 12 hour light/dark cycle
-Bedding: Wood based bedding
-Enrichment: Chew devices and plastic shelters for hiding in, provided
- Diet (e.g. ad libitum): Teklad 2014C Diet (ad libitum)
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles
with sipper tubes.(ad libitum, except during urine collection)
- Acclimation period: 14 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:
Diet: Certificates of analysis scrutinized and approved before release for use.
Water: Certificate of anaylsis routinely provided by the water supplier


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24C
- Humidity (%): 40-70%.
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: To:
From 21 January 2021 (animal arrival) to 07 May 2021 (final necropsy day)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The dose volume was 5 mL/kg.
The dose volume was based on the most recent body weight measurement. Doses were given using a syringe with attached gavage cannula.

Vehicle:

methylcellulose

Remarks:

1% (aq)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
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 weighings of the test item.
Formulations prepared weekly
Stored at 2 to 8C, up to 8 days or at ambient temperature for 24 hours.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction and dilution in acetonitrile/water 10/90 v/v
followed by reverse phase high performance liquid chromatographic analysis with
evaporative light scattering detection. Sample concentrations were determined with reference
to external standards prepared in the concentration range 25 μg/mL to 250 μg/mL.

Week 1 and 12 formulations were sampled. For all groups, including the control, 4 × 1 mL
(accurately weighed) was sampled from the middle of the formulation

For Week 12, no results were generated due to chromatography deterioration during the injection sequence. This caused failure to meet the acceptance criteria for the system suitability tests defined in the method. An additional occasion of formulation preparation and analysis was carried out to mimic those intended for the Week 12 timepoint. The mean concentrations for the additional formulation preparation were within 7% of the nominal concentration, confirming the accuracy of formulation.

The homogeneity and stability of formulations during storage was confirmed under another study.

Duration of treatment / exposure:

Minimum 90 days

Frequency of treatment:

daily, 7 days each week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
1000 mg/kg/day was selected as the high dose for this study based on findings from previous studies (described below).

- Dose range finding studies:
In a 14-day dose range finding (DRF) study, treatment with Dipentaerythritol, by oral gavage, at 0, 500, 1000, or 1500 mg/kg/day to male and female rats (4/sex/group), did not elicit any obvious adverse reaction. It was therefore concluded that 1000 mg/kg/day (“limit” dose) could be used as the high dosage for a future repeat dose and reproductive/developmental toxicity screening test.

Additionally, in a preliminary screening test for reproductive and general toxicity, male and female rats (10/sex/group) were treated with Dipentaerythrito,l by oral gavage, at 0, 500,1000 mg/kg/day, for a minimum of 4 weeks. Apart from a reduced body weight gain at 1000 mg/kg/day in females during pregnancy, compared to controls, which recovered post partum, it was concluded that there were no other findings.

In a prenatal developmental toxicity study (OECD TG 414), female rats (24/group) were dosed orally by gavage at 0, 100, 300, 1000 mg/kg/day with Dipentaerythritol on Days 6-19 of gestation. At dose levels up to and including 1000 mg/kg/day, no test item-related effects on clinical observations, body weight gain, food consumption, gross pathology findings, pregnancy performance parameters, embryofetal survival or fetal weights, fetal abnormalities and variants, were observed. The maternal and embryofetal no observed effect levels (NOEL) were considered to be 1000 mg/kg/day.

