Reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3-diol and 2-ethylpropane-1,3-diol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Preliminary Toxicity Study by Oral Gavage Administration to Crl:CD(SD) Rats for 7 days

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 Jun 2021-5 July 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Principles of method if other than guideline:

The purpose of the study was to assess the systemic toxicity of the study in a 7-day oral gavage study in Crl:CD(SD) rats, to select a suitable high dose for a subsequent repeated dose studies. The study was not designed to meet any particular regulatory requirements.

Parameters analysed in the study are detailed in relevent sections below.

GLP compliance:

no

Remarks:

The study was conducted in a GLP facility, however no claim of GLP compliance was made. The study was a preliminary dose-range-finding study prior to an GLP study.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3-diol and 2-ethylpropane-1,3-diol

EC Number:

938-572-7

IUPAC Name:

Reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3-diol and 2-ethylpropane-1,3-diol

Details on test material:

The registered substance, Reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3-diol and 2-ethylpropane-1,3-diol is also known as Dimethylolpropane technical.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at start of treatment: 34 to 40 days
- Weight at start of treatment: Males: 158 to 196 g; Females: 138 to 160 g
-Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
-Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable.
-Number of animals per cage: 3 of the same sex.
-Bedding: Wood based bedding which was changed at appropriate intervals each week.
- Diet :Teklad 2014C, pelleted diet. Availability not-restricted
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Availability not-restricted
- Acclimation period: 5 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Certificates of analysis for the diet were scrutinized and approved before any batch of diet
was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminant was known that may have interfered with or prejudiced the outcome of the study and, therefore, no special assay was performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24°C
- Humidity (%): 40-70%.
- Air : Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: From: 23 Jun 2021 To: 5th July 2021

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gavage, using a suitably graduated syringe and a flexible cannula inserted via the mouth. Prior to the dosing of each individual animal, the cannula was dipped into a container filled with 5% glucose solution to aid intubation.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

-VEHICLE: Purified water
- Concentration in vehicle: 50, 75, and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg b.w.
-Method of preparation The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were
prepared in ascending order.
-Frequency of preparation: Weekly.
-Storage of formulation: Refrigerated (2 to 8°C).
-Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

no

Remarks:

The homogeneity and stability of formulations in the concentration range 1 to 250 mg/mL during storage were confirmed as part of another study, but formulation analysis was not performed on this study

Duration of treatment / exposure:

7 days

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3 animal/sex/dose group

Control animals:

no

Details on study design:

- Dose selection rationale:

The acute toxicity of the substance was investigated in a previou study where a single oral administration to the Sprague Dawley rat followed by a 14-day observation period (RTC Study No.: A2908). A first group of three female animals was initially dosed at 2000 mg/kg body weight (Step 1).
No mortality occurred. Clinical signs were observed in all animals on the day of dosing;
hunched posture and ataxia occurring from 30 minutes up to four hours after dosing,
piloerection occurring from two up to four hours after dosing and decreased activity
occurring only at two hours after dosing were recorded. In addition, one animal showed râles
from 30 minutes up to two hours after dosing. A second group of three female animals was
then dosed at the same dose level (Step 2). No death occurred. Decreased activity was
recorded in all animals from 30 minutes up to four hours after dosing, ataxia from 30 minutes
up to two hours after dosing and hunched posture and piloerection four hours after dosing.
These signs recovered on Day two of study. Body weight changes recorded during the study
were within the expected range for this strain and age of animals. No abnormality was
observed at necropsy examination performed at the end of the observation period on animals
of both groups. The lack of death and the complete recovery of the observed clinical signs demonstrate the acute toxicity estimates to be greater than 2000 mg/kg body weight. Accordingly, dose levels of 500, 750 and 1000 mg/kg/day were selected for this study.

- Animal assignment: Randomly allocated on arrival.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

-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
occupants. 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 acclimatization period, observations of the animals and their cages were recorded
at least once per day.

-Signs Associated with Dosing
Detailed observations were recorded daily at the following times in relation to dose
administration:
 Pre-dose.
 As each animal was returned to its home cage.
 At the end of dosing
 One to two hours after dosing.
 As late as possible in the working day.

Clinical Signs
A detailed weekly physical examination was performed on each animal to monitor general
health.

BODY WEIGHT:
- The weight of each animal was recorded immediately before treatment, once during the treatment week and on Day 8.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week of 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: No.

HAEMATOLOGY: NO

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

Necropsy
Animals were killed on Day 8 following seven days of treatment. All 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.

Organ Weights
The liver, lungs (including bronchi), kidneys and spleen were weighed for each animal. For
bilateral organs, left and right organs were weighed together. Requisite organs were weighed
for animals killed at the scheduled interval.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin pending any future
requirement for further examination.


HISTOPATHOLOGY: No

Optional endpoint(s):

Optional endpoints: No

Statistics:

No statistical analysis was performed as the small sample size and absence of controls
precluded meaningful statistical evaluation

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test article-related clinical sign. Post-dosing chin rubbing was apparent in
males and females treated at 1000 mg/kg/day; this was considered to be a response to the
taste of the dose rather than toxicity

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related alteration in body weight or body weight gain was evident. Female
animals treated at 750 mg/kg/day showed reduced body weight gain throughout the treatment
period; since a similar effect was not apparent in females at 1000 mg/kg/day or in males, it
was considered to represent biological variation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related alteration to food consumption was noted. Female animals treated at
750 mg/kg/day showed marginally reduced food consumption; since a similar effect was not
apparent in females at 1000 mg/kg/day or in males, it was considered to represent biological
variation.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There was no visual effect of treatment on the amount of water consumed

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:

effects observed, non-treatment-related

Description (incidence and severity):

The weights of the livers from females treated at 1000 mg/kg/day were considered to be
slightly higher than expected; since a similar effect was not apparent in males at
1000 mg/kg/day and there was no macroscopic correlate, it was considered to represent
biological variation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There was no treatment-related necropsy finding

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In this preliminary dose-range finding study, treatment was well tolerated at all doses (500, 750 or 1000 mg/kg/day) to Sprague Dawley rats for seven days with no overt toxicity being evident. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day and this was considered to be a suitable high dose for subsequent repeated dose toxicity study. 

Executive summary:

The purpose of this study was the assessment of the systemic toxic potential of the substance in a seven day oral gavage study in Crl:CD(SD) rats, to select a suitable high dose for a subsequent repeated dose toxicity study. Three groups, each comprising three male and three female Crl:CD(SD) rats, received the substance by oral gavage at doses of 500, 750 or 1000 mg/kg/day. During the study, clinical condition, body weight, food consumption, water consumption (by visual assessment), organ weight, and macropathology investigations were undertaken.

There was no death and no test article-related clinical sign. No test article-related alteration in body weight or body weight gain was evident and there was no effect on food or water consumption. It was considered that there was no test article-related effect of treatment on organ weights or macroscopic appearance of organs at necropsy. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was
considered to be 1000 mg/kg/day and this was considered to be a suitable high dose for a subsequent repeated does toxicity study.