2-ethylhexanoic acid, monoester with propane-1,2-diol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2015 to 28 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in an accredited GLP compliant laboratory according to current OECD test Guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxypropyl 2-ethylhexanoate

EC Number:

261-499-0

EC Name:

2-hydroxypropyl 2-ethylhexanoate

Cas Number:

58921-10-1

Molecular formula:

C11H22O3

IUPAC Name:

2-hydroxypropyl 2-ethylhexanoate

Constituent 2

Reference substance name:

1-hydroxypropan-2-yl 2-ethylhexanoate

Molecular formula:

C11H22O3

IUPAC Name:

1-hydroxypropan-2-yl 2-ethylhexanoate

impurity 1

Reference substance name:

Monoesters of 2-ethylhexanoic acid and dipropyleneglycol

IUPAC Name:

Monoesters of 2-ethylhexanoic acid and dipropyleneglycol

impurity 2

Reference substance name:

1-methylethylene 2-ethylhexanoate

EC Number:

301-185-3

EC Name:

1-methylethylene 2-ethylhexanoate

Cas Number:

93981-97-6

Molecular formula:

C19H36O4

IUPAC Name:

propane-1,2-diyl bis(2-ethylhexanoate)

impurity 3

Reference substance name:

2-ethylhexanoic acid

EC Number:

205-743-6

EC Name:

2-ethylhexanoic acid

Cas Number:

149-57-5

Molecular formula:

C8H16O2

IUPAC Name:

2-ethylhexanoic acid

impurity 4

Reference substance name:

Propane-1,2-diol, propoxylated

EC Number:

500-039-8

EC Name:

Propane-1,2-diol, propoxylated

Cas Number:

25322-69-4

IUPAC Name:

Propane-1,2-diol, propoxylated

impurity 5

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

liquid: viscous

Specific details on test material used for the study:

- Name of test material (as cited in study report): RD 15134
- Physical state: Liquid
- Analytical purity: 95.8%
- Lot/batch No.: 85114-00-7
- Expiration date of the lot/batch: 01-June-2018
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Reputable commercial supplier
- Age at study initiation: 41-47 days
- Weight at study initiation: 190-244g, Males: 146-191g, Females
- Fasting period before study:diet removed overnight before blood sampling for haematology and blood chemistry
- Housing:Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals, Five of the same sex per cage
- Diet (e.g. ad libitum): non-restricted
- Water (e.g. ad libitum): non-trestricted
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23ºC
- Humidity (%): 40-70%
- Air changes (per hr):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: To: 08 July to 19/20 October 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Chosen to be consistent with previous studies
- Concentration in vehicle: 50, 100 or 200 mg/mL as appropriate
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): not specified
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of formulations during storage were determined as part of another study, Envigo Study Number NUG0005

Samples of each formulation prepared for administration in Weeks 1, 6 and 13 of treatment were analysed for achieved concentration of the test substance. The details of the method of analysis and results are presented in the report

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily at approximately the same time each day

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Randomly allocated on arrival
- Rationale for selecting satellite groups: N/a
- Post-exposure recovery period in satellite groups: N/a
- Section schedule rationale (if not random): N/a

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: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.

DETAILED CLINICAL OBSERVATIONS: Yes see above

BODY WEIGHT: Yes
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy


OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:
Occasion Animals
Pretreatment All animals (including spares)
Week 12 All animals of Groups 1 and 4

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 ocular fundus were examined.

HAEMATOLOGY: Yes
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:
Occasion Animals
Week 13 All animals
Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant

CLINICAL CHEMISTRY: Yes
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:
Occasion Animals
Week 13 All animals
Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein, collected into tubes containing lithium heparin anticoagulant

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength and motor activity assessments were performed (before dosing) on all animals during Week 12 of treatment
- Battery of functions tested: sensory activity, grip strength and motor activity

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slight effect of treatement at 1000 mg/kg/day

Food consumption and compound intake (if feeding study):

no effects observed

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 were no ophthalmic findings that were considered to be related to treatment

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity responses, grip strength and motor activity were unaffected by treatment