- Fasting period before blood sampling for clinical biochemistry:
Animals were fasted overnight prior to blood sample collection for blood chemistry analysis

Positive control:

Not required for this study type.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Pretreatment period = at least once daily; Treatment period = at least twice daily
Animals were inspected visually for evidence of ill-health or reaction to treatment. Cages were inspected 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Week 1 = daily; Weeks 2-4 = twice weekly; Week 5 to 13 = once weekly
At these timepoints during the day:
*Pre dose
*Immediately after dosing (on return to home cage)
*On completion of dosing the group
*1-2 hours after dosing
*As late as possible in the working day

DETAILED PHYSICAL EXAMINATION AND ARENA OBSERVATIONS (general health monitoring):
- Time schedule: Once weekly starting from Pretreatment period
Animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
- Time schedule for examinations: Week -1, first day of treatment, weekly thereafter, on day of necropsy

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment period & Week 12
- Dose groups that were examined: Pretreatment = All groups examined; Week 12 = Groups 1 and 4
Eyes examined using binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: All animals
-Sample volume/route: nominally 0.5 mL into EDTA containing tubes/sublingual vein
-Analyzer: Bayer Advia 120
- Parameters examined:
*Hematocrit (Hct)#
* Hemoglobin concentration (Hb)
* Erythrocyte count (RBC)
* Absolute reticulocyte count (Retic)
* Mean cell hemoglobin (MCH)#
*Mean cell hemoglobin concentration (MCHC)#
* Mean cell volume (MCV)
* Red cell distribution width (RDW)
* Total leucocyte count (WBC)
* Differential leucocyte count:
* Neutrophils (N)
* Lymphocytes (L)
*Eosinophils (E)
* Basophils (B)
* Monocytes (M)
* Large unstained cells (LUC)
* Platelet count (Plt)
# Derived values calculated in ClinAxys.

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. In the presence of platelet clumping a manual count of the differential white blood cell parameters was performed.

COAGULATION
-Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: All animals
- Sample volume/route: nominally 0.5 mL into citrate anticoagulant containing tubes/sublingual vein
-Analyzer: Stago STA Compact Max
- Parameters examined:
*Prothrombin time (PT) - using IL PT Fibrinogen reagent.
*Activated partial thromboplastin time (APTT) - using IL APTT reagent.
*Clauss fibrinogen concentration (Fib) - using IL PT Fibrinogen reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: All animals
- Sample volume/route: nominally 0.7 mL into lithium heparin containing tubes/sublingual vein
- Analyzer: Roche Cobas 6000
- Parameters examined:
*Alkaline phosphatase (ALP)
*Alanine aminotransferase (ALT)
* Aspartate aminotransferase (AST)
* Total bilirubin (Bili)
* Urea#
* Creatinine (Creat)
* Glucose (Gluc)
* Total cholesterol (Chol)
* Cholesterol (HDL)
* Cholesterol (LDL)
* Sodium (Na)
* Potassium (K)
* Total protein (Total Prot)
* Albumin (Alb)
# Numerically equivalent to Blood Urea Nitrogen (BUN)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

PLASMA/SERUM HORMONES/LIPIDS: Yes - Thyroid hormone analysis
- Time of blood sample collection: at termination
- Anaesthetic used for blood collection: Yes, isoflurane, with no recovery
- Animals fasted: No
- How many animals: All animals
- Sample volume/route: 2.0mL into Grenier tubes with clotting activator/sublingual vein
- TSH Analytical technique: Luminex Milliplex MAP Assay
- T3 and T4 total concentration determination: LC-MS/MS
- Parameters examined:
*Triiodothyronine (T3)
*Thyroxine (T4)
*Thyroid stimulating hormone (TSH)

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Animals placed in metabolism cages overnight without food or water
- Parameters examined:
Using manual methods:
*Clarity and Color (App) - by visual assessment
*Volume (Vol) - using a measuring cylinder
*pH - using a pH meter
*Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek 500 instrument:
*Bile pigments (Bili)
*Blood pigments (UBld)

Using a Roche Cobas 6000 Analyzer:
*Protein - total (T-Prot) and concentration (Prot)
*Glucose - total (T-Gluc) and concentration (U-Gluc)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sensory Reactivity Observations and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges.
Three trials were performed.
At any point during the observations, additional comments were made as free text where
considered appropriate.

Motor Activity
During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.


