2-(2-ethoxyethoxy)ethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 January 2007 to 11 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A well reported GLP study conducted under a guideline equivalent protocol.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-ethoxyethoxy)ethanol

EC Number:

203-919-7

EC Name:

2-(2-ethoxyethoxy)ethanol

Cas Number:

111-90-0

Molecular formula:

C6H14O3

IUPAC Name:

2-(2-ethoxyethoxy)ethan-1-ol

Details on test material:

- Name of test material (as cited in study report):Transcutol HP
- Physical state: clear, colorless liquid
- Analytical purity: 100%
- Lot/batch No.: 450449013; 450449013
- Storage condition of test material: Room temperature, under nitrogen

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ridglan Farms, Inc., Mt. Horeb, Wisconsin
- Age at study initiation: 5-6 months old
- Weight at study initiation: males - 6.3 to 9.1kg; females - 5.3 to 7.9kg
- Fasting period before study: overnight prior to blood collection for serum chemistry and prior to necropsy.
- Housing: Individually in stainless steel cages elevated above stainless steel cage pans. Animals were allowed regular opportunity for exercise and social interaction in accordance with the standard operating procedures.
- Diet : Offered once daily. Approximately 400g of PMI Nutrition International, LLC, Certified Canine LabDiet 5007. On dosing days feed bowls from the previous day were removed approximately 1 hour prior to dosing and new feed was offered after the 2-hour post-dosing clinical observations. In addition, at the discretion of the study director, the daily diet of individual animals was supplemented appropriately with Alpo or Science Diet A/D to maintain the health status of the animals.
- Water : ad libitum, mains water.
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+/-3
- Humidity (%): 50+/-20
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2007, February 5 To: 2007 May 8

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Group 2 - 400mg/kg/day - test article concentration: 80mg/ml
Group 3 - 1000mg/kg/day - test article concentration 200mg/ml
Group 4 - 2000/1500mg/kg/day - test article concentration 400/300mg/ml

The test article formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated, protected from light, under nitrogen. The pH was measured: On 2 February 2007, the pH measurements for the 80, 200 and 400mg/ml formulations were 9.09, 9.16 and 9.47, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Two sets of duplicate samples for concentration analysis were collected from the study week 0, 3, 6, 9 and 12 formulations at the time of preparation from each dosing formulation (including the vehicle formulation administered to the control group). The analyses were conducted by gas chromatography.

Duration of treatment / exposure:

91 days

Frequency of treatment:

continuous

Doses / concentrations

Remarks:

Doses / Concentrations:
400, 1000, 1500 and 2000 mg/kg/day
Basis:
other: gavage, 5ml/kg

No. of animals per sex per dose:

Control, and 1500/ 2000mg/kg/day groups: 6 males and 6 females
400 and 1000mg/kg/day groups: 4 males and 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the results of a previously conducted escalating and 7-day repeat dose range-finding study (WIL-261016; Padgett, 2007).
Initial dosages were 400, 1000 and 2000 mg/kg/day for groups 2-4, respectively. However, within approximately the first 2 weeks of dose administration, individual dogs in the 2000mg/kg/day group were noted with dose-limiting toxicity. Subsequently, the high dose was lowered from 2000mg/kg/day to 1500mg/kg/day. Dose administration was suspended for the 2000mg/kg/day group from study days 16 to 20 (males) and 15 to 19 (females). Dose administration at 1500mg/kg/day resumed on study day 21 for the remaining males and study day 20 for remaining females.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily except on nondosing days during dose suspension for animals in the 2000mg/kg/day group, when it was once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, beginning approximately 2 weeks prior to test article administration and prior to the scheduled necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the initiation of dose administration (Week -1) and near the end of the treatment period (Week 13).
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: From all dogs, prior to the initiation of dose administration (Week -1) and the day of the scheduled necropsy (Week 13)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: From all dogs, prior to the initiation of dose administration (Week -1) and the day of the scheduled necropsy (Week 13)
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: From all dogs, prior to the initiation of dose administration (Week -1) and the day of the scheduled necropsy (Week 13)
- Metabolism cages used for collection of urine: Yes, for an approximate 24-hour period
- Animals fasted: No
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:

TOXICOKINETICS:
Blood samples were collected at 0.5, 1, 2, 4, 8 and 24 hours after dose administration on study days 0 and 86.
Urine samples were collected for an approximate 24-hour period overnight on study days 0 and 86.

ELECTROCARDIOGRAPHIC DATA:
Multilead ECGs were recorded for all animals prior to the initiation of dose administration (Week -2) and during study week 12 using the Multi Lead ECG equipment with the Ponemah platform from LDS Test and Meaurements, LLC. During the treatment period, ECGs were recorded approximately 2 hours after dose administration.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes in all animals. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including viscera. The following organs were weighed from all animals at the scheduled necropsy: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, prostrate, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix.
HISTOPATHOLOGY: Yes (see table 4)

Other examinations:

no data

Statistics:

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Statistical analyses were not conducted if the number of animals was <=2.
Body weight, body weight change, food consumption, clinical pathology, heart rate and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Following 6 and 12 consecutive days of dose administration, 2 males and 1 female, respectively, in the 2000mg/kg/day group were euthanised in extremis on study days 7 and 15. These three animals showed clinical findings (hypoactivity, diarrhea and emesis), a marked decrease in food consumption, as well as pronouced body weight losses. The most probably cause of morbidity, based on histologic findings was moderate to severe renal tubular degeneration.
All other animals survived to the scheduled necropsy. There were no test substance-related clinical observations in the 400 or 1000mg/kg/day groups and no test substance-related clincial observations noted for the remaining animals in the 1500mg/kg/day group.

BODY WEIGHT AND WEIGHT GAIN
Body weight effects were noted in the 1000mg/kg/day female group, 2000mg/kg/day group, and the 2000/1500mg/kg/day female group.
Body weights in the 400mg/kg/day group, 1000mg/kg/day group males and 2000/1500mg/kg/day group males were generally similar to the control group values throughout the dose administration.

FOOD CONSUMPTION
Test substance-related effects on food consumption were noted for the 2000mg/kg/day group males and females.

OPHTHALMOSCOPIC EXAMINATION
No effect observed.

HAEMATOLOGY, CLINICAL CHEMISTRY and URINALYSIS
Slight, non-adverse differences from controls in hematology (MCV and MCH), serum chemistry (alkaline phosphatase, albumin, A/G ratio, chloride, bicarbonate) and urine chemistry (specific gravity, osmolality, pH and electrolyte balance) parameters were noted in the 400, 1000 and/or 2000/1500mg/kg/day groups at week 13. These differences from controls were relatively small and may represent the residual effects of a regenerative response in the case of the red blood indices or compensatory/adaptive mechanisms in the case of slightly elevated alkaline phosphatase and/or differences in urine parameters to eliminate the test article.

ORGAN WEIGHTS
Higher liver weights were noted in the 1000mg/kg/day group females and the 2000/1500mg/kg/day group males and females at the scheduled necropsy. The slightly higher liver weights for these groups may be associated with the somewhat elevated alkaline phosphatase serum levels observed for these groups at the study week 13 clinical chemistry evaluations. There were no histologic correlates related to the increase in liver weights. The liver weight differences from control may be a reflection of an adaptive response related to the metabolism of the test article and the differences from control were not considered adverse.

GROSS PATHOLOGY
Gross necropsy observations findings in the animals euthanized in extremis included pale kidneys in the males; enlarged kidneys with reddened cortico-medullary junction in the female; dark red areas in the esophagus, stomach, duodenum and/or ileum for both sexes, and reddened mucosa and or intussusception in the ileum for both sexes. Microscopic correlates for these observations were renal tubular degeneration int he kidney; ulceration or erosion in the esophagus, stomach and duodenum and hemorrhage and muscle degeneration in the ileum.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance-related organ-specific microscopic findings of consequence at any dosage level for the animals surviving to the scheduled necropsy, and the histologic alterations in the unscheduled death animals associated with dosage at 2000mg/kg/day were considered reversible, since there was no histologic evidence of injury in the animals continued to be dosed at 1500mg/kg/day.

OTHER FINDINGS
No test substance-related effects were noted in the electrocariographic data at the study week 12 evaluation.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: This is the highest dosage level administered for the entire duration of the study and it corresponds to plasma levels of 3159-6346ug*h/ml.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analytical chemistry: the analysed dosing formulations were found to contain the amount of test article prescribed in the protocol (90 -110% of target concentration) with a few exceptions that the study director deemed not to be significant.

Table 5. Mean toxicokinetic results for DEGEE

Plasma						% of Dose eliminated in urine over 24 hours
	Oral Dosage (mg/kg/day)	AUC (ug*h/ml)	Cmax (ug/ml)	Tmax (h)	T1/2 (h)
Males
Day 0	400	875	434	0.50	0.94	0.31
	1000	4088	1280	0.63	0.89	5.2
	2000/1500	12890	2516	1.1	2.1	4.3
Day 86	400	1366	504	0.63	1.2	0.73
	1000	6346	1443	0.50	1.7	1.8y
	2000/1500	10479yy	2248yy	0.63yy	2.1yy	2.9yy
Females
Day 0	400	904	434	0.63	0.89	0.50
	1000	3159	1124	0.63	0.80	1.4
	2000/1500	10756	2501	0.75	1.6	3.5
Day 86	400	958	469	0.50	1.0	0.6
	1000	5195	1421	0.50	1.1	2.4
	2000/1500	8877#	2093#	0.50#	1.5#	2.4#
N=4 at 400 and 1000mg/kg/day; N=6 at 2000/1500mg/kg/day, exceptyN=3;yy=N=4; #N=5
*AUC = AUC0-inf on Day 0, or AUC0-24 on Day 86

Table 6. Mean toxicokinetic results for EEAA

Plasma						% of Dose eliminated in urine over 24 hours
	Oral Dosage (mg/kg/day)	AUC (ug*h/ml)	Cmax (ug/ml)	Tmax (h)	T1/2 (h)
Males
Day 0	400	1396	301	2.0	1.4	31
	1000	5628	667	4.0	1.9	45
	2000/1500	13276	983	6.7	2.2	57
Day 86	400	2140	345	4.0	2.0	45
	1000	6952	632	4.0	1.9	52y
	2000/1500	9602yy	789yy	4.0yy	2.2yy	41yy
Females
Day 0	400	1944	339	2.0	2.0	53
	1000	4782	620	3.5	1.9	49
	2000/1500	12059	972	4.0	2.2	50
Day 86	400	2031	347	2.5	2.1	44
	1000	6201	787	4.0	1.8	57
	2000/1500	8143#	730#	4.0#	1.9#	47#
N=4 at 400 and 1000mg/kg/day; N=6 at 2000/1500mg/kg/day, exceptyN=3;yy=N=4; #N=5
*AUC = AUC0-inf on Day 0, or AUC0-24 on Day 86

Applicant's summary and conclusion

Conclusions:

A NOAEL of 1000mg/kg/day can be concluded from this study

Executive summary:

In a GLP 13 -week study in male and female dogs, 2 -(2 -ethoxyethoxy)ethanol was administered at 400, 1000, or 2000mg/kg/day by oral gavage. A fourth control group was given the vehicle water. Based on the results of this study, the initial high dose of 2000mg/kg/day was not well tolerated as evidenced by clinical findings, pronounced body weight losses and decreased food consumption which resulted in mortality for 2 males and 1 female within the first 2 weeks of dose administration. The primary histologic alteration contributing to the morbidity for these dogs was severe renal tubular degeneration in the kidney. The dosage level was subsequently lowered to 1500mg/kg/day for the remaining animals. Slight, non-adverse differences from controls in hematology, serum chemistry and urine chemistry parameters were noted in the 400, 1000 and/or 2000/1500mg/kg/day groups. Higher liver weights were observed in the 1000mg/kg/day female group and the 2000/1500mg/kg/day group at the scheduled necropsy, which may be associated with the slightly elevated srum ALP levels resulting from an adpative response related to the metabolism of the test article. There were no organ-specific microscopic findings of consequence at any dosage level for the animals surviving to the scheduled necropsy, and the histologic alterations in the unscheduled death animals associated with dosage at 2000mg/kg/day were considered reversible, since there was no histologic evidence of injury in the animals continued to be dosed at 1500mg/kg/day. The 1500mg/kg/day dose was well tolerated. Based on the results of this study the NOAEL was considered to be 1000mg/kg/day (3159 -6346 ug*h/ml) for both male and female dogs.