3,3'-[methylenebis(oxymethylene)]bisheptane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 April 2020 to 17 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[methylenebis(oxymethylene)]bisheptane

EC Number:

244-815-1

EC Name:

3,3'-[methylenebis(oxymethylene)]bisheptane

Cas Number:

22174-70-5

Molecular formula:

C17H36O2

IUPAC Name:

3,3'-[methylenebis(oxymethylene)]diheptane

Test material form:

liquid

Specific details on test material used for the study:

Sponsor’s identification: 2-Ethylhexylal
Alternative names: 3,3’-[methylenebis(oxymethylene)]bisheptane
Batch No.: 1912191500R
CAS No. 22174-70-5
Purity: 99.9180%
Appearance: Clear liquid
Expiry date: 19 December 2021
Storage conditions: Ambient storage (15 to 25°C), in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

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 Sprague-Dawley
[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) Ltd.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 36 to 42 days
- Weight at study initiation: males: 173 to 215 g; females: 120 to 153 g
- Fasting period before study: no
- Housing: Up to five of the same sex unless reduced by mortality
- Diet ad libitum Teklad 2014C, pelleted diet - Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water ad libitum Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals - Non-restricted (except during the period of urine collection).
- Acclimation period: Eight days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY: 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 contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 12 hours light: 12 hours dark

IN-LIFE DATES: From: 22 April 2020 To: 11 June 2020

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was chosen to simulate the conditions of human exposure

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test item was weighed into a suitable container. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test item and was magnetically stirred until uniformly mixed. A further amount of vehicle was added to make up to the required volume and further mixing, using a magnetic stirrer, was performed until the formulation was homogeneous. The formulation was transferred to the final containers, via syringe, whilst magnetically stirring. The remaining concentrations were then formulated in ascending order of concentration. Prepared weekly

VEHICLE
- Concentration in vehicle: 0, 66, 120 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Remarks:

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

Details on analytical verification of doses or concentrations:

The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M031/20).
The analytical method involved extraction in and dilution in tetrahydrofuran followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 5 μg/mL to 100 μg/mL.
A suitable volume (100 μL) of the prepared internal standard solution (0.5 mg/mL tetradecane in tetrahydrofuran) was added to each vial containing 1 mL calibration standard solution to provide a final concentration of ca. 50 μg/mL of tetradecane.
The formulations for Week 1 and Week 4 were sampled, 1 × 10 mL (accurately weighed), from the middle of the formulation by Pharmacy personnel. Two aliquots from the first sample were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

Duration of treatment / exposure:

28 days followed by a 14 day recovery period. The last dose was administered on Day 28.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery group

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Recovery group

Doses / concentrations 3

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

600 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 330, 600 and 1000 mg/kg/day) were selected in conjunction with the Sponsor.
The preliminary study DF09ND, rats dosed at 125, 250, 500 and 1000 mg/kg/day did not show any treatment-related effect at 125, 250 and 500 mg/kg bw/day. At 1000 mg/kg/day, overall body weight gain and food consumption was low in males and liver weights were increased in males and females. Therefore, for this study, a low dose of 330 mg/kg/day was chosen as an intermediate dose between 250 and 500 mg/kg/day and a high dose of 1000 mg/kg/day was chosen to induce toxicity but not death or findings of a severity to compromise interpretation of the results. The intermediate dose (600 mg/kg/day) was the approximate geometric mean of the high and low dose levels.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes (overnight withdrawal of food)
- Post-exposure recovery period in recovery groups: 2 weeks
- Dose range finding studies: 2-Ethylhexylal: Preliminary Toxicity Study by Oral Gavage Administration to CD Rats for 7 Days.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period),

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded six days before treatment commenced, on the day that treatment commenced (Day 1), twice during Week 1, weekly throughout the remainder of the study (last scheduled body weight during the treatment period was recorded on Day 28 and last scheduled body weight during recovery was recorded on Day R14) and before necropsy.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at the following occasions: day 29 for all main study animals, week R3 for all recovery animals
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all main study and recovery animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at the following occasions: day 29 for all main study animals, week R3 for all recovery animals
- Animals fasted: Yes
- How many animals: all main study and recovery animals
- Parameters checked in table 2 were examined.

THYROID HORMONE ANALYSIS: Yes
- Time of blood sample collection: Blood samples were collected at necropsy as follows: Day 29 for all main study animals; Week R3 for all recovery animals
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight at the following occasions: Day 29 for all main study animals; Week R3 for all recovery animals. During Week 4 of treatment (before dosing), the motor activity of all Main study animals in Groups 2 and 3 and all Recovery phase animals was measured
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity and grip strength assessments were performed (before dosing) on all Main study animals in Groups 2 and 3 and all Recovery phase animals in Groups 1 and 4 during Week 4 of treatment.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Tail pinch response, Auditory startle reflex, Pinna reflex, Approach response

IMMUNOLOGY: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 4)

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The appearance and behaviour of the animals was unaffected by treatment

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two deaths occurred during the study, neither of which was attributed to treatment. Male No. 16 (600 mg/kg/day) was found dead on Day 9 and female No. 64 (1000 mg/kg/day) was euthanized for welfare reasons on Day 15. Macroscopic findings of pericardial fluid, abnormal thoracic contents, thoracic adhesions, inflammation of the lung, thymus and thoracic cavity and esophageal perforation were observed at necropsy and/or on microscopic evaluation and these findings were consistent with a dose administration error (accidental perforation of the esophagus during the dosing procedure). Consequently, both deaths were considered accidental.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The overall bodyweight gain by males receiving 1000 mg/kg/day was approximately 11% lower than controls, but the difference from controls did not attain statistical significance. There was no evidence of any improvement during the two-week recovery period. As the lower bodyweight gain over Days 1-28 was confined to one sex and within the historical control range (2019-2021 (7 studies): 95th percentile: 100 to 197 grams: n=50) this is not considered adverse.
Bodyweight gain was unaffected in males and females receiving 330 or 600 mg/kg/day and in females receiving 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was not adversely affected by treatment.
Food consumption tended to be higher than pre-treatment and control values at all doses in females, though there was no dose-response. This finding was not evident during the recovery period.
A lower food consumption was observed in one cage of Group 4 males (006) in comparison with the control. This lower consumption was observed in the pre-treatment period and consequently is not considered to be related to treatment.

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

no effects observed

Description (incidence and severity):

Water consumption was unaffected by treatment

Haematological findings:

no effects observed

Description (incidence and severity):

The haematological examination of peripheral blood after four weeks of treatment did not reveal any differences from control that were attributable to treatment.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the small reduction of prothrombin time in males receiving 1000 mg/kg/day since small reductions are considered of no toxicological significance and there was no similar trend in females. They also included the slight reductions of hematocrit and hemoglobin concentrations at all doses in females, since the extent of the differences from controls was small and lacked dose-relationship, and the low reticulocyte count at 1000 mg/kg/day in females since there was no consequential decrease of the erythrocyte counts of these animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The biochemical examination of the blood plasma after four weeks of treatment indicated a dose-dependent increase of urea concentration in males and females receiving 330, 600 or 1000 mg/kg/day, though the difference from controls was not statistically significant in females receiving 330 mg/kg/day. There was no associated increase of plasma creatinine concentration. By the end of the two week recovery period the effect on plasma urea concentration showed full recovery.
Males and females receiving 600 or 1000 mg/kg/day had lower plasma glucose concentrations than controls, with the extent of the decrease being dose-related. By the end of the two week recovery period the glucose concentrations of males previously given 1000 mg/kg/day remained low, though there had been slight recovery, whilst in females there was full recovery.
The plasma triglyceride concentrations of females receiving 600 mg/kg/day and of males and females receiving 1000 mg/kg/day were higher than those of the controls, though statistical significance was attained only in the high dose females. These findings showed full recovery.
In males there was a small reduction of plasma albumin concentration at all doses which, at 600 and 1000 mg/kg/day associated with a small increase of the globulin fraction resulting in higher albumin to globulin ratios in these animals. These trends, which were not observed in females, showed full recovery.
All other inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the small reductions of plasma aspartate amino-transferase activity at 600 and 1000 mg/kg/day in females, the decreased total bilirubin concentrations in females receiving 1000 mg/kg/day since such reductions are considered of no toxicological importance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The composition of the urine was unaffected by treatment.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the slightly low pH at all doses in females since there was no dose-response and no similar finding in males.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength were unaffected by treatment.
Motor activity was unaffected by treatment.
All inter-group differences from control, including those that attained statistical significance, were minor or transient differences that were attributed to natural variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

2-Ethylhexylal-related organ weight changes were observed in the liver.
For the liver of males and females administered 330, 600 or 1000 mg/kg/day, mean group weight values were increased after 4 weeks and this correlated with the portal hepatocellular vacuolation observed microscopically. At the recovery sacrifice liver weights for males previously administered 1000 mg/kg/day were similar to control values, indicating that complete recovery had occurred whilst for females liver weights remained higher than control values, but the percentage difference from controls was less than at the end of the treatment period indicating that partial recovery had occurred.
All other differences in organ weights, statistically significant or not, were consistent with normal variation and were considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; lack of a dose relationship or correlative findings; the magnitude was considered small.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No 2-Ethylhexylal-related macroscopic findings were observed.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for this animal age and/or strain; therefore, they were considered not test article related.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

2-Ethylhexylal-related microscopic findings were confined to the liver.
Minimal to moderate periportal hepatocellular vacuolation was observed after 4 weeks of treatment in males administered 1000 mg/kg/day and females administered 330, 600 or 1000 mg/kg/day. The incidence and severity were higher in females than males. The contents of the vacuoles were confirmed to be lipids by Oil-Red-O staining. This finding correlated with the increased liver weight in males and females administered 1000 mg/kg/day. Periportal hepatocellular vacuolation was not seen after 2 weeks of recovery, indicating that complete recovery had occurred. There was no hepatocellular necrosis, inflammation or increased liver enzymes associated with the hepatocellular vacuolation and, consequently, these findings in the liver were considered non-adverse.
All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this animal age and/or strain. Consequently, they were considered not test article related.

Effect levels

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:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

It is concluded that oral (by gavage) administration of 2-Ethylhexylal to Sprague-Dawley rats for four weeks at doses up to 1000 mg/kg/day was well tolerated. Periportal hepatocellular vacuolation was observed at all doses in females and at 1000 mg/kg/day in males however this was fully reversible and considered a non-adverse finding. In view of the findings on this study, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 1000 mg/kg/day in males and females.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of 2-Ethylhexylal when administered orally, by gavage, to Sprague-Dawley rats for four weeks. Recovery from any effects was evaluated in a 2 week recovery period. Three groups, each comprising five males and five females, received 2-Ethylhexylal at doses of 330, 600 or 1000 mg/kg/day and a similarly constituted control group received the vehicle (corn oil) at the same volume does as treated groups. A further five males and five females were assigned to each of the control and high dose groups  and these animals were treated for four weeks, followed by a two week period without treatment to assess the potential for any treatment-related change to recover.

During the study, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food and water consumption, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

Results

The appearance and behaviour of the animals was unaffected by treatment and there was also no effect on sensory reactivity responses, grip strength or motor activity. There were no treatment-related deaths (the two deaths that occurred in this study were due to gavage-related injuries).

Body weight and bodyweight gain, food and water consumption was unaffected in both sexes.

There were no treatment-related haematological findings.

Biochemical changes in the blood plasma after four weeks of treatment comprised: a dose‑dependent increase of urea concentration at all doses and in both sexes; low glucose concentrations in males and females receiving 600 or 1000 mg/kg/day, the extent of which was dose-related; slightly high triglyceride concentrations at 1000 mg/kg/day in both sexes; slightly low albumin concentration at all doses in males which, at 600 and 1000 mg/kg/day associated with a small increase of the globulin fraction resulting in higher albumin to globulin ratios in these animals. By the end of the two week recovery period the glucose concentrations of males previously given 1000 mg/kg/day remained low, though there had been slight recovery, whilst all other treatment-related findings showed full recovery.

The composition of the urine was unaffected by treatment.

Liver weights were high after four weeks at all doses and in both sexes, though only in males was the extent of the increase dose-related. This finding showed partial recovery in females and full recovery in males.

There were no treatment-related macroscopic findings.

Minimal to moderate periportal hepatocellular vacuolation was observed after four weeks of treatment in males administered 1000 mg/kg/day and females administered 330, 600 or 1000 mg/kg/day, with the incidence and severity being greater in females than males. There was, however, no associated hepatocellular necrosis or inflammation. The contents of the vacuoles were confirmed to be lipids by Oil‑Red‑O diagnostic staining. Periportal hepatocellular vacuolation was not present after two weeks of recovery, indicating that complete recovery had occurred.

Conclusion

It is concluded that oral (by gavage) administration of 2-Ethylhexylal to Sprague-Dawley rats for four weeks at doses up to 1000 mg/kg/day was well tolerated. Periportal hepatocellular vacuolation was observed at all doses in females and at 1000 mg/kg/day in males however this was fully reversible and considered a non-adverse finding. In view of the findings on this study, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 1000 mg/kg/day in males and females.