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An increase in absolute and body weight adjusted liver and kidney weights in males and females given 500 or 1000 mg/kg/day, attaining statistical significance in the majority of the body weight adjusted groups.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes in the kidneys of male rats only considered test-item related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Test item-related effects on the cortex of the kidney consisted of minimal to moderate hyaline droplets, as intracytoplasmic droplets and eosinophilic inclusion round bodies surrounded by a clear halo on males receiving 250 mg/kg/day or above, although one control male was also observed with this change. Many xenobiotics bind to alpha-2u-globulin and decrease effectiveness of lysosomal degradation in male rats. These findings are likely to be alpha-2u-globulin nephropathy, which is a sex and species specific entity. In addition, minimal to slight tubular basophilia with interstitial fibrosis was recorded in males given 250, 500 and 1000 mg/kg/day, which was predominantly characterized by multifocal clusters of basophilic tubules, nuclear crowding, tubular dilatation, interstitial inflammatory cell infiltrates and thickening of the basal membranes (demonstrated by the PAS stain), which in one male progressed to tubular necrosis. These findings correlated with increased kidney weights and increased levels of urea in males given 500 or 1000 mg/kg/day after 13 weeks of treatment. The test-item changes observed in the kidneys were considered adverse in males given 500 or 1000 mg/kg/day as the findings reflected dose-dependent changes that correlated with elevated urea and increased kidney weights.
In the liver, minimal centrilobular eosinophilia/hypertrophy was observed in males and females given 1000 mg/kg/day and females given 500 mg/kg/day. This finding correlated with increased liver weights in both males and females after 13 weeks of treatment and was expected for this compound class. The test item, RD 15134 (2-Ethylhexanoic acid, monoester with propane 1,2–diol) is related to ethylhexanoic acid, which is considered a peroxisome proliferator that typically produces hypertrophy of the centrilobular hepatocytes due to the increase of the volume of peroxisomes, accompanied by lipid reduction and increased liver weights (Sundberg et al., 1994; Keith et al., 1992). There are considerable species differences in sensitivity to peroxisomal proliferation and humans appear to be less susceptible to many peroxisomal proliferators (Chevalier et al., 1998). The test-item related
changes in the liver were not considered adverse as these changes represented an adaptive response.
The changes in the thyroid are considered secondary to the induction of liver metabolism enzymes. There was an increased incidence of minimal follicular cell hypertrophy observed in all treated groups, with a higher incidence in the males. The incidence in the males given 500 mg/kg/day was slightly higher than 1000 mg/kg/day, which reflects a degree of biological variation between treated males. Some studies have shown that peroxisome
proliferators lead to enzymatic induction in the liver, which can induce increased breakdown of thyroid hormones and also displace thyroid hormones from serum carrier proteins. This will consequently lead to lower circulating levels of thyroid hormones, resulting in a release from negative feedback inhibition and compensatory increased secretion of TSH from the pituitary gland, resulting in follicular cell hypertrophy in the thyroid (Richardson and Klaasen, 2010, Miller et al., 2001). Again, there are considerable species differences in thyroid hormone synthesis, transport and metabolism between rodents and humans. The thyroid changes are not considered adverse as the follicular cell hypertrophy reflects a rodentspecific
adaptive response.
In the absence of histopathological correlate the following changes were not considered to be of toxicologically significance; the decrease in haematocrit, haemoglobin concentration and red blood cells counts in males receiving 250, 500 or 1000 mg/kg/day, a decrease in haematocrit and haemoglobin concentration in females at 1000 mg/kg/day. In addition, there was no correlate for low mean cell haemoglobin and mean cell volume in females receiving 500 or 1000 mg/kg/day and changes were seen in neutrophils, lymphocytes, and basophils, with an associated reduction in overall low white blood cell count or the low large unstained stain cell counts or shorter activated partial thromboplastin time in females receiving 1000 mg/kg/day. Similarly, total protein and albumin concentration were low in treated males, with an associated high A/G ratio in high dose males were not considered to be toxicologically significant.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based primarily on the changes seen in the kidneys

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No test substance effects seen at 1000 mg/kg bw/day the highest dose level tested

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It is concluded that oral administration of RD 15134, a coalescing agent, to Sprague-Dawley Crl:CD(SD) rats at 250, 500 or 1000 mg/kg/day for 13 weeks was relatively well-tolerated at doses in males at 250 mg/kg/day and in females up to 1000 mg/kg/day. In males, a dose of 500 mg/kg/day or above resulted in hyaline droplets and tubular basophilia with interstitial fibrosis in the kidney, with an associated increase in kidney weight and urea concentration.
It is therefore considered that the no-observed-adverse- effect-level (NOAEL) was 250 mg/kg/day in males, based primarily on the changes seen in the kidneys, and 1000 mg/kg/day in females.