Poly(oxy-1,2-ethanediyl), α-butyl-ω-hydroxy-

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well reported guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA TSCA Consent Order

Deviations:

not applicable

Principles of method if other than guideline:

This study was conducted according to the EPA TSCA Test Guidelines (EPA, 1987) as modified in the Section 4 Testing Consent Order for TGME (EPA, 1989).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

112-5-6

IUPAC Name:

112-5-6

Constituent 2

Reference substance name:

2-(2-(2-methoxyethoxy)ethoxy)ethanol

EC Number:

203-962-1

EC Name:

2-(2-(2-methoxyethoxy)ethoxy)ethanol

Cas Number:

112-35-6

Molecular formula:

C7H16O4

IUPAC Name:

2-[2-(2-methoxyethoxy)ethoxy]ethanol

Details on test material:

- Name of test material (as cited in study report): Triethylene glycol monomethyl ether (TGME)
- Molecular formula: CH3OCH2CH2OCH2CH2OCH2CH2OH
- Molecular weight: 164.2
- Physical state: Clear liquid
- Analytical purity: 99.23% prior to star of study (Spratt and Fish, 1989)
- Composition of test material, percentage of components:
- Supplier: Union Carbide Corporation, South Charleston, WV.
- Vapor Pressure: 0.01mm Hg@ 20C
- Specific Gravity: 1.052

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were randomly assigned into exposure groups using a computer-generated randomization procedure based on individual animal body weights. Animals were uniquely identified with an alphanumeric metal ear tag.

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, MI>
- Age at study initiation: 6 weeks old
- Weight at study initiation:
- Fasting period before study:
- Housing: animals were housed individually in stainless-steel cages with wire bottoms in a room designed to maintain adequate environmental conditions.
- Diet: Certified Laboratory Rodent chow #5002, Ralston Purina Company, St. Louis, MO ad libitum
- Water: ad libitum
- Acclimation period: >7 days prior to dosing, and to elastic bandage, used to hold test material in place, at least four times prior to dosing

Administration / exposure

Type of coverage:

occlusive

Details on exposure:

TEST SITE
- Area of exposure: 12cm2 on the back sides of each rat clipped free of hair.
- % coverage: (>10% of the total body surface area)
- Type of wrap if used: Test material was uniformly spread over the clipped area using a syringe and blunt-tipped needle, covered with at least one absorbent gauze patch, and held in place using an elastic bandage. Elastic bandage consisted of a two-inch wide strip of Vetrap cut to a length suitable for wrapping 1-2 times around the body of the animal. The Vetrap was held in place with Elastikon® elastic tape (1 inch wide) cut to lengths similar to the Vetrap.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): application area was wiped with a water-dampened towel to remove any residual test material.
- Time after start of exposure: 6 hours.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

no further data available

Duration of treatment / exposure:

13 weeks.

Frequency of treatment:

6 hours/day, 5 days/week, (excluding holidays).

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (sham control), 400, 1200 and 4000 mg/kg/bw/day.
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

- Frequency of observations and weighing: Cage-side at least once daily for morbidity, mortality, availability of food and water, and treatment-related effects. An additional observation was made each day of the work-week (typically in the morning) and two observations daily were made on weekends and holidays by animal care personnel for morbidity, mortality and the availability of food and water.
- Necropsy of survivors performed: yes, the day following the last application of test material using methyoxyflurane. The animals were fasted overnight prior to sacrifice. The heart, brain, liver, kidneys, adrenals, spleen, thymus, ovaries and testes from each animal were weighed and recorded. The necropsy included a supplemental in situ examination of the eyes by a glass-slide technique with fluorescent illumination. A complete set of tissues was collected from each animal and preserved in neutral, phosphate-buffered 10% formalin with the exception of the testes, epididymides and ovaries which were fixed in Bouin’s solution.
- Other examinations performed:
- Clinical Observations: Detailed clinical examinations on all animals prior to the start of the study, weekly thereafter throughout the study duration. Thorough evaluations of the skin, fur, mucous membranes, respiration, nervous system function, salivation, diarrhea and behavior patterns were made.
- Body weights and feed consumption: All animals were weighed prior to the first treatment and weekly thereafter. Feed consumption was determined weekly for all animals in the main study group.
- Clinical Pathology: Blood samples for hematologic and clinical chemistry analyses were taken at necropsy from the orbital sinus of fasted rats anesthetized with methoxyflurane. The samples for clinical chemistry analyses were chilled with crushed ice or refrigerated until analyzed.
- The following hematologic parameters were evaluated for each animal:
HCT, HGB, RBC, WBC, PLAT, MCV, MCH, MCHC. Evaluations were made using an ELT-8. Slides for differential leukocyte counts and red blood cell morphology were prepared and evaluated by light microscopy for each animal. Smears for reticulocyte counting were prepared for each animal at the scheduled necropsy. Reticulocyte counts were performed for each animal in the high dose and control groups.
- Urinalysis: animals in main study were housed overnight in metabolism cages for the collection of urine prior to initiation of dosing, after 31 days on test and during the final week of exposure. The following parameters were evaluated using a Clinitek 200: color, appearance, volume, specific gravity, glucose, ketones, blood, pH, protein, urobilinogen and a semiquantitative estimate of bilirubin. In addition, a microscopic examination of the microsediment of the urine from each animal included an evaluation of the presence of erythrocytes, leukocytes, and renal tubular cells.
- Estrous Cyclicity: Daily vaginal smears were obtained from all females in the main study group during the final two weeks of the study. Vaginal smears were sampled via a saline rinse and evaluated microscopically for predominant cell types according the method of Zarrow et al. (1964).

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observationsincluded: To the extent possible, these observations included evaluation of the skin, fur, mucous membranes, respiration, nervous system, and behavior pattern. An additional observation was made each day of the workweek (typically in the morning) and two observations daily were made on weekends and holidays by animal care personnel for morbidity, mortality and the availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were conducted on all animals prior to the start of the study and weekly thereafter throughout the duration of the study. Examinations included thorough evaluations of the skin, fur, mucous membranes, respiration, nervous system function (e.g. observations of tremors and convulsions), salivation, diarrhea and behavior patterns.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: During the first five weeks of the study, the condition of the skin at the application site was subjectively evaluated prior to each application of the test material using this laboratory's modification of the acute dermal irritation scoring system recommended by the Organization for Economic Co-operation and Development (OECD, 1981) and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed prior to the first treatment and weekly thereafter.

FOOD CONSUMPTION:
- Feed consumption was determined weekly for all animals in the main study group.

HAEMATOLOGY: Yes
- Parameters checked: hematocrit, hemoglobin, erythrocyte count, total leukocyte count, platelet counts, and red blood cell indices (MCV, MCH, MCHC). Slides for differential leukocyte counts and red blood cell morphology were prepared and evaluated by light microscopy for each animal. Smears for reticulocyte counting were prepared for each animal at the scheduled necropsy. Reticulocyte counts were performed for each animal in the high dose and control groups.

CLINICAL CHEMISTRY: Yes
- Parameters checked: alkaline phosphatase, alanine aminotransferase activity, aspartate aminotransferase activity, total protein, albumin, globulin, total bilirubin, glucose, urea nitrogen, cholesterol, triglycerides, creatinine, phosphorus, calcium, sodium, potassium (K) and chloride. All analyses with the exception of globulin, Na, K and CI assays were conducted using a CentrifiChem automated chemistry analyzer (Centrifichem System Methods File, Union Carbide Corp., Rye, NY). Globulin values were calculated as the difference between total protein and albumin levels while a Beckman E4A flame photometer (Beckman Instruments Inc., Brea, CA) was used to determine Na, K and CI levels.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes
- Parameters checked: parameters were evaluated using a Clinitek 200 (Ames Division, Miles Laboratory, Elkhart, Indiana): color, appearance, volume, specific gravity, glucose, ketones, blood, pH, protein, urobilinogen and a semiquantitative estimate of bilirubin. In addition, a microscopic examination of the microsediment of the urine from each animal included an evaluation for the presence of erythrocytes, leukocytes, and renal tubular cells.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals in the main study group were necropsied the day following the last application of test material. The animals were fasted overnight prior to sacrifice. Each animal was weighed, anesthetized with methoxyflurane, and blood was collected via orbital sinus puncture for hematologic and clinical chemistry evaluations. The trachea was exposed and clamped prior to decapitation. The heart, brain, liver, kidneys, adrenals, spleen, thymus, ovaries and testes from each animal were weighed and recorded. All animals were examined for gross pathological alterations by a veterinary pathologist. The necropsy included a supplemental in situ examination of the eyes by a glass-slide technique with fluorescent illumination.
HISTOPATHOLOGY: Yes, a complete set of tissues (Table 3) was collected from each animal and preserved in neutral, phosphate-buffered 10% formalin with the exception of the testes, epididymides and ovaries which were fixed in Bouin's solution. The lungs were infused with buffered formalin to their approximate normal inspiratory volume and the nasal cavity flushed via the pharyngeal duct to insure rapid fixation of the tissue. Bone marrow smears were prepared from each animal from the shaft of the femur and stained with May-Grinwald. The testes and epididymides were examined for males in the intermediate and low dose levels. The presence of microscopically visible morphologic abnormalities were graded based on a subjective assessment of the degree to which a specific section of tissue was involved; in the event of mulitiple sections the grading was based upon a composite assessment. All tissues, except testes and associated reproductive organs, evaluated histologically were processed by conventional techniques, sectioned at approximately 6 um, stained with hematoxylin and eosin and evaluated by a veterinary pathologist using a light microscope. A cross section through the approximate center of each testis was obtained, dehydrated through a series of graded ethanols and infiltrated with glycol methacrylate resin. The testes were then sectioned at 3 um and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the 14 stages of spermatogenesis were evaluated following the guidance of Clermont and Perey (1957). Microscopic evaluation included an assessment of the relationships between spermatogonia, spermatocytes, spermatids and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations define the cycle of spermatogenesis. Alterations in these cell associations indicate the presence of some degree of arrest in the maturation of the normal spermatogenic cycle. In addition, the testes were examined for the presence of degenerative changes e.g. vacuolization of the germinal epithelium, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization and fibrosis. The variable degrees of arrest of the spermatogenic cycle and the degenerative changes were graded as previously stated.

Other examinations:

Estrous cyclicity: Daily vaginal smears were obtained from all females in the main study group during the final two weeks (14 days) of the study. Vaginal cells were sampled via a saline rinse and evaluated microscopically for predominant cell types according to the method of Zarrow et al. (1964).

Statistics:

Descriptive statistics (means and standard deviations) were reported for differential leukocyte counts, red blood cell indices and feed consumption. Body weights, absolute and relative organ weights, clinical chemistry data, urine specific gravity and volume, and appropriate hematology data were evaluated by Bartlett’s test for equality of variances. Based on the outcome of Barlett’s test, exploratory data analyses were performed by a parametric or non-parametric analysis of variance. (ANOVA), followed respectively by Dunnett’s test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons when appropriate. Statistical outliers were identified by a sequential test but were not excluded from analyses.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals in the 13-week study survived to termination of the study. All animals in the satellite group, except one control female and one female from the high dose group, survived to the scheduled one-month termination. These two animals did not recover from the anesthesia used during the 48-hr blood collection for hematology and were replaced with two animals from the same shipment. No treatment-related changes were observed in rats administered TGME dermally during daily in-life observations, weekly clinical observations or in ophthamological examinations. A few sporadic observations, principally chormodacryorrhea, were noted in controls as well as TGME-treated groups and are common observations for this age and strain of rat. A few animals were noted to have occasional bleeding from the nose and/or mouth. This was attributed either to overgrown incisors or trauma associated with the extensive handling of the animals that is required in a dermal study of this design. Local dermal irritation was observed in all dose groups except controls. Clinical signs of irritation consisted of very slight to well-defined erythema, very slight-to well-defined edema, slight to moderate/sever scaling, scabbing or scarring.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights and feed consumption for male and female rats administered TGME dermally were comparable to control throughout the 13-week study.

HAEMATOLOGY
No treatment-related effects were noted in hematologic data from male rats. In female rats, a statistically significant decrease (15%) in the mean platelet count from animals in the high dose group was observed when compared to the mean control value. The mean platelet counts for the high dose females were slightly lower than the range of laboratory historical control mean values and were considered to be of no toxicological significance.

CLINICAL CHEMISTRY
No treatment related effects were noted in clinical chemistry data following 13 weeks of treatment.

URINALYSIS
The only effect noted in urinalysis was a statistically significant decrease in urine volume from male rats in the high dose group versus controls following one moth but not 13 biological variability in this parameter and was considered to be of no toxicological significance.

GROSS PATHOLOGY
In particular, all treated and control male rats had light brown discoloration of the dermal test site. This discoloration was secondary either to the clipping procedure or the dermal wrapping technique and was clearly not related to application of the test material. All dermal observations noted prior to necropsy were not evident following exsanguination. Moreover, microscopic examination of tissue from the dermal test site did not indicate pathologic changes in the epidermis, dermis, or subcutis.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no clearly defined treatment-related microscopic changes in either male or female rats. The vaginal cytology of a control rat suggested persistent estrus. Microscopic examination of the ovaries indicated numerous bilateral follicular cysts and cornified vaginal epithelium which supported the cytologic diagnosis of prolonged estrus. The reproductive tracts from all other control and treated female rats had no pathological alterations. Bilateral microscopic changes were observed in the testes and epididymides from one high dose male and one male in the 1200 mg/kg/day group. The testes weights of these two males were the lowest in their respective groups; however, the mean testes weights for these two groups were not statistically different from that of the control mean. The testes of the high dose male had bilateral decreased spermatogenesis in seminiferous tubules which was graded as severe, indicating a complete lack of mature spermatids in greater than 41% of tubules in each testicle. In addition, few spermatids beyond stage 12 of the cycle of seminiferous epithelium were observed in the plastic-embedded, periodic acid-Schiffshematoxylin-stained sections. The epididymides had decreased spermatic elements in the head and tail of greater than 41% of the tubules and ducts. The major component within the ducts was eosinophilic debris and immature spermatids. The testes from the 1200 mg/kg/day group rat had bilateral multifocal degeneration of spermatocytes as well as spermatids which was graded as very slight. The degenerative changes were characterized by loss of spermatocytes and spermatids form germinal epithelium and by the presence of multinucleated spermatocytes. All stages of the cycle of the seminiferous epithelium were observed in morphologically normal tubules. The epididymides from this rat had bilateral decreased spermatic elements in the head and tail of approximately 1-5% of ducts. Some of these ducts also contained immature spermatids. The testes from all other male rats in the 1200 and 4000 mg/kg/day dose groups, as well as the 400 mg/kg group, were morphologically comparable to control rat testes.

ESTROUS CYCLICITY
No treatment related effects were observed on the estrous cycle. One female in the control, two females in the 400 mg/kg/day group and one female in the 1200 mg/kg/day group did not show a complete estrous cycle over the 14 day period. All remaining females in these dose groups and all females in the 4000 mg/kg/day dose group exhibited at least one complete cycle. Most animals exhibited 2-3 cycles that were 4-5 days in duration.

Effect levels

Dose descriptor:

NOEL

Effect level:

4 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

All animals, except those in the satellite groups, were examined by the laboratory veterinarian before the start of the study (test day 3) and during the week prior to sacrifice (test day 89) using a mydriatic solution and an indirect ophthalmoscope.

Applicant's summary and conclusion

Conclusions:

There were no target organs positively identified following 13 weeks of dermal administration of extremely high dose levels of TGME. In addition to evaluating the systemic toxicity of TGME via the dermal route of administration, this study was specifically designed to assess the potential for TGME to induce hematological and testicular effects that have been observed in rats exposed to a structural analogue, 2-methoxyethanol. The only treatment-related effects noted in this study consisted of focal areas (<2mm) of dermal irritation in nearly all animals administered TGME. The dermal irritation was secondary to small abrasions induced by repeated clipping of the fur. Areas of the skin that were not abraded by clipping were unaffected by treatment with TGME. All dermal observations noted prior to necropsy were not evident following exsanguination. Moreover, microscopic examination of tissue from the dermal test site did not indicate pathologic changes in the epidermis, dermis, or subcutis. There were no clearly defined indications of systemic toxicity following 13 weeks of treatment, even with the additional emphasis on lymphoid, hematopoietic and reproductive organs. Therefore, the no-observed effect level (NOEL) for systemic toxicity was the highest dose level, 4000 mg/kg body weight/day.

Executive summary:

In a guideline and GLP study, dermal exposure to triethylene glycol methyl ether (TGME at >10% of total body surface area, shaved, occluded) at doses up to 4000 mg/kg bw/day for 6 hours/day, 5 days/week (except holidays) for 13 weeks produced no clearly-defined indications of systemic toxicity, even with additional emphasis on lymphoid, hematopoietic and reproductive organs. Parameters evaluated included in-life clinical observations, dermal irritation, body weights, feed consumption, hematology, clinical chemistry, urinalysis, estrous cyclicity, selected organ weights, gross pathology and histopathology. The only treatment-related effects noted in this study consisted of focal areas (<2 mm) of dermal irritation in nearly all animals administered TGME. The dermal irritation was considered secondary to small abrasions induced by repeated clipping of the fur. Areas of the skin that were not abraded by clipping were unaffected by treatment with TGME. All dermal observations noted during the course of the study were not evident at necropsy. Moreover, microscopic examination of tissue from the dermal test site did not indicate pathologic changes. Based on these findings, 4000 mg/kg bw/day is considered a subchronic dermal NOEL for TGME systemic toxicity. The substance "Ethanol, 2-butoxy- ,manufacture of, by-products from", the subject of this dossier which is primarily a mixture of triethylene and tetraethylene glycol butyl ether, is expected to exhibit very similar toxicity due to its close structural similarity to TEGME. Comparable metabolism would occur. On a molecular weight scaled basis, the NOAEL would be ~5000mg/kgbw/day.