IMMUNOLOGY: No

OTHER:
VAGINAL SMEARS FOR ASSESSMENT OF ESTROUS CYCLES:
Wet smears were taken from the vagina of all surviving females using pipette lavage for four days before scheduled termination. The last smear was taken on the morning of necropsy. Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) at termination and were used to assist in the histological evaluation of estrogen sensitive tissues.

Sacrifice and pathology:

METHOD OF KILL:
Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY:
Time Schedule: Following 13 weeks of treatment
All study animals were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues was performed.
All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.

GROSS PATHOLOGY: Yes - see TABLE 1 attached for details
HISTOPATHOLOGY: Yes - see TABLE 1 attached for details

BONE MARROW
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period and from animals killed prematurely during the study.
*Fixation: Smears were air dried and subsequently fixed in methanol.
*Analysis: No examinations were performed, however, the smears were retained for possible future examination.
*Retention: The smears were transferred to archives and will be retained for the same period as the study raw data.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together, unless specified above.
Requisite organs were weighed for study animals killed at scheduled intervals.

TISSUE FIXATION
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
*Testes: In modified Davidson’s fluid.
*Eyes: In Davidson’s fluid.
*Bone marrow: Smears were air dried and subsequently fixed in methanol.

SPERM ANALYSIS
Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed. The following tests were performed:

*Sperm motility - all groups
A sample of sperm was expressed from the vas deferens into pre-warmed (37C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 m depth cannula by capillary action and at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS Computer Assisted Sperm Analyzer (CASA). The percentages of motile and progressively motile sperm were reported.

*Left vas deferens - all males:
sperm sample was assessed for motility using a computer assisted sperm analyzer (CASA).
Samples from all animals were retained in neutral buffered formalin pending any future requirement for additional morphology analysis.

*Left cauda epididymis:
The left cauda epididymis of each male was weighed, then retained deep frozen (10 to 30°C) pending any future requirement for additional analysis.

*Left testis:
The left testis of each male was weighed, then retained deep frozen (10 to 30°C) pending any future requirement for additional analysis.

HISTOLOGY
*Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

*Full List: All animals killed or dying prematurely.

*All animals of Groups 1 and 4 killed at a scheduled interval.

*Abnormalities only: All animals of Groups 2 and 3 killed at a scheduled interval.

*Eyes: Particular attention was paid at scheduled termination to any clearly or potentially test item related Ophthalmoscopy findings, there were none to note, which was communicated to Histology by the Study Director in advance of the procedure. In addition, special attention was paid to the orientation of the eyes with respect to the ophthalmic abnormalities of interest.

*Routine staining: Sections were stained with hematoxylin and eosin.

LIGHT MICROSCOPY:
Tissues preserved for examination were examined as follows:
*Premature deaths: all animals form all groups - All specified tissues in Table 1 (attached)
*Scheduled kill: Group 1 and 4 - All specified tissues in Table 1 (attached)
*Scheduled kill: Group 2 and 3 - abnormalities only
*Ophthalmoscopy findings: Affected animals at scheduled sacrifices - Particular attention was paid at scheduled termination to any clearly or potentially test item related findings.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Optional endpoint(s):

Optional endpoints: Yes
Prolactin ICH staining of pituitary - all female groups
This additional examination is being performed in order to investigate (pre)neoplastic findings in high dose females.
Please see letter (ANNEX 1 attached), informing ECHA of this additional work.

Other examinations:

Not required for this study type

Statistics:

All statistical analyses were carried out separately for males and females. the analyses were carried out using the individual animal as the basic experimental unit, except for food consumption data which were analysed on a cage basis.
The following comparisons were performed: Group 1 vs 2, 3 and 4

Grip strength, motor activity, body weight, organ weight and clinical pathology data - the sequence of statistical tests were as follows:
-Parametric analysis performed if Bartlett's test for variance homogeneity (Bartlett, 1937) was not significant at 1% level. 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 (Dunnett, 1955; 1964) was performed instead.

-Non-parametric analysis performed if Bartlett's test was still significant at 1% level following both logarithmic and square-root transformations. 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 (Steel, 1959) was performed instead.

