2-[(2-ethylhexyl)oxy]ethanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-ethylhexyl)oxy]ethanol

EC Number:

216-323-7

EC Name:

2-[(2-ethylhexyl)oxy]ethanol

Cas Number:

1559-35-9

Molecular formula:

C10H22O2

IUPAC Name:

2-[(2-ethylhexyl)oxy]ethan-1-ol

Specific details on test material used for the study:

Test material information
Ethylene Glycol 2-Ethylhexyl Ether
Specific details on test material used for the study
Test item: Ethylene Glycol 2-Ethylhexyl Ether.
Test item identity (including alternative names): Eastman EEH
CAS number: 1559-35-9.
Intended use: Substance used in industry.
Appearance: Liquid.
Storage conditions: In a refrigerator (2 to 8C).
Supplier: Sponsor
Batch number: TXEG.
Expiry date: Two years from receipt.
Purity: 100%.

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 Crl:CD(SD) rat (virgin) 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:

Environmental Control
Rodent facility Limited access - to minimize entry of external biological and chemical agents and to
minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70
%. There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropr
iate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing,
treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used
during pairing. These were suspended above absorbent paper which was changed daily during p
airing.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, enviro
nmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed
at appropriate intervals each week.
Number of animals per cage Pre-pairing: up to five animals of one sex, Pairing: one male and one
female, Males after mating: up to five animals, Gestation: one female, Lactation: one female + litter
Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study
[(except during pairing and lactation (returned on Day 13 of lactation)] and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation) and
replaced at the same time as the cages.
Diet Supply
Diet SDS VRF1 Certified pelleted diet. A sample (100g) of each batch of diet used was retained
within Pharmacy (frozen -10 to -30ºC) until finalization of the report. Samples were discarded after
finalization of the report. The diet contained no added antibiotic or other chemotherapeutic or prop
hylactic agent.
Availability Non-restricted (removed overnight before blood sampling for hematology and blood
chemistry investigations).
Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles
were changed at appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Details on route of administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This bal
ance was compared with the expected usage
stirred using a magnetic stirrer before and throughout the dosing procedure.
Vehicle
corn oil
Details on oral exposure
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This bal
ance was compared with the expected usage
as a check of correct administration. No significant discrepancy was found. Formulations were
stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations
yes
Duration of treatment / exposure
Rats were exposed for 2 weeks of pre-mating, two weeks for mating (until signs of mating are observe
d), gestation and through lactation day 14. Females and their litters are sacrified on lactation day 14.
Males are killed on gestation day 35.
Frequency of treatment
Daily.

Details on mating procedure:

Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Females treated from 2 weeks pre-mating, 2 weeks of mating, gestation and through lactation day 14.
Males treated from 2 weeks pre-mating, 2 weeks of mating and through week 5 of gestation.

Duration of treatment / exposure:

n/a

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Rats were treated for 2 weeks prior to mating, 2 weeks for mating. Females were treated through lactation day 15 while males were sacrificed in week 5 of gestation. An FOB was conducted at the start and end of treatment. At necropsy, tissues were weighed and fixed for histopathology, gross observations were taken, blood was collected for hematology and clinical chemistry.

For the repro screen, rats were mated and the females delivered, and litters were kept to lactation day 14. At necropsy, males and females had reproductive organs kept for histopathology. The pups were given a gross necropsy and litter values were recorded.

Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.

After pairing until mating.

For four days before scheduled termination (nominally Days 11 to 14 of lactation).

Litter observations:

Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring.

Postmortem examinations (parental animals):

All adult 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.

Postmortem examinations (offspring):

Premature deaths Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
F1 offspring on Day 4 of age Blood sampling was undertaken (See Section 3.6.11).
Externally normal offspring were discarded without examination.
Externally abnormal offspring were examined, and retained pending possible future examination.
F1 offspring on Day 13 of age Blood sampling was undertaken (See Section 3.6.11).
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Thyroid glands were preserved from one male and one female in each litter.

Statistics:

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Reproductive indices:

The incidence and percentage females showing the following classifications of estrous cycles before pairing and during treatment are presented:
Regular: All observed cycles of 4 or 5 days
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that show estrus during this period and the cycle stage at termination.


Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.


Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating
Conception rate
Fertility index

Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day

Offspring viability indices:

The viability of the offspring was recorded.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were four decedent animals on study; all of these deaths were considered incidental and were not attributable to treatment. Details were as follows:
Group 1 Male No. 25 (Control) was killed for reasons of animal welfare due to physical injury; this male had two open wound areas on the dorsal body surface and on the scrotum. Scabs/encrustations were also seen on the dorsal body surface. These lesions were considered to be ‘fight wounds’ following observed aggression in the home cage.
Group 2 Male No. 16 (50 mg/kg/day) was killed for reasons of animal welfare reasons due to physical injury; this male had a deep open wound and encrustations on the dorsal body surface. These lesions were considered to be ‘fight wounds’ following observed aggression in the home cage.
Group 4 Female No. 72 and No. 78 (500 mg/kg/day) were killed for reasons of animal welfare due to dosing trauma. Female No. 72 was found on Day 5 of treatment to be underactive with irregular breathing, partially closed eyelids and was seen to be uncoordinated. Macroscopic examination revealed perforation of the esophagus and clear aerated fluid in the thorax; result of a dosing trauma. Female No. 78 was found on Day 23 of gestation to be hunched with piloerection, irregular breathing, pale eyes and skin. Macroscopic examination similarly revealed perforation of the esophagus with pale, viscous fluid in the thorax. In addition thoracic adhesions and thickened pericardium were observed; result of a dosing trauma.

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological evaluation in males treated at 50, 150 or 500 mg/kg/day, revealed mean cell hemoglobin concentration was high when compared with controls attaining statistical significance at all levels; however the increases observed were slight and there was no relationship to treatment. A slight increase was observed in the number of large unstained cells seen in males treated at 150 or 500 mg/kg/day.
In females treated at 500 mg/kg/day mean haematocrit and haemoglobin values were low when compared with controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination performed after 5 weeks of treatment or on Day 14 of lactation revealed no test item-related lesions . The incidence and distribution of all other findings were considered to be unrelated to treatment.
Lesions in the kidneys (cortical tubular degeneration/necrosis, mineralisation) were seen in several females, including in control animals, with no relationship to test item. Microscopic findings seen in the kidneys correlated with pale areas seen at necropsy.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

Executive summary

This study was designed toassess the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of Ethylene Glycol 2-Ethylhexyl Ether (hereafter referred to as EEH) by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received EEH at doses of 50, 150 or 500 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy

after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their

offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received

the vehicle, corn oil, at the same volume dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood),

blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations

were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also recorded. Nipple counts were performed on male offspring

on Day 13 of age.

Results

There were no treatment related deaths and noadverse effects of treatment on clinical condition, sensory reactivity, grip strength, motor activity, body weight gain, food intake, haematology, blood chemistry or

organ weight measurements in males and females. In addition, the reproductive endpoints assessed which were unaffected by treatment included estrus cycles, pre-coital interval, gestation length, mating

performance, fertility, litter size, offspring survival or offspring sex ratio. Offspring clinical condition, body weight, body weight gain, ano-genital distance and external

development were also unaffected by treatment. Body weight gain was unaffected by treatment in males and in females before pairing. During gestation, overall weight gain in females treated at 500 mg/kg/day was low, as was food intake during Days 14-19 when compared with controls. During lactation body weight gain and food consumption were unaffected by treatment.

Hematological evaluation in males treated at 50, 150 or 500 mg/kg/day, revealed mean cell hemoglobin concentration was slightly high when compared with controls attaining statistical significance at all levels;

however the increases observed were minor and there was norelationship to treatment. A slight increase was observed in the number of large unstained cells seen in males treated at 150 or 500 mg/kg/day. In females treated at 500 mg/kg/day mean haematocrit and haemoglobin values were low when compared with controls.

Biochemical evaluation revealed no clear effect of treatment in males or females treated with EEH. Group mean adjusted epididymides, testes and prostate weights were slightly high in males treated with

EEH at all dose levels (50, 150 or 500 mg/kg/day) although there was no clear relationship to treatment with EEH; Mean adjusted liver weights were high and spleen weights were low in males treated at 500

mg/kg/day. Group mean spleen weights were slightly high in females treated with EEH at all dose levels (50, 150 or 500 mg/kg/day) although there was no clear relationship to treatment with EEH.

At macroscopic examination pale areaswere seen in the kidneys of several females, including in control animals; this correlated to lesions in the kidneys (cortical tubular degeneration/necrosis, mineralisation)

however there was no relationship to test item. No macroscopic or microscopic changes were detected in the full list of tissues examined. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.

Conclusion

Based on the results of this study it is concluded that the No-observed-adverse-effect-level (NOAEL) of Ethylene Glycol 2-Ethylhexyl Ether (EEH) for systemic toxicity and for reproductive/developmental

effects is 500 mg/kg/day.