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Toxicological information

Repeated dose toxicity: dermal

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Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For detailed read across justification that covers all repeat dose toxicity routes of ingestion, see the read across object in chapter 7.5.1 (oral route of exposure.)
Cross-referenceopen allclose all
Reason / purpose:
read-across source
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: TSCA: 40CFR 798.2250 as amended by 40CFR 799.1560
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (reported as 'CD' rats)
- Age at study initiation: 6 wks
- Housing: individual in suspended stainless steel cages, except during mating and lactation period. During latter, solid steel pan fitted and hardwood shaving bedding added.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (ad libitum): Certified Purina lab chow #5002 mash diet
- Water (ad libitum): tap
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 8 - 77
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: back
- % coverage: 3x3cm
- Type of wrap if used: polyethylene (PE) patch held in place by adhesive bandage wrapped around trunk of animal. Gauze pad added beneath PE patch after start of study
- Time intervals for shavings or clipplings:


REMOVAL OF TEST SUBSTANCE
- Washing (if done): wiped only.
- Time after start of exposure: 6hrs


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2ml/kg
- Concentration (if solution): neat
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analysis of diluted dosing solutions showed that 10% dose within 99.5(+/-3.1)% and 30% dose within 99.8(+/-2.6)%.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
200 mg/kg bw/day
Remarks:
applied as 10% solution in water to give constant application volume per unit body weight.
Dose / conc.:
600 mg/kg bw/day
Remarks:
applied as 30% solution in water to give constant application volume per unit body weight.
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
applied neat to give constant application volume per unit body weight.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Rationale for animal assignment (if not random): randomised but to keep mean body weights of control and treatment groups equal.
- other: Post-exposure period: none
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily checks for morbidity/mortality and overt toxic effects

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily qualitative analysis at treatment (dosing) time and after wrapping removed.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, 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: Yes / No / No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-study and near end study

HAEMATOLOGY:
- Time schedule for collection of blood: Pre-study and at 4 and 13 weeks. Collected by venipuncture of orbital sinus.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight and not dosed until after blood collection.
- Parameters examined: Hb, Hct, RBC, MCV, MCH, MCHC, total and differential WBC
- How many animals: no data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-study and at 4 and 13 weeks. Collected by venipuncture of orbital sinus.
- Animals fasted: Yes, overnight
- How many animals: No data
- Parameters examined: ALP, AST, ALT, BUN, glucose, total protein, albumin, globulin, creatinine, bilirubin, Na, K, Cl, Ca, inorganic P.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: Pre-study and at 4 and 13 weeks
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: appearance, colour, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, 16hr volume, microscopic analysis.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: During a 14 day period near the end of the study, daily vaginal smears were collected to dermine if normal oestrus cycle was occuring.
Sacrifice and pathology:
Sacrifice by ether anesthesia.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, including adrenals, brain, kidneys, pituitary, prostate, testes, epididymides, seminal vesicles, liver, ovaries and spleen. All weighed and fixed. Other tissues preserved. High dose and control animal tissues sectioned for histology.
Other examinations:
none
Statistics:
Body weights, feed consumption, organ weights using Bartlett's test to determine if variances equal. If variances equal, parametric procedures used (ANOVA followed by Dunnett's test if appropriate). Alternative non-parametric procedures were Kruskal-Wallis test follwed by Dunn's summed rank test if appropriate. Tests for trend using regression analysis and Jonckheere's test. Walsh's test used to determine equality of means. Comparisons made at 1 and 5% signficance levels (two sided.)
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Concentration dependent increase in irritation worse in females. In females it was evident (erythema) from week 1 at the high dose and week 8 at the mid and high dose, with increasing numbers of animals affected with time. In males, it was only seen at the end of the study and and never effected more than 50% of animals. In males the severity was slight or very slight whilst in females there was necrosis and eschar in some animals at high doses.
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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Occult blood was noted in females treated at 600 or 2000  mg/kg but there was no evidence of urinary casts or significant numbers of erthyrocytes.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
< 200 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: skin irritation
Dose descriptor:
NOAEL
Sex:
male/female
Basis for effect level:
other: all end points other than local skin irritation
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no

Females appeared to be more susceptible than males to concentration-dependent irritation at the application site. Effects at the highest dosage were desquamation,  atonia, eschar and necrosis.

Conclusions:
The only effect of note was dermal irritation at the site of repeated application which occured at all doses, albeit very slight at the low dose and only in males at towards the end of the study. Irritancy was more marked in females than males and produced some necrosis at the highest dose. There were no other adverse findings noted.
Executive summary:

In a well conducted study designed to assess the sub-chronic and reproductive toxicity of 2 -(2 -butoxyethoxy)ethanol to rats, the test substance was administered by the dermal route for 13 weeks to the maximum practical concentration attainable of 2ml/kg. The only effect of note was dermal irritation at the site of repeated application which occured at all doses, albeit very slight at the low dose and only in males at towards the end of the study. Irritancy was more marked in females than males and produced some necrosis at the highest dose. There were no other adverse findings noted.