-For grip strength, motor activity and clinical pathology data, if 75% of the data (across all
groups) were the same value, Fisher’s exact tests (Fisher, 1973) were performed.

-For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The
treatment comparisons were made on adjusted group means in order to allow for differences
in body weight which might influence the organ weights.

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

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical signs and arena observations recorded were considered to be unrelated to treatment of Dipentaerythritol.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female receiving Dipentaerythritol at 300 mg/kg/day (Animal number 139) was killed for welfare reasons during Week 6 of treatment because of marked breathing impairment.
The in-life findings responsible for the early sacrifice of this animal were related to the esophageal perforation, and this accidental death was considered to be due to an intubation error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was considered to be no effect of treatment on body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was considered to be no effect of treatment on food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There was considered to be no effect of treatment during ophthalmic examination.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared to control, mean hematocrit and mean cell hemoglobin concentration were statistically significantly higher for males receiving 1000 mg/kg/day but there was no effect for other erythrocytes parameters or for females.

Additionally, mean white blood cell count was higher than control, for females receiving 300 or 1000 mg/kg/day, although this was associated with no statistical significance or dosage relationship. Mean lymphocytes were higher than control for females receiving 300 or 1000 mg/kg/day, however again, there was no statistical significance or dosage relationship.

Higher mean neutrophils, compared to control, were evident for treated males at all dose levels, and reflected a high mean control value due to an atypically high individual value for one control male. Marginally higher basophils were observed for males receiving 300 or 1000 mg/kg/day and for females at 100 or 300 mg/kg/day but not for females at 1000 mg/kg/day.

Finally, mean Large Unstained Cell concentration was slightly low, compared to control, for males receiving 300 or 1000 mg/kg/day but conversely high for females at these dose levels, with no dose relationship apparent.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed during Week 13 of treatment revealed, when compared to control, lower group mean values for alanine amino-transferase and aspartate amino-transferase in all groups of treated males. Whereas, both of these parameters were noted to be dose dependently higher than control in females treated with 100 and 300 respectively, however, no such effect was evident in Group 4 females.

High-density lipoproteins and low-density lipoproteins were lower than control in all treated groups of males. However, these changes from control lacked a dose dependent relationship and statistical significance. Additionally, such an effect was not evident in the females.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

For TSH male rats showed a slight dose-related response to the test item but the responses were highly variable and not statistically significant. These results indicate that there is no dose related effect on serum TSH concentrations from administering Dipentaerythritol to rats.

For T3 data, no statistically significant difference (p-value > 0.05) was observed between the treatment groups (2 to 4) and control group (Group 1). For T4 data, no statistically significant difference in males (p-value > 0.05) was observed between the treatment groups (2 to 4) and control group (Group 1). For T4 data, no statistically significant difference in females (p-value > 0.05) was observed between the treatment group 3 and control group (Group 1), although there was a statistically significant difference between females in treatment Group 2 (p-value <0.05) and Group 4 (p-value <0.01) and control Group 1.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis investigations carried out during Week 13 of treatment revealed, when compared to control, lower urinary protein and total protein values for males of all treatment groups. Whereas, only females of Groups 2 and 3, treated with 100 and 300 mg/kg/day respectively, were noted to have similarly low values for urinary protein, and Group 4 females treated with 1000 mg/kg/day had similarly low values for total protein.

Total glucose was evidently higher than control only in females of Group 2, treated with 100 mg/kg/day. Additionally, urinary glucose was lower than control in both females Groups 2 and 3, treated with 100 or 300 mg/kg/day respectively.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was considered to be no effect of treatment on sensory reactivity, grip strength or motor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Body weight adjusted pituitary weights were statistically significantly higher in all treated females, compared to controls.

Body weight adjusted thymus weights were statistically significantly lower in males receiving 1000 mg/kg/day compared to controls (2/10 males with very low values).

See TABLE 2 attached, for Group Mean organ weight data

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Small thymus was reported at necropsy for two males receiving 1000 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal involution/atrophy of the thymus was seen in four males receiving 1000 mg/kg/day, correlating with the necropsy findings and organ weight changes in this group.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mammary adenocarcinoma was seen in two females receiving 1000 mg/kg/day, and lobuloalveolar hyperplasia was seen in an additional two females of the same dose group.

Other effects:

no effects observed

Description (incidence and severity):

VAGINAL SMEARS
There was considered to be no effect of treatment on oestrus in females

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Preliminary NOAEL, to be confirmed once study report is finalised

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: This NOAEL is preliminary. Additional investigations with regards to the neoplastic findings are currently in progress.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: mammary gland

Organ:

mammary gland

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Any other information on results incl. tables

FORMULATION ANALYSIS:

For Week 1 results, mean concentrations were within 10% of the nominal concentration,
confirming the accuracy of formulation. 

Applicant's summary and conclusion

Conclusions:

Daily oral gavage administration of Dipentaerythritol at doses up to 1000 mg/kg/day were well tolerated, in both males and females, with regards to inlife findings: with no exhibition of any behavioural or clinical signs, or effects on food consumption or body weight.

Pathological findings were noted at 1000 mg/kg/day. In females these consisted of mammary adenocarcinoma (2/10 females) and lobuloalveolar hyperplasia (additional 2/10 females), which were considered to be of adverse severity. In addition, statistically significant increases in pituitary weights were noted for all female treatment groups, compared to the control.

In males at 1000 mg/kg/day, minimal involution/atrophy of the thymus was noted in 4/10 males, correlating with lower relative thymus weight and small thymus reported at necropsy, in 2/10 males.

Additional IHC staining of pituitary (in all female groups) is still ongoing with the aim to understand any potential Prolactin changes and its correlation with the pituitary weight change and histopathology changes in the mammary gland.
As a result of this ongoing work, a preliminary no observed adverse effect level (NOAEL) of 300 mg/kg/day has been identified for females. This NOAEL may be revised following the conclusion of the this additional work.

For males a preliminary NOAEL of 1000 mg/kg/day has been considered.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of Dipentaerythritol, a chemical intermediate, in a 13-week daily administration oral gavage study in Crl:CD(SD) rats at doses of 100, 300 and 1000 mg/kg/day, to aid selection of doses for subsequent longer duration studies.

The animals were examined for clinical signs, body weight, body weight gain, food consumption, ophthalmology, functional observations, clinical pathology parameters which comprised haematology, coagulation, clinical chemistry and urinalysis, thyroid hormone analysis, gross necropsy, organ weights and histopathological analysis.

Daily oral gavage administration of Dipentaerythritol at doses up to 1000 mg/kg/day were well tolerated, in both males and females, with regards to inlife findings:  with no exhibition of any behavioural or clinical signs, or effects on food consumption or body weight. The administration of Dipentaerythritol did not result in any unscheduled deaths considered related to treatment.

Pathological findings were noted at 1000 mg/kg/day. In females these consisted of mammary adenocarcinoma in 2 out of 10 females and lobuloalveolar hyperplasia in an additional 2 out of 10 females, which were considered to be of adverse severity.  In addition, statistically significant increases in pituitary weights were noted for all female treatment groups, compared to the control.

In males at 1000 mg/kg/day, minimal involution/atrophy of the thymus was noted in 4/10 males,  correlating with lower relative thymus weight and small thymus reported at necropsy, in 2/10 males.

Additional IHC staining of pituitary (in all female groups) is still ongoing with the aim to understand any potential Prolactin changes and its correlation with the pituitary weight change and histopathology changes in the mammary gland.

As a result of this ongoing work, a preliminary no observed adverse effect level (NOAEL) of 300 mg/kg/day has been identified for females. This NOAEL may be revised following the conclusion of the this additional work. For males a preliminary NOAEL of 1000 mg/kg/day has been considered.