Reason / purpose:
read-across source
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well reported guideline study. Rationale for using a read across substance is included in overall remarks section
Qualifier:
according to
Guideline:
other: EPA TSCA Consent Order
Deviations:
not applicable
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)
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. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
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).
Dose / conc.:
400 mg/kg bw/day
Dose / conc.:
1 200 mg/kg bw/day
Dose / conc.:
4 000 mg/kg bw/day
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
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.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before start and during week prior to sacrifice (test day 89)
- Dose groups that were examined: 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.

NEUROBEHAVIOURAL EXAMINATION: No.
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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in rats administered TEGME dermally during daily in-life observations. A few sporadic observations, principally chormodacryorrhea, were noted in controls as well as TEGME-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.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
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. 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.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
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.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and feed consumption for male and female rats administered TGME dermally were comparable to control throughout the 13-week study.
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:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
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 biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment related effects were noted in clinical chemistry data following 13 weeks of treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
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.
Behaviour (functional findings):
not examined
Immunological 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
Description (incidence and severity):
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. Microscopic examination of tissue from the dermal test site did not indicate pathologic changes in the epidermis, dermis, or subcutis.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
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.
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.

Dose descriptor:
NOAEL
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Relevant for humans:
yes

Bilateral microscopic changes were observed in the testes and epididymides of one male (of 10) receiving 4000 mg/kg and one male (of 10) receiving 1200 mg/kg.  The mean testes weights for these two groups were not statistically significant from controls.  The authors of the study commented that these degenerative changes were not typical of the lesions associated with the glycol ether 2-methoxyethanol.  The authors also reported that the background incidence of this lesion from contemporary historical control data for this rat species was in the range 0-17%, concluding that these findings were not attributable to TEGME exposure.  The lack of biological relevance of the findings is reinforced by the fact that no similar effects were seen in the oral study at similar doses.

Conclusions:
There were no target organs positively identified following 13 weeks of dermal administration of extremely high dose levels of TEGME. In addition to evaluating the systemic toxicity of TEGME via the dermal route of administration, this study was specifically designed to assess the potential for TEGME 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 TEGME. 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 TEGME. 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 TEGME (>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 TEGME. 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 TEGME. 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 TEGME systemic toxicity.

Reason / purpose:
read-across source
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptably documented study which meets basic scientific principles and contains sufficient detail to be able to judge the results reliable as a contribution to the understanding of the repeat dose toxicity of this substance.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
, limit test - single dose used, not all parameters examined.
GLP compliance:
not specified
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products Inc, Denver, PA
- Age at study initiation: 2-3 months
- Housing: standard conditions
- Diet (e.g. ad libitum): no data
- Acclimation period: 51 days
- Other: animals pre-checked for body weight, haematology, clinical chemistry and skin integrity.

ENVIRONMENTAL CONDITIONS
- no data
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: back: shoulder to rump, 15cm wide strip
- Type of wrap if used: Gauze bandaging and Dermiform tape
- Time intervals for shavings or clipplings: Before testing (time not specified) and repeated as necessary during the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes. Tepid water followed by paper towel to dry.
- Time after start of exposure: after each 6 hour occlusive exposure

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1g/kg/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hour exposures over a period of 21 days
Frequency of treatment:
Daily
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, general appearance, behaviour, dermal toxicity

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily immediately prior to dosing

BODY WEIGHT: Yes
- Time schedule for examinations: no data

WATER AND FOOD CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: beginning and end of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters checked : WBC, RBC, platelet count, reticulocytes, differential counts, MCV, Hgb, mean corpuscular Hgb, mean corpuscular Hgb conc, hematocrit.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: beginning and end of study
- Animals fasted: No data
- How many animals: all
- Parameters checked : Na, K, Cl, Ca, P, bilirubin, total protein, albumin, glucose, GGT, AST, ALT, OCT, BUN, creatinine, globulin, cholesterol.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Within 1 day of final dose, animals administered sodium pentobarbital followed by exsanguination.
GROSS PATHOLOGY: Yes. Thorough examination of 40 tissues, not named. All preseved in formalin (glutaraldehyde for eyes). Following weighed: adrenals, brain, kidneys, liver, ovaries, testes.
HISTOPATHOLOGY: Yes , tissues as for gross pathology.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Slight erythema and oedema from day 6 of study when appearance seen in one animal. By day 9, all animals affected. Odema disapeared by day 18. Some desquamation was seen from days 10-17 at application site and fissuring was seen between days 8 and 16. Histopathology showed that the changes were due to trace acanthosis and trace to moderate dermatitis.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some haematological measurements were seen that were statistically significant from controls but these were occasional and random and not thought linked to treatment (and were also within historical control ranges.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Male brain absolute but not relative weights were increased by 5.5% relative to controls. Since there were no histopathological changes noted and female brain weights were normal, this finding was not attributed to treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A focal testicular degeneration was seen in one rabbit to a slight degree, but this lesion is known to occur spontaneously in rabbits and was therefore not regarded as biologically significant.
Histopathological findings: neoplastic:
not examined
Details on results:
No other significant findings noted.
Dose descriptor:
NOAEL
Sex:
male/female
Basis for effect level:
other: all effects other than dermal irritation
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
The substance does not seem to have the adverse effects on blood or testes that are associated with some other glycol ethers.
Executive summary:

In a sub-acute repeat dose toxicity study, rabbits were exposed to 2-(2-(2 -methooxyethoxy)ethoxy)ethanol by occlusive dermal application for a period of 21 days. Daily application of a single 1000mg/kg dose did not produce systemic toxicity in males or females, including haematological or testicular effects. The only significant adverse effects seen were dermal irritation associated with repeat application at the same site. These were not manifest for 7 -10 days and exhibited as slight erythema, odema, desquamation and fissuring. The severity of these effects declined towards the end of the study, with only erythema remaining towards the end.

Reason / purpose:
read-across source
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptably documented study which meets basic scientific principles and contains sufficient detail to be able to judge the results reliable as a contribution to the understanding of the repeat dose toxicity of this substance.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
, limit test - single dose used, not all parameters examined.
GLP compliance:
not specified
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products Inc, Denver, PA
- Age at study initiation: 2-3 months
- Housing: standard conditions
- Diet (e.g. ad libitum): no data
- Acclimation period: 51 days
- Other: animals pre-checked for body weight, haematology, clinical chemistry and skin integrity.

ENVIRONMENTAL CONDITIONS
- no data
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: back: shoulder to rump, 15cm wide strip
- Type of wrap if used: Gauze bandaging and Dermiform tape
- Time intervals for shavings or clipplings: Before testing (time not specified) and repeated as necessary during the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes. Tepid water followed by paper towel to dry.
- Time after start of exposure: after each 6 hour occlusive exposure

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1g/kg/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hour exposures over a period of 21 days
Frequency of treatment:
Daily
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Basis: nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, general appearance, behaviour, dermal toxicity

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily immediately prior to dosing

BODY WEIGHT: Yes
- Time schedule for examinations: no data

WATER AND FOOD CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: beginning and end of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters checked : WBC, RBC, platelet count, reticulocytes, differential counts, MCV, Hgb, mean corpuscular Hgb, mean corpuscular Hgb conc, hematocrit.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: beginning and end of study
- Animals fasted: No data
- How many animals: all
- Parameters checked : Na, K, Cl, Ca, P, bilirubin, total protein, albumin, glucose, GGT, AST, ALT, OCT, BUN, creatinine, globulin, cholesterol.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Within 1 day of final dose, animals administered sodium pentobarbital followed by exsanguination.
GROSS PATHOLOGY: Yes. Thorough examination of 40 tissues, not named. All preseved in formalin (glutaraldehyde for eyes). Following weighed: adrenals, brain, kidneys, liver, ovaries, testes.
HISTOPATHOLOGY: Yes , tissues as for gross pathology.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Slight erythema and oedema from day 6 of study when appearance seen in one animal. By day 10, all animals affected. Some desquamation was seen from days 10-17 at application site and fissuring was seen between days 8 and 16. Histopathology showed that the changes were due to trace acanthosis and trace to moderate dermatitis
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some haematological measurements were seen that were statistically significant from controls but these were occasional and random and not thought linked to treatment (and were also within historical control ranges.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Male brain absolute but not relative weights were increased by 8.6% relative to controls. Since there were no histopathological changes noted and female brain weights were normal, this finding was not attributed to treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
No other significant findings noted.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: all effects other than dermal irritation
Critical effects observed:
no
Conclusions:
The substance does not seem to have the adverse effects on blood or testes that are associated with some other glycol ethers.
Executive summary:

In a sub-acute repeat dose toxicity study, rabbits were exposed to 2-(2-(2-butoxyethoxy)ethoxy)ethanol by occlusive dermal application for a period of 21 days. Daily application of a single 1000mg/kg dose did not produce systemic toxicity in males or females, including haematological or testicular effects. The only significant adverse effects seen were dermal irritation associated with repeat application at the same site. These were not manifest for 7 -10 days and exhibited as slight erythema, odema, desquamation and fissuring. The severity of these effects declined towards the end of the study, with only erythema remaining towards the end.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For detailed justification see document attached to this record.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: based on a weighted average consideration of the results for the source substances.
Critical effects observed:
not specified

Data source

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
no data

Results and discussion

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All other effects other than local irritation
Remarks on result:
other: based on a weighted extrapolation of the results from the source substances
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Remarks on result:
other: based on a weighted extrapolation of the results from the source substances

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion