Registration Dossier

Administrative data

Description of key information

Data on the components of the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade, diethylene glycol (DEG), triethylene glycol (TEG), and tetraethylene glycol (TTEG), were used to assess its repeated dose toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
prior to or in 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP , no specific information whether a guideline study but appears to follow intent of OECD 407.
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Rats were 6-7 weeks of age at study initiation.
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
Test material was diluted in double distilled water to provide 0, 220, 660, 2000 mg/kg/d
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
ad libitum
Remarks:
Doses / Concentrations:
Concentrations were adjusted to deliver 0, 220, 660, 2000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
Test material was administered in the drinking water to provide 0, 220, 660, 2000 mg/kg/d to groups of Sprague-Dawley rats for 4 weeks. Animals were examined twice a day except on weekends and holidays. Body weights were measured daily and food and water consumption was determined weekly. At the end of the 4 week period, blood was obtained for hematology and clinical chemistry determinations and urine for urinalysis determinations. Organ weights were obtained at necropsy and gross and histopathologic examination of selected tissues conducted.
Positive control:
Not applicable
Observations and examinations performed and frequency:
Clinical observations performed and frequency: 2x/day (clinical signs), except 1x/day on weekends and public holidays; body weights determined daily, food and water consumption weekly. Hematology (5 rats/group in week 4) included erythrocyte count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, leukocyte count, differential blood count and platelet count. Clinical chemistry (5 rats/group in week 4) include determination of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, total bilirubin, cholesterol, creatinine, glucose, calcium, potassium, sodium, inorganic phosphate, urea, and total protein. Urine samples (week two, 20 hr collection from 5 animals per group, and week 5, 24 hour collection; all animals) were cooled during the collection period. Urinalysis included measurement of urine volume, specific gravity, osmolality, N-acetyl beta-D-glucosaminidase, lactate dehydrogenase, alanine aminopeptidase, urea, sodium, calcium, potassium, and inorganic phosphate. Occult blood, protein, glucose, ketones, bilirubin, urobilinogen were estimated semiquantitatively. Sediment of urine samples was microscopically examined for leukocytes, erythrocytes, epithelial cells, amorphous crystals, and bacteria. Additional urine samples collected on day 2 (5 animals/group, 20-h collection) and day 25 (all animals, 24-h collection) for determination of oxalic acid.

Sacrifice and pathology:
Organs examined at necropsy: weights of brain, heart, lungs, adrenals, spleen, ovaries, liver, kidneys, and testes were recorded. Brain, urinary bladder, ureter, testes, epididymes, heart, liver, spleen, kidneys and adrenals of control and high-dose animals were evaluated histopathologically. The following tissues and organs were collected and examined histologically: adrenals, aorta, bone, bone marrow, brain (cerebrum, brain stem, and cerebellum), cecum, cervix, coagulating glands, colon, duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin (normal and treated), spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.
Other examinations:
No additional information available
Statistics:
U-test for significance.

Clinical signs:
no effects observed
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):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
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 specified
Details on results:
No treatment-related effects of toxicological significance were noted in any of the animals treated
Dose descriptor:
NOEL
Effect level:
> 2 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects of toxicological significance were noted in any of the animals treated at 2000 mg/kg/day, the highest dose examined.
Critical effects observed:
not specified

No additional information available.

Conclusions:
NOEL >2000 mg/kg/day
Executive summary:

The subacute toxicity of tetraethylene glycol was examined in Sprague Dawley rats. Test material was administered in the drinking water to provide 0, 220, 660, 2000 mg/kg/d to groups of Sprague-Dawley rats for 4 weeks. Animals were examined twice a day except on weekends and holidays. Body weights were measured daily and food and water consumption was determined weekly. At the end of the 4 week period, blood was obtained for hematology and clinical chemistry determinations and urine for urinalysis determinations. Organ weights were obtained at necropsy and gross and histopathologic examination of selected tissues conducted.

No treatment-related effects of toxicological significance were noted in any of the animals treated. The NOEL >2000 mg/kg/day, the highest dose examined.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented study report, GLP, similar to OECD 408
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Fischer 344 rats were used. A pretest health screen was carried out 2 d after arrival, using 5 males and 5 females from the 14-d study, and 10 males and 10 females from the subchronic study. The screen consisted of clinical examination, examination for fecal parasites, viral screen, necropsy, and histology of multiple organs and tissues. They were housed 2/side of divided stainless steel cages mounted on a stainless steel rack. One to 2 w later, they were housed in similar cages but 1/side and this was maintained throughout the study. They were allowed free access to food and water from an automatic system. Environmental temperature was maintained at 66-77°F and relative humidity at 40-70%. A 12-h photoperiod was used.
Route of administration:
oral: feed
Vehicle:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 748, 1522, 3849 mg TEG/kg/day (= 0, 10000, 20000 or 50000 ppm) for males and 0, 848, 1699, 4360 mg TEG/kg/day (= 0, 10000, 20000, 50000 ppm) for females.
Basis:
nominal in diet
No. of animals per sex per dose:
30/sex/group in the control and high dose and 20/sex/group in the low and mid dose groups
Control animals:
yes, concurrent no treatment
Details on study design:
Fresh diet was prepared and offered to the animals each week. All diet concentrations were prepared by dilution of the premix and mixing for 15 minutes.
Homogeneity of the test substance at each concentration was established prior to the start of the study. Stability of the test material in the diets at 10000 and 50000 ppm was determined prior to dosing after storage in open glass feed jars. Diet concentrations were verified for all dose levels prior to administration of the diets to the animals for the first 4 preparations.
Observations and examinations performed and frequency:
Observations for mortality wer emade twice daily. Detailed clinical observations were performed weekly, and observations for overt clinical signs were performed on other days. Opthalmic examinations were performed prior to the start of dosing and prior to interim (13 week) sacrifice). Body weight and food consumption data were collected for all animals weekly.
Blood was collected on day 30. Prior to termination, hematology ,clinical chemistry and urinalysis were performed.
The following organs were removed and weighed: liver, kidneys, heart, spleen, brain, adrenal glands, testes, and ovaries. A further number of tissues and organs were removed and processed for histological examination.
The following blood parameters were measured or calculated: hemoglobin concentration, erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, total and differential leukocyte count.
The following elements were measured or calculated in serum: glucose, urea nitrogen, albumin globulin, total protein creatinine, bilirubin (total, conjugated and unconjugated), phosphorus, Ca++, Na+, K+, CI-, aspartate and alanine aminotransferases, alkaline phosphatase, gamma-glutamyl transferase, creatine kinase, lactate and sorbitol dehydrogenases.
The following urine values were determined: volume, pH, specific gravity, color, microscopy, blood, protein, ketones, glucose, bilirubin and urobilinogen.
Sacrifice and pathology:
Twenty rats/sex/group were sacrificed at the end of the dosing period, and 10 rats/sex from the control and high-dose groups were sacrificed after the recovery periods and subjected to necropsy examination for any signs of gross pathology.
Statistics:
Data for continuous, parametric variables were intercompared for the dose and control groups using Levene's test for homogneity of variances, by analysis of variance, and by pooled variance t-tests. Frequency data were compared using Fisher's exact tests where appropriate. All statistical tests, except the frequency comparisons were performed using BMDP Statistical Saftware. The fiducial limit of 0.05 was used as the critical level of significance for all tests.
Details on results:
Treatment of rats with TEG for 13 weeks did not result in abnormal or biologically significant clinical signs, ophthalmological changes, changes in food consumption, alterations in clinical chemistry measurements, necropsy findings, or histologic findings. Body weight depression compared to controls occurred in males from the high dose group throughout the study and in females during the latter weeks of the study. Body weights for males from the recovery group were similar to controls after the 6-week period but females did not show recovery of body weight. In fact, the largest difference from the control value occurred during the recovery period for the females. Based on the larger magnitude of change in the high dose group than was observed for the other dose groups, the body weight differences were considered related to treatment. A transient decrease from control in cumulative body weight gain was observed for the animals from both sexes in the middle weeks of the study. Due to the unusual pattern of weight differences, the relationship to treatment of these decreases was unclear. Hematology measurements, including decreased erythrocytes and hematocrit in males from the high and mid dose groups and decreased hemoglobin and increased MCV in the high dose group only, were altered at the 13-week measurement period. These changes were considered to be of questionable biological significance based on a lack of similar effect in the females, the small magnitude of the changes, and the lack of corresponding effects in other cell indexes. Decrease in urine pH at all dose levels in males and the mid and high dose levels in females and an increase in urine volulme in males from the high dose group were considered to be related to TEG treatment. Observations of small increases in kidney weight (high dose group females) and kidney weight relative to body weight (all groups of males and mid and high dose group females) were also considered to be probably treatment related. Based on the lack of any other significant toxic effects, particularly the lack of histologic evidence of renal injury, hyperplasia, or hypertrophy, the altered urine measurements were considered to be most likely related to excretion of the large amounts of test material (or metabolites) during the course of this study.
Dose descriptor:
NOAEL
Effect level:
20 000 ppm
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
1 522 mg/kg diet
Sex:
male
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
1 699 mg/kg diet
Sex:
female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
In the currently reported study with TEG there was no evidence for any specific organ or tissue toxicity. TEG did not show the repeated exposure toxicity characteristic of the lower molecular weight homologues. In this present study, a NOAEL of 20,000 ppm of dietary TEG for the rat was observed.
Executive summary:

Fischer 344 rats (30/sex/group in the control and high dose groups and 20/sex/group in the low and mid dose groups) were exposed to triethylene glycol (TEG) in the diet at concentrations of 0, 10000, 20000, or 50000 ppm for 13 weeks. The doses corresponded to approximate mean consumption values of 748, 1522 and 3849 mg TEG/kg/day for the males from the 10000, 20000, and 50000 ppm groups, respectively. The extra 10 animals/sex in the control and high dose groups were monitored without additional treatment for a 6-week recovery period following the dosing phase of the study. Observations or measurements for clinical signs of toxicity, ophthalmologic changes, food consumption, body weight, clinical pathology (interim and final), organ weights, necropsy, and histology were made.

Treatment of rats with TEG at doses of 10000, 20000, or 50000 ppm for 13 weeks did not result in abnormal or biologically significant clinical signs, ophthalmologic changes, changes in food consumption, alterations in clinical chemistry measurements, necropsy findings, or histological findings. Body weight depression compared to controls occurred in males from the high dose group throughout the study in females during the latter weeks of the study (starting at approximately week 8) Body weights for males from the recovery group were similar to controls after the 6-week period but females did not show recovery of body weight. In fact, the largest difference from the control value occurred during the recovery period for the females. Based on the larger magnitude of change in the high dose group than was observed for the other dose groups, the body weight differences were considered related to treatment. A transient decrease from control cumulative body weight gain was observed for the animals from both sexes in the middle weeks of the study. Due to the unusual pattern of weight differences, the relationship to treatment of these decreases was unclear. Hematology measurements, including decreased erythrocytes and hematocrit in males from the high and mid dose groups and decreased hemoglobin and increased MCV in the high dose group only, were altered at the 13-Week measurement period. These changes were considered to be of questionable biological significance based on a lack of similar effect in the females, the small magnitude of the changes, and the lack of corresponding effects in other red cell indexes.

Decreases in urine pH at all dose levels in males and mid and high dose levels in females and an increase in urine volume in males from the high dose group were considered to be related to TEG treatment. Observations of small increases in kidney weight (high dose group females) and kidney weight relative to body weight (all groups of males and mid and high dose group females) were also considered to be probably treatment related. Based on the lack of any other significant toxic effects, particularly the lack of histologic evidence of renal injury, hyperplasia, or hypertrophy, the altered urine measurements were considered to be most likely related to excretion of the large amounts of test material (or metabolites) during the course of this study. Due to the alterations in urine pH in the males from the 10000 ppm level and the abnormal body weight measurements, a clear NOEL could not be established.

Endpoint:
chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: basic information given.
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Principles of method if other than guideline:
The present experiments were initiated to provide adequate short-term investigations, with particular reference to effects on urinary oxalate excretion, using a DEG sample with a low (less than 0-01 %) MEG content.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: obtained from a specified-pathogen-free colony
- Housing: five per cage
- Diet: Spillers' Laboratory Small Animal Diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 1°C
- Humidity (%): 40 - 50%


Route of administration:
oral: feed
Duration of treatment / exposure:
98 days (first experiment)
225 days (second experiment)
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
First experiment: 0, 300, 1500 or 3000 mg/kg bw for 98 days (= 0, 0.4 , 2.0 or 4.0 %)
Basis:

Remarks:
Doses / Concentrations:
Second experiment: 0, 64, 128, 300, 1500 mg/kg bw for 225 days (= 0 %, 0.085 %, 0.17 %, 0.4 %, 2.0 %)
Basis:
nominal in diet
No. of animals per sex per dose:
15-20 rats in the first experiment (99 days)
10 rats in the second experiment (225 days)
Control animals:
yes, plain diet
Details on study design:
Groups of 15-20 rats of each sex were given diets containaing 0, 0.4 . 2.0 or 4.0 % diethylene glycol (DEG) containing less than 0 .01 %
monoethylene glycol for 98 days .
In a second experiment groups of 10 rats of each sex were given diets containing 0, 0.085, 0.17, 0.4 or 2 .0 % of the same sample of DEG for 225 days.
Observations and examinations performed and frequency:
The animals were weighed initially, on days 1, 4 and 8 and then at approximately weekly intervals throughout the study.
Food and water intake over a 24-hr period were measured at the same intervals.
Urine samples were collected in week 8, 13 and 19 from the males and in week 9, 14 and 19 from the females over 24-hr periods, during which the rats were denied access to food and water. These samples were rendered strongly acid with hydrochloric acid and analysed for oxalic acid by the method of Hodgkinson & Williams (1972). Urine analyses, renal concentration and dilution tests and urinary cell counts on samples collected during the last week of the study, post mortem examinations, organ-weight analyses and collection of blood for haematological examination were carried out.
Sacrifice and pathology:
In the haematological examination the reticulocyte and differential leucocyte preparations were not examined. Histological examination was confined to the kidneys.
Details on results:
14 week study:
4% - deaths, dpressed body weight, increased water intake, increased urine volume, hematuria, increased kidney weight, abnormal gross/microscopic kidney findings, increased liver weight, and abnormal gross/microscopic liver findings noted
2% - kidney hydropic degeneration in the proximal tubule (1/15 rats)

32 week study:
2% - no hydropic degeneration of the kidneys observed; lower body weights in both males and females observed
Dose descriptor:
NOAEL
Remarks:
(98 days)
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on renal hydropic degeneration.
Dose descriptor:
NOAEL
Remarks:
(225 days)
Effect level:
234 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified
Conclusions:
Fifteen to twenty Wistar rats/sex/dose were fed 0, 0.4, 2.0, or 4.0% DEG (equivalent to 300, 1600, and 3000 mg/kg/day in males and 400, 1800 and 3700 mg/kg/day in females) for 14 weeks (phase 1) or ten male and ten female Wistar rats were fed 0, 0.085, 0.17, 0.4, or 2.0% DEG (equivalent to 50, 100, 230, and 1200 mg/kg/day in males and 60, 130, 290, and 1500 mg/kg/day in females) for 32 weeks (phase 2). Observations and measurements included body weights, food and water intake, urinalysis, hematology, clinical chemistry (14 week), and organ weight and histopathology (kidneys only in 32 week). In the 14 week study, effects included death, decreased body weight, and kidney hydropic degeneration at the high dose of 4% DEG. Oxalic acid observed in the urine in some animals of all dose groups was considered a biomarker and does not indicate toxicity. Since hydropic degeneration of the kidney was observed in one animal exposed to 2% DEG and decreased male and female body weights were observed, the LOAEL is considered 2% for the 14 week exposure. The 14 week NOAEL was therefore 0.4% (300 mg/kg/day). For the 32 week study, reduced body weight was observed at 2%, resulting in a NOAEL of 0.4% (234 mg/kg/day).
Executive summary:

Fifteen to twenty Wistar rats/sex/dose were fed 0, 0.4, 2.0, or 4.0% DEG (equivalent to 300, 1600, and 3000 mg/kg/day in males and 400, 1800 and 3700 mg/kg/day in females) for 14 weeks (phase 1) or ten male and ten female Wistar rats were fed 0, 0.085, 0.17, 0.4, or 2.0% DEG (equivalent to 50, 100, 230, and 1200 mg/kg/day in males and 60, 130, 290, and 1500 mg/kg/day in females) for 32 weeks (phase 2). Observations and measurements included body weights, food and water intake, urinalysis, hematology, clinical chemistry (14 week), and organ weight and histopathology (kidneys only in 32 week). In the 14 week study, effects included death, decreased body weight, and kidney hydropic degeneration at the high dose of 4% DEG. Oxalic acid observed in the urine in some animals of all dose groups was considered a biomarker and does not indicate toxicity. Since hydropic degeneration of the kidney was observed in one animal exposed to 2% DEG and decreased male and female body weights were observed, the LOAEL is considered 2% for the 14 week exposure. The 14 week NOAEL was therefore 0.4% (300 mg/kg/day). For the 32 week study, reduced body weight was observed at 2%, resulting in a NOAEL of 0.4% (234 mg/kg/day).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
22 January - 5 February 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to GLP and sufficient data is available for the interpretation of results.
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Qualifier:
no guideline available
Principles of method if other than guideline:
Test material was administered either by oral gavage or in the drinking water to set dose levels for a dominant lethal study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
other: One group was administered the test material in the drinking water while another group was administered the test material via oral gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
14 days
Frequency of treatment:
Drinking water was available ad libitum while rats were orally gavaged daily.
Remarks:
Doses / Concentrations:
0, 1000, 5000, 25000 or 50000 ppm in drinking water or 0, 500, 1000, 2500 or 5000 mg/kg via oral gavage (see Table 1)
Basis:
other: nominal in drinking water and actual ingested in oral gavage
No. of animals per sex per dose:
5 males and 5 females for each dose level and route
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOEL
Effect level:
50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects noted at the highest dose, 50,000 ppm in the drinking water, which was equivalent to 6387 mg/kg/day.
Dose descriptor:
NOEL
Effect level:
5 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects noted in rats administered 5000 mg/kg via oral gavage, 5 day/week for 2 weeks.
Critical effects observed:
not specified

Dosing was initiated on January 22, 1990. The final day of sacrifice was February 5, 1990. Body weight range at first dose was 220.9 to 275.0 grams for the drinking water animals and 220.1 to 273.6 grams for the peroral gavage animals .

Drinking Water

Analytical Chemistry

Homogeneity studies performed on samples from three regions (top, middle, and bottom) of the mixing vessel for each dose level indicated that the test substance was uniformly distributed in the test solutions. The range of the analyses for the homogeneity tests was 89.3 to 100.0% of nominal (Mean + SD = 93.7% + 4.3) for the 1000 ppm dose solution, 93.5 to 98.3% of nominal (Mean + SD = 95.7% + 2.1) for the 5000 ppm dose solution, 95.8 to 98.2% of nominal (Mean + SD = 97.0% + 1.3) for the 25000 dose solution, and 95.9 to 99.5% of nominal (Mean + SD = 97.9% + 1.7) for the 50000 ppm dose solution.

Stability studies were conducted on formulations (1000 and 50000 ppm) stored at room temperature in Nalgenea carboys and also in glass water bottles with rubber stoppers equipped with stainless steel sippers. The mean measured concentrations for the carboy stability study ranged from 93.5 to 98.0 and 97.9 to 106.3 percent of nominal for the 1000 and 50000 ppm solutions, respectively. These results indicated that TTEG in processed tap water was stable at room temperature in Nalgene carboys used for the study for at least 7 days. The mean measured concentrations for the water bottle stability study ranged from 92.3 to 97.9 and 96.3 to 100.6 percent of nominal for the 1000 and 50000 ppm solutions, respectively. These results indicated that TTEG in processed tap water stored at room temperature in the bottles used for the study was stable for at least 13 days. The mean measured concentrations of the dosing solutions prepared for Study Week 1 ranged from 93.5 to 97.9 percent of nominal. The mean measured concentrations of the dosing solutions prepared for Study Week 2 ranged from 93.2 to 98.9 percent of nominal. All solutions were found acceptable for the study.

Clinical Observations and Mortality

No clinical signs related to test substance administration were noted for any dosage group, and no animals died during the study.

Water Consumption

Statistically significant increases in water consumption were noted in animals from the 25000 ppm group at Day 4 and Day 14 (15.8% and 15.2% increases, respectively), and in animals from the 50000 ppm group at all intervals (increases ranged from 42.8% to 70.7%). The mean amount of TTEG consumed was 92, 391, 2355, and 6387 mg/kg/day for the 1000, 5000, 25000 and 50000 ppm dosage levels, respectively.

Food Consumption

No treatment-related differences in food consumption values were noted in any treatment group.

Body Weights

No treatment-related differences in absolute body weights or body weight gains were observed in any treatment group.

Necropsy Findings, Organ Weights, and Final Body Weights

No treatment-related differences were noted in these measurements for any treatment group.

There were no deaths during the conduct of the study and all animals were sacrificed at termination. There appeared to be no consistent changes that could be attributed to treatment with the test chemical, although several of the rats in the drinking water treatment groups did have some discoloration of the stomach mucosa. However, there appeared to be no dosage relationship with respect to the incidence of the discoloration.

Gavage

Analytical Chemistry

Homogeneity studies performed on samples from three regions (top, middle, and bottom) of the mixing vessel for each dose level indicated that the test substance was uniformly distributed in the test solutions. The range of the analyses for the homogeneity tests was 96.4 to 98.8% of nominal (Mean + SD = 97.5% + 0.7) for the 50 mg/ml solution, 94.2 to 100.1% of nominal (Mean + SD = 98.1% + 2.4) for the 100 mg/ml solution, 94.3 to 96.6% of nominal (Mean + SD = 95.4% + 1.3) for the 250 mg/ml solution, and 94.6 to 99.3% of nominal (Mean + SD = 97.3% + 1.6) for the 500 mg/ml solution.

Stability studies were conducted on formulations (50 and 500 mg/ml) stored at room temperature in glass Erlenmeyer flasks. The mean measured concentrations ranged from 97.2 to 97.6 and 93.1 to 97.3 percent of nominal for the 50 and 500 mg/ml solutions, respectively. These results indicated that TTEG in Milli-Q water stored at room temperature in glass Erlenmeyer flasks was stable for at least 14 days. The mean measured concentrations of the dosing solutions prepared for the study ranged from 93.8 to 97.4 percent of nominal, and all solutions were found acceptable for the study.

Clinical Observations and Mortality

No clinical signs related to test substance administration were noted for any treatment group, and no animals died during the study. Summary tables for clinical signs have been omitted from this report because of the lack of findings.

Food Consumption

No treatment-related differences were observed in food consumption values for any treatment group.

Body Weights

No treatment-related differences in absolute body weight or body weight gain were observed for any treatment group.

Necropsy Findings, Organ Weights, and Final Body Weights

No significant treatment related effects on final body weights and organ weights were noted for any group. No treatment-related effects were observed at necropsy.

Conclusions:
No treatment-related effects were noted in any of the observations or monitors for animals treated with tetraethylene glycol via the drinking water for 14 consecutive days or via oral gavage 5 days/week for two weeks.
Executive summary:

Fischer 344 male rats (5/group) were dosed with tetraethylene glycol (TTEG; CAS Number 112-60-7) in drinking water (0, 1000, 5000, 25000, and 50000 ppm) or by gavage (0, 500, 1000, 2500, and 5000 mg/kg), in order to establish the most appropriate mode of administration, and to select doses for a dominant lethal study with TTEG. Animals given TTEG in drinking water were dosed for 14 consecutive days. A dose-related increase in water consumption measurement was observed for the 25000 and 50000 ppm groups. A subsequent probe study confirmed that this increase was due to a real increase in water intake (as opposed to increased spillage), which was confirmed by an increase in urine output. Based on the lack of effects on body weights, food consumption, or other measurements, the increased water consumption was not considered to be a result of toxicity of the TTEG but more likely the result of an osmotic diuresis. The doses corresponded to actual mean intake levels of 92, 391, 2355, and 6387 mg/kg/day for the drinking water animals in the 1000, 5000, 25000, and 50000 ppm groups, respectively. Animals dosed via gavage received treatment for 5 days/week for 2 consecutive weeks. No effects considered to be treatment-related were observed for any monitor for animals treated via drinking water or peroral gavage. The results of this study indicate that either method of chemical administration would be suitable for the dominant lethal study.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
Prior to December 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well documented study published in peer-review journal
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Principles of method if other than guideline:
One month repeat dose toxicity study
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: HanWistar (RccHan:WIST)
Details on species / strain selection:
The rat was selected as the species (rather than the other default regulatory species, the dog), as it was considered more ethically acceptable to use a species of lower sentience for initial explorations.
Sex:
male/female
Details on test animals and environmental conditions:
HanWistar rats (RccHan:WIST), approximately 9–10 weeks of age, were obtained from Harlan UK Ltd. (Derby, UK) Females weighed approximately 160 to 220 g and males 250 to 310 g on Day -7 before the first day of dosing. They were acclimatized for at least 10 days and were housed up to three per cage in transparent plastic cages. Sizzle nest, plastic tunnels and aspen chew blocks were provided. Bedding material consisted of aspen wood chips. The animals had free access to pelleted RM1 (E) SQC diet (Special Diets Services, Witham, UK) and to water from the site drinking water supply, except during urine collection when access to food was temporarily withdrawn. Target values for temperature and relative humidity were 19 to 23 °C and 40% to 70%, respectively. Recorded temperature values remained within the set limits. The animal room was illuminated by artificial light from fluorescent tubes on a 12-hlight/dark cycle.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The rats were dosed daily for at least 28 consecutive days in the morning, at a dose volume of 10 ml kg–1.
30% and 40% solutions were prepared by (v/v) and made up to volume with water, resulting in clear solutions.
Formulations were made up weekly and stored at room temperature.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28-32 days
Frequency of treatment:
Daily
Dose / conc.:
3 380 mg/kg bw/day (nominal)
Remarks:
30% v/v in water
Dose / conc.:
4 500 mg/kg bw/day (nominal)
Remarks:
40% v/v in water
No. of animals per sex per dose:
3 male and 3 female
The dose group sizewas selected based on advice from our laboratory’s statisticians and also on the group sizes used for dose range-finding studies at our laboratory. Larger group sizes (as used on regulatory safety assessment studies) were not deemed appropriate as it was considered that any significant toxicities could be identified using three males and three females, and that the use of more animals would not be ethically justified.
Control animals:
yes, concurrent vehicle
Details on study design:
Three male and three female HanWistar rats were dosed daily for 28-32 days and clinical observations, body weight, food consumption, water consumption, haematology, clinical chemistry, urinalysis, liver CYP induction, gastric emptying, organ weights (kidneys, liver, lung, stomach), and organ histopathology (eyes, liver, kidneys, urinary bladder, lungs, heart, thymus, sternum, upper jaw, lower jaw, stomach/duodenum, jejunum, ileum, colon, caecum, rectum, mesenteric lymph node, tongue) were performed

Initially, one male and one female rat from each group were dosed daily for 4 days to assess tolerance. The data generated from Days 1 to 4 were reviewed, and as no adverse findings were observed, all animals in each group (three males and three females) were then dosed daily for the following 28 days. Hence, all animals were dosed for between 28 to 32 days. Control group animals were given water.
Observations and examinations performed and frequency:
All animals were inspected at least twice daily, and observations were recorded. A physical examination was performed at least once weekly. Body weights of all animals were recorded when random allocation by weight was conducted and then twice weekly from 1week prior to the start of dosing, including Day 1 and on the day of termination. Food consumption was recorded daily for
each cage of animals from 1 week prior to the start of dosing until termination. Water consumption was recorded daily for each cage of animals from 1 week prior to the start of dosing until termination, except when urine samples were being collected.

Blood was collected from non-fasted animals. Blood for haematology and plasma chemistry was taken fromthe vena cava at necropsy (plasma chemistry: up to 2.0ml into lithium heparin tubes; haematology: 0.5ml into EDTA tubes). Water was freely available during urine collection, but food was withdrawn. Individual urine samples were collected over ice, during the last week of dosing, for about 6 h starting immediately post-dose.

Haematology parameters assessed: erythrocytes, haemoglobin, haematocrit, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, red cell distribution width, reticulocytes, platelets, leucocytes, neutrophils, lymphocytes, monocytes, basophils, easoinophils, large unstained cells

Plasma chemistry parameters assessed: albumin, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin (total), calcium, cholesterol, creatinine, globulin, glucose, glutamate dehydrogenase, phosphate (inorganic), potassium, sodium, total protein, triglycerides, urea, abnormal colour and lipaemia

Urine analysis parameters: volume, specific gravity, pH, blood (dipstick), bilirubin (dipstick), ketones (dipstick), N-acetyl glucosaminidase, NAG (quantitative), protein (quantitative), glucose (quantitative), abnormal color was noted
Sacrifice and pathology:
Animals were sacrificed 1 day after the final dose. Their body weight was recorded at scheduled necropsy. For all necropsies, external features were inspected, and the cranial, thoracic and abdominal cavities and contents were examined. Macroscopic abnormalities were recorded. The following organs were weighed intact and unfixed at scheduled necropsy: kidneys, liver, lungs and stomach. Gastric emptying was assessed by taking each stomach with approx 1 cm of duodenum attached, and fixing them during the necropsy. These were then weighed with the contents and then again without contents prior to necropsy completion. Samples of the following tissues were taken at necropsy: eyes, liver, kidneys, urinary bladder, lungs, heart, thymus, sternum, upper jaw (with nares and nasal turbinates), lower jaw with skin, stomach/duodenum, intestine (jejunum, ileum, colon, caecum, rectum), mesenteric lymph node and the tongue. Tissues were fixed in buffered formalin except for the eyes which were fixed in Davidson’s solution. Samples were processed by wax block and then sectioned, stained with haematoxylin and eosin (H&E), and examined microscopically by a board certified registered pathologist.
Other examinations:
CYP1A1/2, CYP2B1, CYP3A and CYP4A were determined from liver microcosms using enzyme-linked immunoassays.
Statistics:
For statistical analysis, each group that received a vehicle to be tested was compared with the control group (Group 1) which had been given water. Group sizes were small (three animals/sex/group where data are complete) and therefore caution was exercised when interpreting the results of statistical testing. Body weight and food consumption data were not statistically analysed. Haematology, blood and urine clinical chemistry were assessed for differences from the Control group using a combined sex analysis of variance. Organ weights were analysed using analysis of covariance (using terminal body weight as the covariate), separately for males and females. A two-sided testing approach was used at the 5% level. No adjustment for multiple testing was performed and, therefore, as the significance level of each test was set at 5%, there was a 5% chance of a statistically significant result arising by chance alone. Due to the large number of comparisons that were made, isolated significant differences, not supported by similar differences in related parameters, were, therefore, generally not considered to be real effects.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Not statistically analyzed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Not statistically analyzed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A higher AST (two-fold) was seen for one male given 30% (v/v) TTEG but not for animals given 40% (v/v) TTEG.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
With 40% (v/v) TTEG, increased urinary volume (males: three-fold; females: two-fold), slightly higher specific gravity and lower pH (by 25% when compared with water-dosed control, females only) were observed.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There was no induction of CYP1A1/2, CYP2B1, CYP3A and CYP4A protein levels under the conditions of this study.
There were no apparent effects on gastric emptying with any of the vehicles tested.
Dose descriptor:
LOEL
Effect level:
4 500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
urinalysis
Dose descriptor:
NOEL
Effect level:
3 380 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects observed
Critical effects observed:
not specified

Table 1: Liver enzymes (individual animal data)

 

ACT (IUI-1)

ALT (IUI-1)

GLDH (IUI-1)

Male

Female

Male

Female

Male

Female

Control

61

60

55

41

6

5

76

91

47

48

7

6

78

64

70

40

6

8

30% (v/v) Tetraethylene glycol

65

74

48

56

4

5

80

70

62

49

4

15

156*

-

76

-

4

-

*Value considered raised compared with water-dosed controls

-No data available as animal was terminated early for reasons unrelated to toxicity.

 

Table 2: Urine volume, urinary specific gravity and urinary pH

 

Urinary volume (mean±SD)

Urine-specific gravity (mean±SD)

Urinary pH (mean±SD)

Male

Female

Male

Female

Male

Female

Control

7.0 (±1.7)

4.0 (±1.7)

1.010 (±0.003)

1.007 (±0.002)

8.0 (±0.9)

8.0 (±0.5)

40% (v/v) Tetraethylene glycol

19.3 (±3.8)

7.7 (±0.6)

1.032 (±0.002)

1.033 (±0.001)

6.8 (±0.3)

6.0 (±0.0)

Conclusions:
While 30% (v/v) tetraethylene glycol was well tolerated without any vehicle related findings, animals given 40% (v/v) tetraethylene glycol had increases in urinary volume and specific gravity, along with lower urinary pH in the females. These changes though did not correlate with any histological findings in the kidneys. The authors conclude that that tetraethylene glycol at doses of 4.5 g/kg/d or more has the potential to affect the kidneys in the HanWistar rat, however, to a much lesser extent than ethylene glycol. Hence, the use of 40% (v/v) TTEG as a vehicle should be carefully considered, particularly for test compounds which could themselves affect the renal system and also for studies of longer than 1month’s duration.
Executive summary:

Potential new drugs are assessed in pre-clinical in vivo studies to determine their safety profiles. The drugs are formulated in vehicles suitable for the route of administration and the physicochemical properties of the drug, aiming to achieve optimal exposure in the test species. The availability of safety data on vehicles is often limited (incomplete data, access restricted/private databases). Nineteen potentially useful vehicles that contained new and/or increased concentrations of excipients and forwhich little safety data have been published were tested. Vehicles were dosed orally once daily to HanWistar rats for a minimum of 28days and a wide range of toxicological parameters were assessed. As 40% (v/v) tetraethylene glycol affected urinary parameters, its use should be carefully considered, particularly for compounds suspected to impact the renal system and studies longer than 1 month. No effects were observed with 30% (v/v) tetraethylene glycol.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
14 day
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
1988-1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Principles of method if other than guideline:
Fourteen day dietary toxicity study in male and female Fischer 344 rats to set dosage levels for a ninety-day feeding study.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Triethylene glycol
CAS 112-27-6
Clear, nonviscous liquid
Stored at room temp
Purity: 99.9%
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Two hundred and one Fischer 344 rats (100 males and 101 females) were purchased from Charles River Breeding Laboratories, Inc. (Kingston, NY). Animals tested free of infectious disease and parasites. The females were nulliparous and non-pregnant. The animals were received on October 17, 1988 and were approximately 28 days of age. The rats were approximately 6 weeks of age at first dose.
Upon arrival at BRRC, the rats were housed two animals per side of divided stainless steel cages (solid sides with wire mesh floors) mounted in a stainless steel Maxi-Rack® (Hazleton Systems, Inc., Aberdeen, MD). The purpose of the double housing was to help acclimate the rats to the automatic watering system. Approximately two weeks later, the animals were individually housed in similar caging and remained housed one animal per cage side throughout the study. Water was available ad libitum. The water (Municipal Authority of Westmoreland County, Greensbjrg, PA) was provided by an automatic watering system with demand control valves mounted on each rack. Ground Purina Certified Rodent Chow® #5002 (Ralston Purina Co., St. Louis, MO) was available ad libitum. Temperature and relative humidity were monitored continuously by a Cole-Parmer Hygrothermograph® Seven-Day Continuous Recorder, Model #8368-00 (Cole-Parmer Instrument Company, Chicago, IL). Temperature was routinely maintained between 66° and 77°F, relative humidity between 40 and 70%, A 12-hour light/dark cycle each day was used (0500 to 1700 light).
Body weight ranges at first dose were 89.5-120.0 g for males and 81.6-100.3 g for females.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Test diets were prepared by direct addition of triethylene glycol to ground rodent feed. A concentrated premix was prepared to ensure complete distribution during the original mixing time of 1 hour. All diet concentrations were prepared by dilution of the premix using a Hobart mixer for 15 minutes. Dietary concentrations were based on the test substance as supplied. Diets were stored in polyethylene containers at room temperature. Fresh diet was prepared and offered to the animals each week.
Dietary concentrations of the test substance were not corrected for purity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed using a gas chromatographic procedure developed at BRRC. Homogeneity of triethylene glycol for each diet concentration was established prior to the start of the study. Stability of the test material in diets at the 10000 and 50000 ppm concentrations was determined in open glass feed jars and in the polyethylene storage containers prior to dosing. Diet concentrations were verified for all doses prior to administration of the diets to the animals.
Duration of treatment / exposure:
Two weeks
Frequency of treatment:
Daily (feed)
Dose / conc.:
10 000 ppm
Remarks:
Nominal
Dose / conc.:
20 000 ppm
Remarks:
Nominal
Dose / conc.:
50 000 ppm
Remarks:
Nominal
No. of animals per sex per dose:
20/sex/dose
Control animals:
yes
yes, concurrent no treatment
Details on study design:
Animals were assigned to test groups, based on body weight, by a computer generated weight non-stratified randomization procedure. Group size was 20 males and 20 females. Only rats with body weights within ± 202 of the population mean for each sex were used in the study. Rats not selected for the study were removed from the room.
The study was designed to evaluate the potential toxicity of TEG over a 14-day exposure period with sacrifice on Day 15. The high dose was selected as the maximum concentration that can be administered to rats without significant nutritional deprivation. The lower doses were chosen to help in the selection of diet concentrations for the 90-day study.
Observations and examinations performed and frequency:
During the 2-week, exposure period, observations for mortality were made twice daily (a.m. and p.m.). Detailed clinical observations were performed and recorded daily. Body weights were collected on Days 0. 7, and 14; food consumption data were collected on Days 7 and 14, After 14 days of dosing, the first 10 animals/group/sex were placed in metabolism cages and urine was collected for approximately 24 hours. These same animals were bled for hematology measurements the next day. No additional measurement were performed on these animals. The remaining 10 animals/sex/group were fasted approximately 15 hours and bled for clinical chemistry measurements prior to sacrifice and necropsy. All blood was obtained via the retroorbital sinus. The following hematologic, clinical chemistry, and urinalysis parameters were measured or calculated:
Hematology: erythrocyte count, hemoglobin, hematocrit, erythrocyte indices, platelet count, total leukocyte count, differential leukocyte count
Clinical Chemistry: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALK), gamma-glutamyl transpeptidase (GGT), creatine kinase (CK), lactate dehydrogenase (LDH), sorbitol dehydrogenase (SDH), glucose, albumin, globulin (calculated), creatinine, total bilirubin, direct bilirubin, indirect bilirubin (calculated), urea nitrogen (UN), total protein, phosphorus, calcium, sodium, potassium, chloride
Urinalysis: total volume, specific gravity, protein, ketone, blood, microscopic elements, color and appearance, pH, glucose, bilirubin, urobilinogen
Sacrifice and pathology:
The surviving rats were weighed an necropsied on Day 15. A complete necropsy was performed on 10 animals/group/sex. The liver, kidneys, heart, spleen, brain with stem, adrenals, testes, and ovaries were weighed for all sacrificed animals. Tissues were saved in formalin and the following were subject to histopathological examination: gross lesions, brain, liver, kidneys, pancreas, testes, ovaries, stomach, duodenum, jejunum, ileum, cecum, colon, urinary bladder, peripheral nerve.
Statistics:
Data for continuous, parametric variables were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests. Non-parametric data were analyzed by the Kruskal-Wallis test followed by, if necessary, the Wilcoxon rank sum test as modified by Mann-Whitney. Frequency data were compared using Fisher's exact tests where appropriate. Allstatistical tests, except the frequency comparisons were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal, R. R. and Rohlf, F. J., W. H. Freeman and Company: San Francisco, 1969). The fiducial limit of 0.05 was used as the critical level of significance for all tests.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Rats from the 50000 ppm dose level showed statistically significant increases in food consumption at Day 14 (6.3% for males only) and Day 7 (5.0% for females only) when compared to controls. These increases were not considered to be biologically significant and no treatment-related effects on food consumption were observed.
The test material consumption measured at weekly intervals was calculated as the mean amount (mg) of triethylene glycol/kg body weight/day.The mean triethylene glycol intake over the entire study was 1132, 2311, and 5916 mg/kg/day for the males and 1177, 2411, and 6209 mg/kg/day for the females, respectively for the 10000, 20000, and 50000 ppm groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats exposed to 50,000 ppm triethylene glycol had elevated MCV and MCH. This change was not seen in the male rats or any other exposure group. There were no other differences seen in any hematologic parameters for any exposure groups of male or female rats compared to control animals.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Male rats had decreased AST and creatine kinase activities in the serum of animals in the 10,000 and 20,000 ppm groups. Males in the 50,000 ppm group also had decreased serum creatine kinase activity. A decrease in serum chloride was also noted in the 10,000 and 50,000 ppm groups of male rats. Female rats in the 50,000 ppm group had a decrease in serum alkaline phosphatase activity. None of these decreases in male or female rats was considered biologically significant. There were no other changes in either male or female rats exposed to triethylene glycol.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinalysis results from male rats in the 20,000 ppm group indicated a slight increase in urine volume and decrease in urine bilirubin. Males and females in the 50,000 ppm groups had decreased urine pH and increased urine volume compared to respective control groups. Male rats also had statistically significant decreases in urine ketone, bilirubin, triple phosphate crystals, and a color change in the 50,000 ppm group. Females in the 50,000 ppm group had a statistically significant decreased incidence of urobilinogen compared to the control animals. Both males and females showed a trend of decreased urine pH and increased urine volume with increasing dose levels. The decreases in urinary component concentrations are related to increased volume and pH changes and are not biologically significant or related to triethylene glycol exposure.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were noted for absolute or relative organ weights for either sex at any dosage level when compared to controls. Female rats from the 20000 ppm dosage level showed a statistically significant decrease in ovarian weights (28% decrease) when compared to controls. Ovaries were also significant for these females when expressed as percent of brain or percent of final body weight but this finding was considered spurious based on the lack of dose response.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related findings were observed at necropsy or microscopically in either sex at any dosage level when compared to control findings.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEL
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
urinalysis
Conclusions:
There were no treatment-related clinical signs of toxicity in either male or female rats exposed to triethylene glycol in the diet at concentrations of 10000, 20000 or 50000 ppm for 14 consecutive days. No mortality occurred in either sex during this study. No treatment-related effects were observed in food consumption, body weights, body weight gains, hematology, clinical chemistry, organ weights, gross pathology, or histologic findings. Treatment-related differences occurred in urinalysis measurements in the high dose group. Increased urine volume and decreased pH were observed for both sexes at the 50000 ppm dietary concentration. Associated with the decreased pH was a reduction in triple phosphate crystals. In addition, the males from the mid dose group had a siightly higher urine volume than the controls. These changes were, however, not accompanied by any alterations in serum chemistry measurements or histopathological changes related to the kidneys.
Executive summary:

Fischer 344 rats (20/sex/group) were exposed to triethylene glycol in the diet at concentrations of 0, 10000, 20000, or 50000 ppm for 2 weeks. The corresponding mean ingested doses were 1132, 2311 and 5916 mg/kg/day for the males and 1177, 2411 and 6209 mg/kg/day for the females. No mortality occurred with either sex, and no treatment-related clinical signs were observed. No treatment-related effects were observed in food consumption, absolute body weights, body weight gains, hematology, clinical chemistry, gross pathology, histology, or organ weight data for either sex at any dosage level when compared to controls. The only treatment-related findings in this study were in urinalysis measurements. Increased urine volume, decreased urine pH, and decreased triple phosphate were observed in males and females from the high dose group.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 42 days
- Weight: males group mean 163 g; females 143 g
-Housing: single housing in wire mesh cages (area about 900 cm2)
- Diet: Kliba-Haltungsdiaet Ratte/Maus/Hamster 343 meal of Firma Klingentalmühle AG, Kaiseraugst, Switzerland, ad libitum
- Water: water, ad libitum
- Acclimatization: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark/hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test substance was administered daily in the diet for about 4 weeks. Control animals received food without test substance. At the end of the administration period the main groups were sacrificed. The recovery groups were maintained for another three weeks without test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A test substance characterization was done before study start. The stability of the test substance was also investigated after the end of the study.
Duration of treatment / exposure:
4 weeks; post-exposure period: 3 weeks
Frequency of treatment:
daily; ad libitum
Remarks:
Doses / Concentrations:
0, 500, 2500, 10000, 40000 mg/kg diet
Basis:
nominal in diet
No. of animals per sex per dose:
control and high dose group: 10;
other test groups: 5
Control animals:
yes, plain diet
Details on study design:
Based on dietary doses used in a carcinogenicity study of 10000, 20000 and 40000 mg/kg bw (Fitzhugh, 1946), the following doses were used for the present study: 500, 2500, 10000 and 40000 mg/kg in the diet. In the Fitzhugh study, bladder stones were found in all 3 dose groups and bladder tumours were reported in the mid dose.
Observations and examinations performed and frequency:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period and the recovery period the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals. Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.
The animals were examined for evident signs of toxicity or mortality twice a day. Additionally, further general clinical examinations were determined weekly.

Sacrifice and pathology:
Clinical Pathology: Blood was taken from the retroorbital venous plexus in the morning from fasted, not anesthetized animals. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer using a random number generator. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. Furthermore liver samples were taken at necropsy. The following examinations were carried out in all animals per test group and sex at the end of the application period. At the end of the recovery period only urinalyses (sediment of males) and clinical chemistry examinations were carried out in the surviving animals.
Clinical Chemistry: An automatic analyzer was used to examine the clinicochemical parameters. The values obtained were transferred to a computer. The following parameters were determined: - sodium - potassium - chloride - inorganic phosphate - calcium - urea - creatinine - glucose - total bilirubin - total protein - glutamat-pyruvat-transaminase - glutamat-oxalacetat-transaminase - alkalische phosphatase
Hematology: The following parameters were determined in blood: - leukocytes - erythrocytes - hemoglobin - hematocrit - mean corpuscular volume - mean corpuscular hemoglobin concentration - platelets The clotting analyses were carried out using a ball coagulometer (KC 10 A model; Amelung, Lemgo, FRG) and the results transferred to the computer. The following parameter was determined: - prothrombin time (Hepato Quick's test)
Urinanalysis: With the exception of sediment examination and Oxalic acid all the urine constituents were determined serniquantitatively using test strips (Combur-9-test M, Roche, Mannheim, FRG). The sediment was evaluated microscopically. The following examinations were carried out: - volume - ph - protein - glucose - ketones - urobilinogen - bilirubin - blood - sediment - nitrite - oxalic acid.
Pathology/Necropsy: All animals were sacrificed by decapitation under C02-anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Weight parameters: Weight assessment was carried out on all animals sacrificed at scheduled dates. The weights of the following organs were determined: - Liver - Kidneys - Testes - Epididymides - Brain - Heart
Organ/Tissue preservation list: The following organs / tissues were preserved in neutral buffered 4% formaldehyde: - All gross lesions - Brain - Heart - Liver - Spleen - Kidneys - Adrenal glands - Testes - Urinary bladder - Ureter - Prostate
Statistics:
The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft (laboratory data processing, responsible:Dr. H.D. Hoffmann). Dunnett´s test was used for statistical evaluation of clinical datas whereas William´s test was used for the pathological results. 70 animals (35 for each sex) were used and exposed to diethylene glycol over a 4 week period.
Clinical signs:
no effects observed
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Urinanalysis:
male:
1) A significant increase of oxalic acid concentration (mg/l) and -amount (µg/16 hours) in the 40000 mg/kg treatment group was observed after an exposure time of 13 and 23 days (days of urinanalysis) in males. The higher levels of oxalic acid in urine on reading time day 23 in the 2500 and 10000 mg/kg dose groups seems not to be substance-related, because there was not an increase in the exposure time window between day 13 and day 23 and no higher oxalic acid amount (µg/16 hours) were observed. The urinanalysis of the recovery animals (43 days after the last application) shows a reversibility to the levels of control animals in the highest dose (40000 mg/kg).
2) In the animals of the 40000 mg/kg calcium-oxalate-crystals were found.

female: A significant increase of oxalic acid concentration (mg/l) and -amount (µg/16 hours) in the 40000 mg/kg treatment group was observed only after an exposure time of 23 days (days of urinanalysis) in females. The females shows a noticable fewer amont of oxalic acid than the males. The female recovery group shows also a reversibility of this effects.

Absolute organ weight:
The high dose of females shows a decrease in absolute brain weight. This effect seems not to be a substance related effect, male animals shows no difference between control and DEG exposure animals. The animal number (n=5) was very small for statistical analyses and the weight data of the individual animals shows a larger variance for control animals (1.64 g - 1.81 g) than for the treated animals (1.59 g - 1.67 g). All other analyzed organs show no statistical significant weight changes.

In summary, increased oxalic acid concentration and amount in both sexes and calcium oxalat crystals in the urine of male animals were reported for the 40000 mg/kg group. The findings were reversible within the recovery, treatment-free period.
Dose descriptor:
NOAEL
Effect level:
10 000 mg/kg diet
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
936 other: mg/kg bw./day
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Effect level:
40 000 mg/kg diet
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Documentation is sufficient for interpretation of results
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Principles of method if other than guideline:
32 or 90 day feeding study
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
One quart of each of TEG was received from Fellowship 155 in September, 1948.
Species:
rat
Strain:
Sherman
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
During a period of 32 days, groups of 5 male and 5 female Sherman strain, Rockland rats were fed the Food Research Laboratory solid diet 2C containing 1.0, 0.25, 0.062, and 0.015% triethylene glycol. The following concentrations of TEG was also fed to rats for 90 days; 0.25 and 0.062% triethylene glycol. Control groups of 10 males and 10 females were run concurrently with both the 32 and 90 day glycol groups.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
32 or 90 days
Frequency of treatment:
daily (feed)
Dose / conc.:
1 other: %
Remarks:
TEG - 32 days
Dose / conc.:
0.25 other: %
Remarks:
TEG - 32 days
Dose / conc.:
0.062 other: %
Remarks:
TEG - 32 days
Dose / conc.:
0.015 other: %
Remarks:
TEG - 32 days
Dose / conc.:
0.062 other: %
Remarks:
TEG - 90 days
Dose / conc.:
0.25 other: %
Remarks:
TEG - 90 days
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, plain diet
Details on results:
In none of the criteria of effects examined did any of the 32-day triethylene glycol groups differ from the controls. Minor pathology, light cloudy swelling cf the liver, was seen in two rats of each of the two lowest dosage levels, 0.047 and 0.011 gm./kg./day. This may be discounted, however, as the same type of damage was seen in a higher percentage of the control rats.

None of the 90-day groups of triethylene glycol differed from the control rats in any way.
Dose descriptor:
NOEL
Effect level:
1 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 32 d
Dose descriptor:
NOEL
Effect level:
0.25 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 90 d
Conclusions:
Groups of rats of both sexes were fed concentrations of 1.0, 0.25, 0.062 and 0.015% of triethylene glycol for 32 days. Other groups of rats received 0.25 and 0.062% of triethylene glycol for 90 days. None of these 90-day dosage groups showed any statistically significant deviations from the rats fed the control diet for a similar period. The highest dosage levels fed that produced no significant effect for 32 days was the high dose of 0.83 gm/kg/day for triethylene glycol.
Executive summary:

Groups of rats of both sexes were fed concentrations of 1.0, 0.25, 0.062 and 0.015% triethylene glycol for 32 days. Other groups of rats received 0.25 and 0.062% of triethylene glycol for 90 days. None of these 90-day dosage groups showed any statistically significant deviations from the rats fed the control diet for a similar period. The highest dosage levels fed that produced no significant effect for 32 days was the high dose of 0.83 gm/kg/day for triethylene glycol.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Documentation is sufficient for interpretation of results
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Principles of method if other than guideline:
32 or 90 day feeding study
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
One quart of DEG was received from Fellowship 155 in September, 1948.
Species:
rat
Strain:
Sherman
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
During a period of 32 days, groups of 5 male and 5 female Sherman strain, Rockland rats were fed the Food Research Laboratory solid diet 2C containing 1.0, 0.25, 0.062, and 0.015% diethylene glycol. The following concentrations of diethylene glycol was also fed to rats for 90 days: 0.062 and 0.015% DEG. Control groups of 10 males and 10 females were run concurrently with both the 32 and 90 day glycol groups.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
32 or 90 days
Frequency of treatment:
daily (feed)
Dose / conc.:
1 other: %
Remarks:
DEG - 32 days
Dose / conc.:
0.25 other: %
Remarks:
DEG - 32 days
Dose / conc.:
0.062 other: %
Remarks:
DEG - 32 days
Dose / conc.:
0.015 other: %
Remarks:
DEG - 32 days
Dose / conc.:
0.062 other: %
Remarks:
DEG - 90 days
Dose / conc.:
0.015 other: %
Remarks:
DEG - 90 days
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, plain diet
Details on results:
In the 32-day experiment, the rats that ate 0.85 gm./kg./day of diethylene glycol (1.0%) had kidneys that were statistically -significantly heavier than those of the control group. Also, one rat that died after 28 dosages had a kidney that showed tubular degeneration upon micropatholagical examination.
None of the 90-day groups of diethylene glycol differed from the control rats in any way.
Dose descriptor:
NOEL
Effect level:
0.25 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 32 d
Dose descriptor:
NOEL
Effect level:
0.062 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 90 d
Conclusions:
Groups of rats of both sexes were fed concentrations of 1.0, 0.25, 0.062 and 0.015% of diethylene glycol for 32 days. Other groups of rats received 0.062 and 0.015% of diethylene for 90 days. None of these 90-day dosage groups showed any statistically significant deviations from the rats fed the control diet for a similar period. However, the highest level of diethylene glycol, 1.0%, produced heavy kidneys in 32 days. The highest dosage level fed that produced no significant effect for diethylene glycol for 32 days was 0.18 gm/kg/day.
Executive summary:

Groups of rats of both sexes were fed concentrations of 1.0, 0.25, 0.062 and 0.015% of diethylene and triethylene glycol for 32 days. Other groups of rats received 0.062 and 0.015% of diethylene and 0.25 and 0.062% of triethylene glycol for 90 days. None of these 90-day dosage groups showed any statistically significant deviations from the rats fed the control diet for a similar period. However, the highest level of diethylene glycol, 1.0%, produced increased kidney weight and renal degeneration in 32 days. The highest dosage level fed that produced no significant effect for diethylene glycol for 32 days was 0.18 gm/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
234 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The database includes oral repeat dose toxicity studies conducted on all the ethylene glycol components and is sufficiently robust to draw conclusions regarding toxicity of repeat oral exposure of these compounds as a formulation.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
4-15 March 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Qualifier:
no guideline available
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sixty-five male and 65 female Sprague Dawley rats were received on February 10, 1991, from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 34 days old (birth date was recorded as January 7, 1991) and to weigh 100-124 g and 75-99 g for the male and female rats, respectively, upon arrival. The females were nulliparous and non-pregnant.

Within 2 days of receipt, the animals were examined by the BRRC Clinical Veterinarian, and representative animals were subjected to a pretest health screen including full necropsy and histologic examination of selected tissues including respiratory organs and serum viral antibody analysis. Based on the results of these data, the Clinical Veterinarian indicated that the animals were in good health and suitable for use on this study.

The animals were housed individually in stainless steel, wire-mesh cages (15 cm x 22 cm x 18 cm). DACB® (Deotized Animal Cage Board; Shepherd Specialty Papers, Inc.) was placed under each cage and changed regularly. An automatic timer was set to provide fluorescent lighting for a 12-hour photoperiod starting at 5 a.m. Temperature and relative humidity were recorded continuously (Cole-Parmer Hygrothermograph® Seven-Day Continuous Recorder, Model No. 8368-00, Cole-Parmer Instrument Co., Chicago, IL). Temperature was routinely maintained at 64-79°F; relative humidity was routinely maintained at 40-70%. Any minor exceptions to these specified ranges were noted in the raw data.

Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was available ad libitum except during exposures. Water was provided by an automatic watering system with demand control valves mounted on each rack, except during the water consumption period. Water analyses were provided by the supplier, the NUS Corporation, Materials Engineering and Testing Co., and Lancaster Laboratories, Inc. at regular intervals. EPA standards for maximum levels of contaminants were not exceeded. Powdered, certified AGWAY® PROLAB® Animal Diet Rat, Mouse, Hamster 3200 (Agway Inc.) was available ad libitum except during exposures. Analyses for chemical composition and possible contaminants of each feed lot were performed by Agway Inc.

Animal Acclimation
The acclimation period was 3 weeks for the animals. During this period, the animals were weighed 3 times at scheduled intervals. Detailed clinical
observations were conducted in conjunction with body weight measurements. Cage-side animal observations were conducted at least once daily, and an additional mortality check was conducted each day (morning). The animals were examined approximately 2 weeks prior to the initiation of the study by a Clinical Veterinarian. Animals considered unacceptable for the study, based on the clinical signs, ophthalmic examination, body weights, or body weight gains, were rejected. The fate of rejected animals and the reasons for rejection were documented in the study record.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
other: report does not state type of exposure but based on chamber size must have been a whole-body exposure
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The particle size distribution was measured using a TSI Particle Aerodynamic Sizer Model APS 3300 (TSI Incorporated, St. Paul, MN). The dilution ratio was 100:1, and the sample collection time was 30 seconds for all target concentrations. These determinations were made at least 2 times per week per chamber. The data collected were analyzed by the method of Hinds (1982) to obtain the MMAD and the geometric standard deviation.

The MMAD for the 500, 2000, and 5000 mg/m3 exposures was 1.92, 2.57, and 2.94 microns, respectively, with a geometric standard deviation (Og)
of 1.56, 1.65, and 1.70, respectively.

Reference:
Hinds, W. C. (1982). Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles. John Wiley & Sons, New York, NY, 85.
Details on inhalation exposure:
Inhalation Chamber Description and Operation
The inhalation chambers (Young & Bertke, Cincinnati, OH) were constructed from stainless steel with glass windows for animal observation, were rectangular (132 x 85 x 91 cm) in shape with a pyramidal top and bottom. The volume of each chamber was approximately 1330 liters, and the airfow rate was approximately 300 liters/minute (13-14 air changes per hour). A Dwyer Magnehelic® pressure gauge (Dwyer Instruments, Inc., Michigan City, IN) was used to monitor chamber airflow. The theoretically-derived time required for each chamber to reach 99% of the target concentration (t99) was calculated to be 20 minutes. Chamber temperature and relative humidity were recorded using a Fisherbrand® dial type thermometer (Fisher Scientific, Pittsburgh, PA) and an Airguide humidity indicator (Airguide Instrument Co., Chicago, IL), respectively. Temperature and relative humidity measurements were recorded approximately 2 times each hour of exposure.

Aerosol Generation
Liquid TEG was metered from a piston pump (RPG-6-l/8" - 500 mg/m3 ; RPG-20-l/4" - 2000 mg/m3; RPG-20-3/8" - 5000 mg/m3; Fluid Metering, Inc., Oyster Bay, NY) into an atomizer (Spraying Systems Co., Wheaton, IL) fitted with a No. 1650 liquid nozzle and a No. 64 air nozzle. The atomizer was inserted into the top of the inhalation chamber turret where the liquid aerosol was dispersed throughout the chamber by filtered chamber supply air. The operating pressure of the atomizer was 20 psi.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber Atmosphere Measurements
Chamber concentrations of TEG were analyzed by gravimetric methods. Six samples were obtained from the TEG aerosol exposure chambers each day. The sample flowrates were 4.25, 1.81, and 0.81 liters/minute for the 500, 2000, and 5000 mg/m3 target concentrations, respectively. The sample collection time ranged from 10 to 20 minutes. A glass fiber filter (47 mm; type A-E; Gelman Instrument Co., Ann Arbor, MI) used to collect the TEG aerosol was connected to a dry gas meter (Rockwell International, Pittsburgh, PAl, a critical orifice, and a vacuum pump (Terracon Corp., Waltham, MA). The nominal concentration was calculated by dividing the total amount of TEG used to generate the exposure atmosphere by the total volume of air delivered to each chamber.

Control animals were exposed to filtered air only. For the target concentrations of 500, 2000, and 5000 mg/m3 , mean gravimetric exposure concentrations (± SD) were determined to be 494 (± 14.2), 2011 (± 94.4) and 4824 (± 182.9) mg/m3 , respectively.
Duration of treatment / exposure:
Animals were exposed for 6 hours per day for 5 consecutive days. After 2 days without exposure, the animals were exposed for an additional 4 consecutive days.
Dose / conc.:
494 mg/m³ air (analytical)
Remarks:
500 mg/m3 (targeted)
Dose / conc.:
2 011 mg/m³ air (analytical)
Remarks:
2000 mg/m3 (targeted)
Dose / conc.:
4 824 mg/m³ air (analytical)
Remarks:
5000 mg/m3 (targeted)
No. of animals per sex per dose:
15 males and 15 females in control and high dose group and 10 males and 10 females in low and intermediate dose groups. (Five animals/sex of the control and high concentration groups were assigned to a 2-week recovery group.)
Control animals:
yes, concurrent no treatment
Details on study design:
The exposures began on March 4, 1991 (Study Day 1). Animals were exposed for 6 hours per day for 5 consecutive days. After 2 days without exposure, the animals were exposed for an additional 4 consecutive days. The 6-hour exposure period for each exposure day was defined as the time when the aerosol generation system was turned on and subsequently turned off. All surviving animals were sacrificed on March 15, 1991, after a total of 9 exposures.

Note The 6-hour exposure period for each exposure day was defined as the time when the aerosol generation system was turned on and subsequently turned off.
Positive control:
No data
Observations and examinations performed and frequency:
In-Life Evaluations
All animals were individually observed for signs of toxic effects except during the exposures. During the exposures, observations were recorded on a group basis. Preceding and following each exposure, observations were recorded for animals exhibiting overt clinical signs. At the time of body weight measurements and just preceding sacrifice, detailed observations were performed on all animals. On nonexposure days, the animals were observed once a day for overt clinical signs and twice a day for mortality.

Body weight data were collected for all animals on the morning prior to initiation of the first exposure (denoted as Day 1 of the study), preceding the second, fifth, sixth, and seventh exposures, and immediately preceding sacrifice.

Food and water consumption measurements were obtained over an approximate 15-hour period following the eighth exposure of male rats and the ninth exposure of female rats (excluding the animals designated for the 2-week recovery period). All animals were housed individually in Nalgene® metabolic cages with stainless steel, wire-mesh bottoms, approximately 20 cm diameter x 11 cm high (Nalge Company, Rochester, NY) during the measurement period.

Prior to the first exposure, the eyes of all rats were examined by a Veterinary Ophthalmologist using indirect ophthalmoscopy following dilation of the pupils with MYDRIACYL® 1% (tropicamide 1.0%) Ophthalmic Solution. Following the ninth exposure, the eyes of all surviving rats (excluding the animals designated for the 2-week recovery period) were again examined by a Veterinary Ophthalmologist by the procedure previously mentioned.
Sacrifice and pathology:
Clinical Pathology Evaluation
Prior to sacrifice, blood was collected from all rats (excluding animals designated for the 2-week recovery period) for hematology and clinical chemistry determinations. Blood was obtained from the orbital sinuses of methoxyflurane-anesthetized animals. Food was removed from the animal cages prior to the start of the blood collection period, but water was supplied ad libitum.

Following the eighth exposure of male rats and following the ninth exposure of female rats, urine was collected from all surviving rats (excluding animals designated for the 2-week recovery period) while the rats were in the metabolism cages (see Food and Water Consumption). Food and water were available ad libitum. Two or three thymol crystals were added as a preservative to the collection tubes.

The following parameters were measured or calculated:
Hematology
hematocrit
hemoglobin
erythrocyte count
mean corpuscular volume (MCV)
mean corpuscular hemoglobin (MCH)
mean corpuscular hemoglobin concentration (MCHC)
total leukocyte count
differential leukocyte count
platelet count
reticulocyte count

Clinical Chemistry
glucose
urea nitrogen
creatinine
total protein
albumin
globulin (calculated)
total bilirubin
direct bilirubin
indirect bilirubin
calcium
phosphorus
sodium
potassium
chloride
aspartate aminotransferase (AST)
alanine aminotransferase (ALT)
creatine kinase (CK) with isoenzymes
lactate dehydrogenase (LDH) with isoenzymes
gamma-glutamyl transferase (GGT)
sorbitol dehydrogenase (SDH)
alkaline phosphatase (ALK)

Urinalysis and Urine Chemistry
osmolality
pH
protein
glucose
ketones
bilirubin
blood
urobilinogen
total volume
color and appearance
microscopic elements
N-acetyl-b-D-glucosaminidase (NAG)

The blood smears for the differential leukocyte counts and reticulocyte counts were prepared for all groups, but were evaluated only for the control and highest exposure concentration group that survived the exposure regimen.

Anatomic Pathology Evaluations
At the end of exposures, all rats (except animals designated for the 2-week recovery period) were anesthetized with methoxyflurane and euthanized by exsanguination via the brachial blood vessels. A complete necropsy was performed on all animals (except animals designated for the 2-week recovery period). The liver, spleen, brain, lungs, kidneys, adrenals, and testes (males) were weighed for all sacrificed animals. The following tissues were collected and saved in 10% neutral buffered formalin:

gross lesions*
lungs*
nasal turbinates (four sections)*
brain
thymus
trachea*
heart*
larynx*
liver*
spleen
kidneys*
adrenals*
testes
ovaries
lymph node
(submandibular)
eyes
bladder*
spinal cord*
sciatic nerve*
tibial nerve*

Tissues with an asterisk for 10 rats per sex from the control group and 2000 mg/m3 exposure concentration group and from all rats (15 per sex) from the 5000 mg/m3 exposure concentration group were processed histologically and examined microscopically. In addition, the respiratory tract (nasal cavities, trachea, larynx, and lungs) was also examined from the 500 mg/m3 exposure concentration group rats.

Other examinations:
No additional information available.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by Levene's test for homogeneity of variances, by analysis of variance, and by t-tests. The t-test was used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated homogeneous variances, the groups were compared by an analysis of variance for equal variances followed, when appropriate, by pooled variance t-tests. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variance followed, when appropriate, by separate variance t-tests. Frequency data were compared using Fisher's exact tests. All statistical tests, except the frequency comparisons, were performed using BMDP Statistical Software (Dixon, 1990). The frequency data tests are described in Biometry (Sokal and Rohlf, 1981). The probability value of p < 0.05 (two-tailed) was used as the critical level of significance for all tests.

References:
Dixon, W. J. (1990). BMDP Statistical Software, University of California Press, Berkeley, CA.

Sokal, R. R. and Rohlf, F. J. (1981). Biometry, W. H. Freeman and Company, San Francisco, CA
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All rats from the high dose group died before the scheduled necropsy.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats from the high dose group died before the scheduled necropsy (between days 2 & 5)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males - 2000 and 5000 mg/m3. Females - 5000 mg/m3.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females - Increased at 500 and 2000 mg/m3.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males - Increased at 2000 mg/m3. Females - Increased at 500 and 2000 mg/m3.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Female - Increased RBC in 2000 mg/m3 group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Male - increase ALT activity/decrease in serum creatinine in 2000 mg/m3 group. Female increases in BUN, ALT, ALK, and inorganic phosphorus and decreases in glucose, creatinine, and chloride in 2000 mg/m3 group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Male - Increased urine volume and decreased urine osmolality and pH at 2000 mg/m3. Female - Increased urine volume and decreased pH.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver and kidney weights
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Unkempt fur, swollen eyelids with periocular and perinasal discharge and crusting, and multifocal or diffuse color change due to congestion and/or hemorrhage of various organs and tissues.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Females - alveolar histiocytosis at 2000 mg/m3.
Histopathological findings: neoplastic:
not specified
Details on results:
All references of differences in group mean values in the following text refer to comparisons between the exposure group and the control group, unless otherwise noted. Repeated reference to the control will not be made in order to simplify the text.

Clinical Observations and Mortality
All rats in the 5000 mg/m3 exposure group died or were sacrificed in a moribund condition on or before the end of exposure day 5. Exposure-related clinical observations included ataxia, prostration, unkempt fur, labored respiration (males only), ocular discharge, swollen periocular tissue, perinasal and periocular encrustation, and blepharospasm in both sexes from the 5000 mg/m3 exposure concentration group. The only significant signs of toxicity in rats from the 500 and 2000 mg/m3 exposure concentration groups were periocular swelling and perinasal encrustation.

Body Weights
Statistically significant decreases in body weights were seen in male rats exposed to 2000 and 5000 mg/m3 of TEG aerosol. Significant decreases in body weight gains were also seen in male rats exposed to 5000 mg/m3 TEG. Female rats also showed statistically significant decreases in body weights and body weight gains, but only in the 5000 mg/m3 exposure concentration group.

Food and Water Consumption
Since the highest exposure concentration of TEG (5000 mg/m3 ) killed all the rats before the end of exposures, food and water consumption measurements were obtained only on the remaining exposures group of animals and controls. Statistically significant increases in food consumption were noted for females from the 500 and 2000 mg/m3 exposure concentration groups, but not in any of the male rats exposed to TEG. Water consumption was significantly increased in females exposed to 500 and 2000 mg/m3 TEG and in male rats exposed to 2000 mg/m3 TEG.

Ophthalmic Examinations
No ophthalmic lesions that could be attributed to the TEG aerosol exposure were noted in rats that survived the exposure regimen.

Clinical Pathology Evaluations
Since all the animals in the 5000 mg/m3 exposure concentration group died before the end of the exposures, blood samples were only collected from the two remaining exposure groups and control rats. Male rats did not show statistically significant changes in the hematology parameters measured. Female rats in the 2000 mg/m3 exposure group had a significant increase in total erythrocyte counts with a significant decrease in MCV.

Since all the animals in the 5000 mg/m3 exposure concentration group died before the end of the exposures, blood samples were only collected from the two remaining exposure groups and control rats. The only statistically significant changes in male rats were an increase in ALT activity and a decrease in serum creatinine in the 2000 mg/m3 exposure concentration group. Female rats from the 2000 mg/m3 exposure concentration group showed statistically significant increases in urea nitrogen, ALT, ALK, and inorganic phosphorus and decreases in glucose, creatinine, and chloride. Females from the 500 mg/m3 exposure concentration group had significant increases in ALK activity, inorganic phosphorus, and total protein.

Since all the animals in the 5000 mg/m3 exposure concentration group died before the end of the exposures, urine samples were only collected from the 2 remaining exposure groups and control rats. Male rats from the 2000 mg/m3 exposure concentration group had statistically significant increases in total urine volume and significant decreases in urine osmolality, pH, and NAG activity. However, the decreased NAG activity was probably due to dilution of the enzyme because of the increased urine volume, since the total NAG enzyme excreted was actually slightly elevated in both male and female rats from the 2000 mg/m3 exposure concentration group. Female rats from the 2000 mg/m3 exposure concentration group had statistically significant increases in total urine volume, and decreases in pH.

Organ Weights, Necropsy Observations, and Microscopic Diagnoses
The only statistically significant changes observed for male rats were increases in liver and kidney weights relative to body weights in the 2000 mg/m3 or the 500 and 2000 mg/m3 exposure concentration groups, respectively. Statistically significant changes in female rats included increases in absolute weights and in weights relative to body and brain weights for liver and kidneys from animals in the 2000 mg/m3 exposure concentration group. Female rats exposed to 2000 mg/m3 TEG also showed a significant increase in adrenal gland weight relative to brain weight.

The most notable gross findings from TEG-exposed rats for both those sacrificed and those which died during the study were unkempt fur, swollen
eyelids with periocular and perinasal discharge and crusting, and multifocal or diffuse color change due to congestion and/or hemorrhage of various organs and tissues. Hyperinflation of the lungs (failure of lungs to collapse when chest cavity was opened) was observed in 5 males and 10 females from the 5000 mg/m3 exposure concentration group. Ocular opacities were also seen in 5 males and 5 females from this latter exposure group.

The most prominent microscopic lesions found in the 5000 mg/m3 TEG-exposed rats which died or were sacrificed moribund involved congestion and, occasionally, hemorrhage of the pituitary, nasal cavities, brain, and lungs of both sexes. Congestion of the kidneys and hemorrhage of the thymus were also relatively common in the females.

The only significant microscopic lesion seen was in female rats sacrificed after the 2000 mg/m3 exposure regimen and was limited to minimal to mild
alveolar histiocytosis. Similar findings were seen in both sexes of rats exposed to 500 mg/m3 TEG, but these latter findings were not significant.
Dose descriptor:
LOEC
Effect level:
494 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minimal liver effects noted (slight increase in liver enzyme levels).
Critical effects observed:
not specified

Among exposure groups, the daily mean chamber temperature and relative humidity ranged from 20.5-24.2°C and 39.9-54.9%, respectively.

Table 1 TRIE'l'HYLENE GLYCOL: NINE-DAY AEROSOL INHALATION STUDY SUMMARY OF URINALYSIS

            Group, mg/m3
 Parameter  0  500  2000  5000
 Day 11 Males        
 Total volume mean (ml)  7.6 (1.97)  9.1 (1.79)  17.1 (6.21)**  N.D.
 Osmolality (mOsm/kg)  2153 (341.9)  2047 (331.4)  1771 (279.2)*  N.D.
 pH  6.9 (0.39)  6.8 (0.24)  6.2 (0.26)**  N.D.
 Day 12 Females        
 Total Volume (mL)  8.0 (4.29)  9.9 (2.64)  16.8 (5.54)**  N.D.
 Osmolality (mOsm/kg)  1894 (612.5)  1695 (320.1)  1696 (296.9)  N.D.
 pH  7.2 (0.88)  6.9 (0.21)  6.2 (0.26)**  N.D.

N=10

N.D. = No Data, all animals died before scheduld sacrifice.

* Significantly different from control group (p < .05)

** Significantly different from control group (p < .01)

TABLE 2

TRIETHYLENE GLYCOL: NINE-DAY AEROSOL INHALATION STUDY IN RATS

SUMMARY OF ABSOLUTE AND RELATIVE ORGAN WEIGHTS (GRAMS)

ANIMALS SACRIFICED AT DAY 12

                        Group, mg/m3
      Control     500     2000     5000
 Parameter  Absolute  Relative  Absolute  Relative  Absolute  Relative  Absolute  Relative
 Males                
 Final Body Weight  297.5 (13.69) ---   302.1 (15.18) ---   286.6 (16.77) ---   N.D.  N.D.
 Liver  12.220 (0.9011)  4.107 (0.2460)  12.938 (1.0778)  4.281 (0.2490)  13.159 (1.3653)  4.585 (0.2921)**  N.D.  N.D.
 Kidney  2.301 (0.1610)  0.773 (0.0451)  2.216 (0.1430)  0.733 (0.0293)*  2.344 (0.2226)  0.817 (0.0516)*  N.D.  N.D.
 Females                
 Final Body Weight  201.3 (10.85)  ---  201.1 (9.32) ---   204.5 (18.55) ---   N.D.  N.D.
 Liver  6.886 (0.5243)  3.418 (0.1303)  7.107 (0.5225)  3.534 (0.1852)  7.869 (1.0877)*  3.836 (0.2401)**  N.D.  N.D.
 Kidney  1.512 (0.0789)  0.751 (0.0195)  1.501 (0.0954)  0.747 (0.0360)  1.639 (0.1079)**  0.804 (0.0399)**  N.D.  N.D.

N=10

N.D. = No Data, all animals died before scheduld sacrifice.

* Significantly different from control group (p < .05)

** Significantly different from control group (p < .01)

Conclusions:
Inhalation exposures to 5000 mg/m3 of TEG aerosol for 6 hours/day produced 100% mortality within 5 days. At lower exposure concentrations over the course of 9 daily exposures, gross evidence of toxicity included periocular and perinasal irritation in both sexes exposed to 2000 mg/m3 and in males at 500 mg/m3. The only statistically significant microscopic finding was a minimal to mild increase in alveolar histiocytosis in female rats exposed to 2000 mg/m3 TEG aerosol. Biochemical findings suggest that the liver may be a target organ for toxicity by repeated inhalation exposure to a high concentration of a respirable aerosol of TEG. Minimal effects (irritation and increased serum ALK activity) were seen at 500 mg/m3 , the lowest exposure concentration tested in this study. Preening of the fur at these high aerosol concentration exposures might hav eled to a confounding factor from the resultant oral intake.
Executive summary:

Four groups, each consisting of 10 Sprague Dawley® rats per sex, were exposed to an aerosol of triethylene glycol (TEG) (CAS No. 112-27-6) at target concentrations of 0 (control), 500, 2000 and 5000 mg/m3 for 6 hours per day for 9 exposures during a 2-week period. An additional 5 animals per sex were added to the high exposure concentration and control groups for planned postexposure recovery observations. Control animals were exposed to filtered air only. For the target concentrations of 500, 2000, and 5000 mg/m3, mean analytical exposure concentrations (± SD) were determined to be 494 (± 14.2), 2011 (± 94.4) and 4824 (± 182.9) mg/m3, respectively. A mean mass median aerodynamic diameter (MMAD) for all 3 exposure groups was 2.48 microns with a mean geometric standard deviation (og) of 1.6. Determinants of toxic effects were clinical observations, ophthalmic examinations, body and organ weights, hematologic and serum clinical chemistry evaluations, urinalysis, and macroscopic and microscopic evaluations.

For all TEG-exposed rats, exposure-related clinical observations occurred. At 4824 mg/m3, clinical observations included ataxia, prostration, unkempt fur, labored respiration (males only), ocular discharge, swollen periocular tissue, perinasal and periocular encrustation, and blepharospasm in both sexes unless noted otherwise. At 494 and 2011 mg/m3, there were swollen periocular tissues and perinasal encrustations. Statistically significant decreases in body weights and body weight gains were also seen in animals exposed to 4824 mg/m3 of TEG. At 2011 mg/m3, there were statistically significant decreases in body weights in males from Exposure Day 5. Females from the 2011 mg/m3 exposure concentration group and rats of both sexes from the 494 mg/m3 exposure concentration group had body weights and body weight gains that were not significantly different from the controls. The most notable gross findings from TEG-exposed rats were unkempt fur, swollen eyelids with periocular and perinasal discharge and crusting, and multifocal or diffuse color change due to congestion and/or hemorrhage of various organs and tissues.

Rats from the 5000 mg/m3 exposure concentration group all died or were sacrificed in a moribund condition on or before the beginning of Exposure Day 5. Therefore, only limited data were obtained from these animals. In addition to the gross pathology findings noted above, hyperinflation of the lungs (failure of lungs to collapse when the chest cavity was opened) was observed during necropsy in 5 males and 10 females from the 5000 mg/m3 exposure concentration group. Ocular opacities were also seen in 5 males and 5 females from this exposure group. The most prominent microscopic lesions found in the 5000 mg/m3 TEG-exposed rats which died or were sacrificed moribund involved congestion and, occasionally, hemorrhage of many organs and tissues. The pituitary, nasal mucosa, brain, and lungs were affected in many of the rats of both sexes. Congestion of the kidneys and hemorrhage of the thymus were also relatively common in the females.

For TEG-exposed rats that survived the exposure regimen, food consumption was statistically significantly increased in an exposure concentration-related fashion for females only at 494 and 2011 mg/m3. Water consumption was statistically significantly increased in both sexes at 2011 mg/m3 and in females at 494 mg/m3. The only statistically significant hematological effects were seen in females from the 2011 mg/m3 group, and included slight increases in erythrocyte count, and slight decreases in mean corpuscular volume. Notable clinical chemistry findings were increased activities of alanine aminotransferase at 2011 mg/m3 and alkaline phosphatase at 494 and 2011 mg/m3 , and slight increases in blood urea nitrogen and inorganic phosphorous in females from the 494 and 2011 mg/m3 exposure concentration groups. Urinalysis showed statistically significant increases in urine volume and decreased osmolality, pH, and N-acetyl-S-D-glucosaminidase activity at 2011 mg/m3 , with a trend for changes in these values at 494 mg/m3. Absolute liver and kidney weights were increased in females from the 2011 mg/m3 exposure concentration, group and increased relative (to body weight) weights were measured for both organs at 2011 mg/m3. There was no histological evidence of liver or kidney injury noted in animals from any exposure concentration group. The only microscopic lesion seen was minimal to mild alveolar histiocytosis, which was in excess of that for the controls at 2011 mg/m3 , but not at 494 mg/m3. The above findings indicate impairment of liver function, but without morphological evidence of organ injury.

This study shows that daily inhalation exposures to TEG aerosol at or above concentrations of 5000 mg/m3 were fatally toxic to rats within 5 days. Similar exposures for up to 9 days at or below concentrations of 2000 mg/m3, however, did not result in life threatening signs of toxic effects. The urinary changes and associated increase in water consumption are consistent with an osmotic diuresis resulting from the excretion of absorbed TEG and its metabolites. There were no consistent findings suggestive of renal injury. The increased serum enzyme activities suggest minimal hepatotoxicity, which, for this study, has a threshold exposure concentration for this toxic response that is near to 494 mg/m3.

A NOEC could not be established. Preening of the fur at these high aerosol concentration exposures might hav eled to a confounding factor from the resultant oral intake.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Principles of method if other than guideline:
Nose only inhalation study
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Triethylene glycol, TEG
CAS 112-27-6
Clear liquid
Stored at room temperature
Purity: 99.9%
Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 257.8-332 g (males), 168.5-242 g (females)
- Fasting period before study: Food available ad libitum except during exposures
- Housing: Individually in stainles stell wire mesh cages
- Diet (e.g. ad libitum): Ground Lab Diet The Richmond Standard Certified Rodent Diet #5002 (PMI Feeds, Inc.) available ad libitum except during exposures
- Water (e.g. ad libitum): Municipal tap water available ad libitum
- Acclimation period: 3 weeks

DETAILS OF FOOD AND WATER QUALITY: Water analyzed at regular intervals. EPA standards for maximum levels of contaminants were not exceeded. Analyses for chemical composition and possible contaminants of each feed lot were performed by PMI Feeds, Inc.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66-77 degrees F
- Humidity (%): 40-70
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: Animals arrived on November 15, 1993 at 35 days old. Exposures began on December 6, 1993. Ten animals/sex/group were sacrificed on December 17, 1993. Five animals/sex of the control and high concentration groups assigned to the recovery group were sacrificed on January 14, 1994.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.2 - <= 1.4 µm
Geometric standard deviation (GSD):
1.32
Remarks on MMAD:
1.2, 1.4, and 1.3 microns for the 100, 500 and 1000 mg/m3 groups, respectively
GSD: 1.25-1.39
Details on inhalation exposure:
A polyvinyl chloride (PVC) exposure chamber was used. These exposure chambers utilize the flow-past, nonrebreathing concept (Cannon et al., 1983) for TEG exposure of the animals. The exposure chambers were composed of separate tiers, with each tier containing a total of 8 exposure ports for exposing 8 animals (1 exposure port/animal). Each animal was housed in a plexiglas tube (5.7 cm diameter tapered front x 19.5 cm length). The control and 1000 mg/m3 exposure chambers had 4 tiers, and the 100 and 500 mg/m3 exposure chambers had 3 tiers. Chambers were provided with air at a flowrate of approximately 16 lpm for the 0 and 1000 mg/m3 and 12 lpm for the 100 and 500 mg/m3 to ensure an adequate oxygen content of at least 19%. The airflow rates were monitored continuously and recorded approximately every 30 minutes. All chambers were maintained at a slightly negative pressure.

Liquid TEG was aerosolized, for all exposure levels, by positioning a single barrel Laskin Aerosol Generator (Enviro-Air Tech, Inc., Goshen, NY) into a glass 3-neck flask containing the TEG. The TEG aerosol was introduced to thenose-only exposure chamber by filtered compressed air passing through the nebulizer and flask and exhausting through 1-inch glass tubing connected to the inlet tube of the chamber. Operating air pressures for the nebulizers ranged from 4 to 6.25 psi.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations in each exposure chamber were determined 3 times during each exposure period by standard gravimetric techniques.
Mean gravimetric concentrations were 102 (+/- 8.2), 517 (+/- 37.9), and 1036 (+/- 27.2) mg/m3 for the target concentrations of 100, 500, and 1000 mg/m3 respectively.

The particle size distribution was measured using a TSI Aerodynamic Particle Sizer (TSI Incorporated, St. Paul, MN) and was determined each day for all exposure groups. The data collected were analyzed by probit analysis (Hinds, 1982) to obtain the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (deltag).
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 consecutive days, 2 days without exposure (weekend), then 4 additional consecutive days
Dose / conc.:
102 mg/m³ air (analytical)
Remarks:
100 mg/m3 (targeted)
Dose / conc.:
517 mg/m³ air (analytical)
Remarks:
500 mg/m3 (targeted)
Dose / conc.:
1 036 mg/m³ air (analytical)
Remarks:
1000 mg/m3 (targeted)
No. of animals per sex per dose:
10/sex/dose (day 12 sacrifice)
5/sex/dose - high dose and control only (4 week recovery sacrifice)
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were exposed for 6 hours/day for 5 consecutive days. The 6-hour exposure period was defined as the time when the animals were connected to and then disconnected from the exposure chamber. After 2 days without exposure (weekend), the animals were exposed for an additional 4 consecutive days. All control animals were exposed only to filtered air using the same exposure regimen. Ten animals/sex/group were sacrificed on December 17, 1993, the day after 9 exposures. The 5 animals/sex of the control and high concentration groups assigned to the recovery group were sacrificed on January 14, 1994, 4 weeks after the final exposure.
Observations and examinations performed and frequency:
All animals were individually observed for signs of toxic effects, except during the exposures. During the exposures, observations were recorded on a group basis. Preceding and following each exposure, observations were recorded for animals exhibiting overt clinical signs. At the time of body weight collection and just preceding sacrifice, detailed observations were performed on all animals. On nonexposure days, the animals were observed once a day for overt clinical signs and twice a day for mortality. Body weights were measured for all animals on the morning prior to initiation of the first exposure (Study Day 1), Study Days 2, 5, 8, 9, and immediately preceding sacrifice. The animals were weighed weekly during the 4-week recovery period. Body weight gains were calculated for the periods between weighings. Food and water consumption were measured over Study Days 1-2, 2-5, 5-8, 8-10 (males) and 8-11 (females). Prior to the first exposure, the eyes of all rats were examined by a veterinarian using direct ophthalmoscopy following dilation of eyes with MYDRIACYL 1% (tropicamide 1.0%) Ophthalmic Solution. Following the eighth exposure for the female rats and following the ninth exposure for the male rats, the eyes were again examined by a veterinarian by direct ophthalmoscopy
following dilation of eyes. An ophthalmology examination was not performed on animals held for the 4-week recovery period.
Sacrifice and pathology:
Prior to the final sacrifice, blood was obtained from all surviving animals, with the exception of those designated for the recovery phase, for hematology and clinical chemistry determinations. Blood samples were collected by retroorbital bleeding from methoxyflurane anesthetized rats on Day 12. Rats were not fasted prior to bleeding. Feed was removed from all animal cages prior to the start of the blood collection period, but water was supplied ad libitum. Blood samples were not collected on the recovery animals. Following the eighth exposure (Study Day 10) for male rats and following the ninth exposure (Study Day 11) for female rats, urine was collected over an approximate 18-hour period from all rats, except those designated for the recovery phase. The rats were housed individually in Nalgene metabolic cages with stainless steel, wire mesh bottoms, approximately 22 cm diameter x 12 cm high (Nalge Company, Rochester, NY), during the collection period. Food and water were available ad libitum. Thymol crystals were added to the collection tube as a preservative. Urine samples were not collected on the recovery animals.

The following were measured or calculated:
Hematology: hematocrit total leukocyte count, hemoglobin differential leukocyte count, erythrocyte count platelet count, mean corpuscular volume (MCV) reticulocyte count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin, concentration (MCHC)

Clinical Chemistry: glucose (nonfasting), urea nitrogen, creatinine, total protein, protein electrophoresis, albumin, globulins, total bilirubin, direct bilirubin, indirect bilirubin (calculated), calcium, phosphorus, sodium, potassium, chloride, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALK)

Urinalysis and Urine Chemistry: osmolality, PH, protein, glucose, ketones, bilirubin, creatinine, creatinine clearance (calculated), blood, urobilinogen, total volume, color and appearance, microscopic elements, N-acetyl-beta-D-glucosaminidase (NAG), alpha2U-globulin

At the end of the exposure regimen, all surviving animals, with the exception of those designated for the recovery phase, were anesthetized with halothane and were euthanized by severing the brachial vessels to permit exsanguination. On the day of sacrifice, body weight was obtained to allow expression of relative organ weights. A complete necropsy was performed on all sacrificed animals. The liver, kidneys, brain, adrenals, lungs, spleen and testes were weighed for all sacrificed animals. An additional 5 rats/sex/group from the control and high exposure groups were euthanized in the same manner following a 4-week recovery period. The recovery animals also received a complete necropsy. The following tissues were collected and retained in 10% neutral buffered formalin: gross lesions, lungs, nasopharyngeal tissue, brain, thymic region, trachea, heart, liver, spleen, kidneys, adrenals, testes, ovaries, urinary bladder, lymph nodes, mesenteric and non-mesenteric, nerve, sciatic, eyes, larynx. Gross lesions, lungs, naspharyngeal tissue, trachea, liver, kidneys, urinary bladder, and layrnx from all animals of the control and high exposure groups from the Day 12 sacrifice were processed histologically and examined microscopically. In addition, the lungs, liver, kidneys, and all gross lesions for all animals from the low and intermediate exposure groups were examined.
Statistics:
The data for quantitative continuous variables were intercompared for the 3 exposure groups and the control group by use of Levene's test for equality ofvariances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant or only one group was compared to the control group. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pairwise comparisons. Nonparametric data were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney test. Incidence data were compared using Fisher's Exact Test. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance. Various models of calculators, computers, and computer programs may have been used to analyze data for this study. Since various models round or truncate numbers differently, values in some tables may differ slightly from those in other tables or from independently calculated data. The integrity of the study and interpretation of the data were unaffected by these differences.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male in the control group died during the first exposure and was replaced with another animal. One male and 1 female were found dead in the 500 mg/m3 group on Days 1 and 2, respectively. These deaths were not considered related to the TEG exposure
Body weight and weight changes:
no effects observed
Description (incidence and severity):
By the end of the exposure period (day 12), both male and female rats of the 1036 mg/m3 group had body weight gains that were numerically lower (circa 15%) in comparison with the controls. In this group, the absolute body weights of males were similar to the controls throughout the study and the absolute weights of females were only slightly lower (4%) than the controls by the end of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The only ophthalmic lesion found in any TEG exposed animals was mild unilateral conjunctivitis in 1 female rat from the 500 mg/m3 group. As no rats were affected from the high concentration group, this is considered to be an incidental finding. Animals saved for a recovery period were not reexamined as they had no eye lesions at the postexposure examination.
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean glucose levels were statistically significantly reduced in high dose females. This was not considered related to treatment.
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the Day 12 sacrifice, no organ weight changes in the males or females were observed. At the Day 40 sacrifice, the absolute and relative (as percentages of body and brain weights) adrenal gland weights were increased in the males in the 1000 mg/m3 group. The relative (as a percentage of body weight) adrenal gland weights in the females increased in the 1000 mg/m3 group. As the organ weight changes occurred 4 weeks after the TEG exposures were terminated, they were not considered related to TEG.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Effect level:
1 036 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No statistically significant, treatment-related changes at the high dose.
Conclusions:
A 9 day repeated aerosol study was conducted, but by nose-only exposure of rats for 6 h/day to TEG aerosol concentrations of 0, 102, 517 and 1036 mg/m3. In this study there were no clinical signs, no effects on food and water consumption, and no biochemical or histological evidence of hepatorenal dysfunction. By the end of the exposure period, male and female rats of the 1036 mg/m3 group had body weights lower than those of the controls, but not with statistical significance. Since there were no statistically significant effects on any monitors, 1036 mg/m3 is considered to be a threshold for toxicity by nose-only exposure to TEG aerosol.
Executive summary:

A 9 day repeated aerosol study was conducted, but by nose-only exposure of rats for 6 h/day to TEG aerosol concentrations of 0, 102, 517 and 1036 mg/m3. In this study there were no clinical signs, no effects on food and water consumption, and no biochemical or histological evidence of hepatorenal dysfunction. By the end of the exposure period, male and female rats of the 1036 mg/m3 group had body weights lower than those of the controls, but not with statistical significance. Since there were no statistically significant effects on any monitors, 1036 mg/m3 is considered to be a threshold for toxicity by nose-only exposure to TEG aerosol.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
22 May 2000 - 24 October 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Principles of method if other than guideline:
Nose only inhalation study
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Name: Diethylene glycol (polyester grade)
Source: Union Carbide Corporation, Texas City Plant
Color: colorless
Physical state: liquid
Purity: 99.9%
Expiry date: March 2001
Storage conditions: ambient temperature
Species:
rat
Strain:
other: Alpk:APfSD (Wistar-derived)
Details on species / strain selection:
The rat was used because it is one of the species generally recommended for the assessment of toxicity. The Alpk:APfSD strain of rat was used because of the substantial background data available for this strain, in this Laboratory, relating to studies of this type.
Sex:
male/female
Details on test animals and environmental conditions:
Approximately 7-8 weeks old on delivery. The weight range at the start of exposure was 291-345g (males) and 200-2458 (females).
On arrival in the inhalation unit, the rats were housed 5 per cage, sexes separately.
Animal room conditions: 22 +/- 3 degrees C, 30-70% relative humidity, 15 air changes/hour, 12/12 hr light/dark
Diet (CT1) supplied by Special Diet Services Limited, Witham, Essex, UK and mains water
supplied by means of water bottles, were available ad libitum except during exposure.
22+3"C
30-70%
At least 15 changedhour
Artificial giving 12 hours light, 12 hours dark
Each batch of diet is routinely analysed for composition and for the presence of contaminants. Water is also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study.
The animals were housed under the experimental conditions for at least 10 days at CTL, prior to the start of the exposure.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1.93 - <= 2.03 µm
Geometric standard deviation (GSD):
1.98
Remarks on MMAD:
MMAD was 3.34, 3.20, 2.76 micron for exposure concentrations of 5, 3, and 0.5, respectively
GSD was 2.03, 1.97, and 1.93 for exposure concentrations of 5, 3, and 0.5, respectively
Details on inhalation exposure:
Each test atmosphere was generated using a glass concentric -jet atomiser. Clean, dry air (dried and filtered usingequipment supplied by Atlas-Copco, Sweden) was passed through the atomiser at nominal flow rates of 24,27 or 25llminute (at normal temperature and pressure) for the 0.5, 3.0 or 5.0mgll concentrations respectively and carried the atmosphere to each of the exposure chambers (internal volume of 46.0 litres), in order to achieve a minimum of 12 air changes per hour. Air flows were monitored and recorded at approximately 30 minute intervals using variable area flowmeters (KDG Flowmeters, Burgess Hill, Sussex, UK) and were altered as necessary to maintain the target concentrations. Nominal generation air flows were 20, 24, 27, 25, 20 and 25 L/minute for Groups 1, 2, 3, 4, 5, and 6 respectively.

The rats were exposed nose-only to the test atmospheres. Animals were restrained in polycarbonate tubes supplied by Battelle, Geneva, Switzerland. These were inserted into a PERSPEX exposure chamber. The chamber was covered with an aluminium cone and stood on an aluminium base. The atmosphere was shown to be acceptably stable over approximately 30 minutes before exposure of the test animals. During this period the Identities of rats holes of the exposure chamber were plugged.

Control animals were exposed to air only, but were otherwise be treated in a similar manner to the test animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The particulate concentration of each test atmosphere, close to the animals' breathing zone, was measured gravimetrically at least three times during exposure. This was done by drawing each test atmosphere, at a known flow rate, for a known time, through a 25mm diameter, polyvinyl chloride (PVC) GLA 5000 filter housed in a Delrin open-faced filter holder (both filters and holders supplied by Gelman Sciences Limited, Northampton, UK). The filter was weighed before and after the sample was taken. The concentration was calculated as follows:
Concentration (mgll) = post wt - pre wt (mg) / time (minute) x airflow (l/minute)
pre wt = weight of filter prior to sampling
post wt = weight of filter after sampling
The aerodynamic particle size distribution of each test atmosphere was measured once per concentration per exposure period, using a Marple Cascade Impactor (supplied by Schaeffer Instruments Limited, Wantage, Oxon, UK) which aerodynamically separates airborne particles into pre-determined size ranges. The amount of aerosol, by weight, in each size range, was then used to calculate the aerodynamic particle size distribution of the aerosol. Using a microcomputer, the data were transformed using a loglprobit transform and a linear regression derived from the cumulative data. Using this regression line, the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated.
Duration of treatment / exposure:
11 days exposure; 28 days recovery (recovery groups only)
6 hrs/day
Frequency of treatment:
5 days for week for 9 days over a period of 11 days (5 days exposre, 2 days off, 4 days exposure)
Dose / conc.:
0 mg/L air (nominal)
Remarks:
2 groups: main & recovery
Dose / conc.:
0.5 mg/L air (nominal)
Remarks:
Main group only
Dose / conc.:
3 mg/L air (nominal)
Remarks:
Main group only
Dose / conc.:
5 mg/L air (nominal)
Remarks:
2 groups: main & recovery
No. of animals per sex per dose:
10 animals/sex/dose (main study)
5 animals/sex/dose (2 recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of ten male and ten female Alpk:APfSD (Wistar-derived) rats were exposed nose-only for 6 hours per day to 0 (control), 0.5,3.0 or 5.0 mg/l diethylene glycol for 9 days over a period of 11 days. Additional groups of five male and female animals were similarly exposed to 0 or 5.0 mg/l diethylene glycol for 6 hours per day, using the same regime, and retained without treatment for a further 28 days (recovery study).

Clinical observations were made daily throughout the main study, then weekly during the recovery period. Bodyweights were measured daily, and food and water consumption was measured continuously throughout the exposure period. Urine samples were obtained from the animals at the end of the ninth day of exposure, and at the end of the scheduled period, the animals were killed and subjected to a full examination postmortem. Cardiac blood samples were taken for clinical pathology from all animals, selected organs were weighed and specified tissues were taken for subsequent histopathology examination.
Observations and examinations performed and frequency:
Prior to the start of the study, all rats were examined to ensure that they were physically normal and exhibited normal activity. During exposure, they were observed frequently and, at the end of the 6-hour exposure period, each rat was examined. Detailed clinical observations, included the finding of no abnormalities detected, were recorded daily immediately prior to exposure. During the recovery period the animals were checked daily and detailed observations were recorded weekly.

The bodyweight of each rat was recorded daily during the exposure phase (prior to exposure), once a week during the recovery phase and prior to termination.

Food consumption was recorded continuously throughout the study for each cage of rats and calculated as a daily mean (g food/rat/day) for each cage.

Water consumption was measured for each cage daily and calculated as a mean value (ml) per rat per day.
Sacrifice and pathology:
Blood samples were taken by cardiac puncture, after the animals had been killed. Urine samples were taken from main study and recovery animals following the final exposure on day 11.

The following haematology measurements were made: RBC count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total white cell count (WBC), differential WBC count, platelet count, reticulocyte count. Blood cell morphology and measured of clotting potential were also measured.

The following measurements were made on blood plasma: urea, creatinine, glucose, total protein, albumin, total bilirubin, alkaline phosphatase activity, alanine, aminotransferase activity, aspartate aminotransferase activity, sodium, potassium, chloride, calcium, phosphorus (as phosphate), gamma-glutamyl transferase activity, creatine kinase activity, cholesterol, tri-glycerides.

Individual urine samples were collected from all main study and recovery animals following the final exposure (i.e. prior to termination for main study animals). Samples were collected over a period of 16- 18 hours during which the rats were housed individually in metabolism cages and denied access to food and water. The following parameters were measured and recorded on each urine sample: volume, specific gravity, appearance, pH, osmolality, nitrite, N-acetyl-beta-d-aminoglucosaminidase (NAG), glucose, ketones, bilirubin, protein, blood, urobilinogen, creatinine. In addition, urine samples were centrifuges and the sediment stained and examined microscopically to identify the components.

At the scheduled termination (day 12 for main study animals and day 40 in recovery animals, after 28 days monitoring in recovery animals), the following organs were removed from all animals, trimmed free of extraneous tissue and weighed: adrenal glands, brain, kidneys, lungs (with trachea attached but larynx removed), liver, testes. All animals were subjected to a full examination post mortem. This involved an external
observation and a careful internal examination of all organs and structures.

All processed tissues from the main study control and high exposure groups were examined by light microscopy in the first instance. Following findings in the kidney at the top dose, kidneys from intermediate groups and recovery groups were processed and also examined by light microscopy.
Statistics:
All data were evaluated using analysis of variance andlor covariance for each specified parameter using the MIXED procedure in SAS (1996) and were carried out separately for main study and recovery animals.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Abnormalities generally associated with restraint (wet fur, stains around the nose, chromodaccryorhoea) were seen in all animals during exposure, though chromodaccryorhoea was less prevalent in the control animals. Slight salivation was also seen in animals exposed to diethylene glycol - this observation showed a concentration-response relationship in that this was seen on more occasions and with earlier time to onset with increasing exposure concentration.

Abnormalities generally associated with restraint (wet fur, stains around the nose, chromodaccryorhoea) were seen in some animals immediately after exposure. Salivation was noted on one occasion only in some animals exposed to 5.06 mg~dl iethylene glycol, indicating that recovery from this symptom was rapid on removal from the causative agent. Most clinical observations seen immediately after exposure had recovered by the start of exposures the following day, with the exception of stains around the nose which persisted in some individual animals, across the groups, until the next morning.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the main study, bodyweights were statistically significantly lower in all treated males and showed a dose response relationship, although bodyweights generally remained with 5% of control values. Bodyweights of main study females exposed to 5.06 mgll diethylene glycol were 2% lower on day 2, but recovered by day 6-7. There were no other changes in bodyweight for females in any other group compared with controls.

In the recovery groups there was no reduction in bodyweight for males or females exposed to 5.06 mgA diethylene glycol either during the exposure phase or the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In main study animals, food consumption was generally lower throughout in males exposed to all concentrations of diethylene glycol (reflecting the lower bodyweights) but only achieved statistical significance on a few occasions. On day 1, food consumption in females exposed to 5.06mgfl was lower than controls, also reflecting the lower bodyweight for these animals from day 2 onwards for several days. No changes in food consumption were noted in male or female recovery animals exposed to 5.06 mgA, reflecting the lack of change in bodyweights for these animals.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was generally high in females exposed to all concentrations of diethylene glycol, though there was no coherent dose response, and water consumption returned to control levels during the recovery phase. There were no consistent effects on water consumption in males at any exposure concentration.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects of haematological significance. Variability was seen in some of the parameters, some of which achieved statistical significance. However, in some cases this was attributable to individual variation in some animals, or in the case of lower reticulocyte count in males exposed to 5.06 mg/l diethylene glycol, this was in part due to some high values in both the control group and the group exposed to 3.0 mgll and was considered to be of no biological or toxicological significance.

The lower neutrophil count in females exposed to 5.06 and 0.53 mg/l diethylene glycol, in the absence of a clear dose response and no evidence of this effect in recovery animals, is considered not to be related to treatment. The higher monocyte count in males exposed to 3.00 mg/l diethylene glycol, and reduced APPT in females exposed to 0.53 mg/l, in the absence of a dose response, were considered not to be related to exposure to the test material.

In recovery animals, raised haemoglobin levels in males and females, higher haematocrit and red blood cell counts in males, and higher platelet counts in females exposed to 5.06 mg/l diethylene glycol - whilst statistically significantly different to the concurrent control values, were biologically very small are therefore considered to be incidental to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the main study, triglycerides in males and total bilirubin values in females exposed to 5.06 mg/l diethylene glycol were slightly higher than control values (though this was due in part to one high individual value for female 8 1). Enzyme activity was slightly higher for alkaline phosphatase in main study females exposed to 5.06 mgll diethylene glycol and for alanine aminotransferase and aspartate aminotransferase in main study males exposed to the same concentration, but they were of limited, if any, biological or toxicological significance and these changes were not seen following the recovery period. Other variations seen in main study females (cholesterol and chloride in females) were confined to intermediate exposure concentration groups so were considered not to be treatment-related. The lower creatinine values and aspartate arninotransferase activity seen in recovery females exposed to 5.06 mgll were due to some high control values in females, were not present in main study groups so were not considered to be treatment-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In main study males, urine osmolality and specific gravity were higher in animals exposed to 3.00 or 5.06 mgll diethylene glycol, and these effects were also in the recovery group exposed to 5.06 mgll diethylene glycol. Urine pH was lowered in both sexes in a dose-dependent manner, in both main study and recovery animals exposed to diethylene glycol.

Variations from mean control values seen in other parameters (urine volume, urine creatinine, urine NAG in main study males) were confined to intermediate concentration groups and thus is considered not to be treatment related.

There were no treatment related effects on the qualitative urinary parameters or urine sediments.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Values for organ weights adjusted for final bodyweight for main study animals should be considered with caution since fasting during urine collection resulted in significant weight loss prior to termination. Higher adrenal weights (absolute and adjusted) in females exposed to 3.00 mgll diethylene glycol and higher brain weight (adjusted) in males exposed to 3.00 mg/l diethylene glycol, in the absence of a dose response are considered to be incidental to treatment. Lower liver and lung weights in males exposed to 5.06 mg/l diethylene glycol, in the absence of any accompanying pathological change, similar effects in females or effect in the recovery groups, are considered to be unrelated to exposure to diethylene glycol. No differences from control weights were seen for any organs examined in treated animals following the recovery period.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of other, non-treatment related findings was similar between treated and control animals and was consistent with the expected incidence of spontaneous background findings for this age and strain of rat: In the kidneys, minimal focal tubular vacuolation was seen in a few rats exposed to 5.06 (1110 males and 2110 females) and 3.00 mgll diethylene glycol (2110 females), and also in a control rat from the recovery phase. Although this change was seen at a slightly higher incidence in these treated groups compared to controls, in all cases it was associated with other spontaneous background pathological changes (hydronephrosis, tubular basophilia and intratubular microlithiasis) and is therefore
considered not to be related to treatment with diethylene glycol.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Effect level:
3 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry

Minor changes in study in hematology and clinical chemistry normally would be considered of no toxicological significant, but the fact that DEG produces toxicologically significant effects in a number of these parameters by other routes and at higher doses suggests that the small changes seen here may progress with longer duration inhalation exposure.

Conclusions:
Nose-only exposure for 6 hours per day for 9 days (5 days exposure, 2 days off, followed by 4 days exposure) to diethylene glycol at concentrations of 0.53, 3.00 or 5.06 mg/l resulted in a bodyweight reduction in males exposed to all concentrations, while there were no differences in bodyweight in recovery males throughout the exposure phase or at the end of the recovery period. Minor changes in some haematological and clinical chemistry parameters were seen at 5.06mg/l. While these would normally be considered of no toxicological significance, the fact that diethylene glycol produces toxicologically significant effects in a number of these parameters by other routes and at higher doses suggests that the small changes seen here may progress with longer duration inhalation exposure.

Therefore, with the possibility of progression of these changes at 5.06mg/17 together with a questionable effect on bodyweight, this concentration is considered to be an effect level for diethylene glycol in this study, and the No Observed Adverse Effect Level (NOAEL) is considered to be 3.00mg/l diethylene glycol.
Executive summary:

Study design: Groups of ten male and ten female Alpk:APfSD (Wistar-derived) rats were exposed nose-only for 6 hours per day to 0 (control), 0.5,3.0 or 5.0 mg/l diethylene glycol for 9 days over a period of 11 days. Additional groups of five male and female animals were similarly exposed to 0 or 5.0 mg/l diethylene glycol for 6 hours per day, using the same regime, and retained without treatment for a further 28 days (recovery study). Clinical observations were made daily throughout the main study, then weekly during the recovery period. Bodyweights were measured daily, and food and water consumption was measured continuously throughout the exposure period. Urine samples were obtained from the animals at the end of the ninth day of exposure, and at the end of the scheduled period, the animals were killed and subjected to a full examination postmortem. Cardiac blood samples were taken for clinical pathology from all animals, selected organs were weighed and specified tissues were taken for subsequent histopathology examination.

Results: The achieved test atmosphere had the following characteristics:

 Target concentration (mg/l)  Total particulate concentration (mg/l)  MMAD (micron)  GSD
 5  5.06 +/- 0.32  3.34 +/- 0.27  2.03 +/- 0.16
 3  3.00 +/- 0.23  3.20 +/- 0.28  1.97 +/- 0.19
 0.5  0.53 +/- 0.05  2.76 +/- 0.34  1.93 +/- 0.18

Abnormalities generally associated with restraint (wet fur, stains around the nose, chromodaccryorhoea) were seen in most animals during and immediately after exposure. Slight salivation was also seen in those animals exposed to diethylene glycol. The time to onset and incidence of the observation was concentration-dependent, and animals recovered rapidly on removal from exposure. Bodyweight was lower throughout the study in main study males exposed to all concentrations of diethylene glycol, and a concomitant reduction in food consumption was seen for all treatment groups. Main study females exposed to 5.06 mg/l diethylene glycol lost weight initially (reduced food consumption on day 1 and bodyweight on day 2), but bodyweight returned to control values by day 6-7. There were no treatment related findings in haematology, blood clinical chemistry, or histopathology. A treatment related, concentration dependent lowering of group mean urine pH was seen in all groups exposed to diethylene glycol.

Conclusion: Nose-only exposure for 6 hours per day for 9 days (5 days exposure, 2 days off, followed by 4 days exposure) to diethylene glycol at concentrations of 0.53, 3.00 or 5.06 mg/l resulted in a bodyweight reduction in males exposed to all concentrations, while there were no differences in bodyweight in recovery males throughout the exposure phase or at the end of the recovery period. Minor changes in some haematological and clinical chemistry parameters were seen at 5.06mg/l. While these would normally be considered of no toxicological significance, the fact that diethylene glycol produces toxicologically significant effects in a number of these parameters by other routes and at higher doses suggests that the small changes seen here may progress with longer duration inhalation exposure. Therefore, with the possibility of progression of these changes at 5.06mgl1, together with a questionable effect on bodyweight, this concentration is considered to be an effect level for diethylene glycol in this study, and the No Observed Adverse Effect Level (NOAEL) is considered to be 3.00mg/l diethylene glycol.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
prior to or in 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.
Qualifier:
according to
Guideline:
EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Fischer 344 rats (approximately six weeks of age) were purchased from Charles River Breeding Laboratories, Inc., Kingston, New York. Extra animals were ordered to ensure that a sufficient number of animals of acceptable health and weight were available to conduct the study as designed. This strain of rat was selected because of its general acceptance and suitability for toxicity testing, the availability of historical data and the reliability of the commercial supplier.

Upon arrival at the laboratory, the rats were examined for health status by the laboratory veterinarian and acclimated to the laboratory environment according to the Standard Operating Procedures of the Subchronic/Chronic Toxicity Section. During acclimation the rats were weighed and randomly assigned one/cage into exposure groups using a computer-generated randomization procedure based on body weights. Identification was accomplished by inserting a unique alphanumeric metal tag in one ear of each rat.

The animal rooms of the testing facility were designed to maintain adequate environmental conditions concerning temperature, humidity, and photocycle and were regulated for the species tested. A basal diet of Purina Certified Rodent Chow #5002 (Ralston Purina Co., St. Louis, MO) and tap water was available ad libitum during the prestudy and study periods. The feed was analyzed by the Ralston Purina Co. to confirm that it was nutritionally adequate and to quantitate the levels of selected contaminants associated with the formulation process. Water analysis was performed according to the Standard Operating Procedures of The Toxicology Research Laboratory.
Type of coverage:
semiocclusive
Vehicle:
other: tetraethylene glycol
Details on exposure:
The test solution was applied as received (undiluted) at a volume of approximately 0.4,1.2 or 4.0 ml/kg body weight/day. The vehicle control was applied at a rate of 3.0 ml/kg/day. Dose volumes were determined weekly and were based on the most recent individual animal body weight.

All rats were acclimated to an elastic wrap (used to hold the test material in dermal contact) at least four times prior to the start of the study. An area
approximately 5 X 5 cm on the back of each rat was clipped free of hair prior to study initiation and as necessary thereafter. An absorbent gauze patch was used to hold the test material/vehicle or vehicle control in dermal contact. The wraps were removed approximately six hours after application and the test site was wiped, if necessary, with a water-dampened disposable towel to remove any residual test material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test formulation was shown to be stable over 116 days. After 116 days, the concentration of DBNPA in TTEG was 96% of day 1 values.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week excluding holidays
Remarks:
Doses / Concentrations:
0.4, 1.2 or 4.0 ml/kg/day of the DBNPA test solution
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
Groups of 20 adult rats (10/sex/dose) received topical applications five days/week for 13 consecutive weeks, excluding holidays. One group was designated a sham control and was wrapped only. The vehicle control received only the TEG (needed to solubilize the DBNPA) at the rate of 3.0 ml/kg/day, which corresponds to a dose level of approximately 3360 mg TEG/kg/day. The remaining dose groups received 0.4,1.2 or 4.0 ml/kg/day of the DBNPA test solution, which corresponds to dose levels of approximately 103,309 or 1031 mg DBNPA/kg/day, respectively, and 327,980 or 3266 mg TEG/kg/day, respectively. The dose volume of 4.0 ml/kg represents the maximum amount of material that could be practically maintained at the application site. The dose levels selected for the 13-week study were chosen based on the rationale delineated in the subsequent section.

Dose Level Selection and Results of Dermal Probe Studies
DBNPA was not sufficiently soluble in water to allow the use of water as the vehicle control. Therefore, TEG, the solvent used in the manufacture and use of formulations containing DBNPA, was used to solubilize the DBNPA (therefore enabled the conduct of this dermal toxicity study).

A probe study was initially conducted with DBNPA in TEG to generate information for the basis of appropriate dose level selection for this 13-week
study. In the probe study, groups of five male and five female Fischer 344 rats initially received ten daily dermal applications of the following preparations at the rate of 2.0 &kg: 2.5%, 5%, 10% and 20% DBNPA. This corresponded to dose levels of 64,128,256 and 512 mg DBNPA/kg/day, respectively.

During the course of these ten daily applications, there were no discernible indications of treatment-related adverse effects at the application site or on clinical in-life parameters being monitored for systemic toxicity (Dow, 1990). Thus, the decision was made to extend the probe study for an additional two weeks, with the same groups of males and females given the same dosing regimen, but at the higher application rate of 4.0 ml/kg. This corresponded to dose levels of approximately 128,256,512 and 1031 mg DBNPA/kg/day, respectively.

The day following the last application, the rats were submitted for termination and gross necropsy. The in-life examinations indicated several male and female rats in various treatment groups had a minimal inflammatory reaction at the application site consisting of erythema and edema. At necropsy, localized scab formation, interpreted as evidence of a minor inflammatory reaction, was noted at the dermal test site of 3/5 males given the 10% and 3/5 males given the 20% DBNPA concentration (supplying 1031 mg DBNPA/kg/day). An increased degree of discoloration of the haircoat at the dermal test site was attributed to the physical effects of repeated dermal application of the DBNPA and/or TEG to all groups of males and the
two highest dose groups of females.

These data were interpreted to indicate the maximum dose for the 13-week study to be 4 ml/kg/day, supplying 1031 mg DBNPA/kg/day and 3266 mg TEG/kg/day.

The maximum dose selected for this 13-week study was based on the following considerations:
(1) relative insolubility of DBNPA in water, precluding the use of water as the vehicle,
(2) the required use of a suitable solvent (TEG) to solubilize the technical grade active ingredient, DBNPA,
(3) the use of a 20% concentration of DBNPA in TEG which represents the approximate concentration of the technical grade material that is manufactured and used,
(4) the maximum dose volume of 4 ml/kg which could be practically maintained at the application site, and
(5) the minor inflammatory reaction that was noted at the application site of some rats in the highest dose level of the probe study which was considered tolerable over the course of a 13week study.

Based on these factors, a maximal dose of 1031 mg DBNPA/kg/day was selected for the 13-week study.
Positive control:
Not applicable.
Observations and examinations performed and frequency:
Body Weights and Feed Consumption.
All animals were weighed prior to the first application and approximately weekly thereafter. Feed consumption was determined weekly for all animals.

Observations and Records,
Ophthalmological Examination. The eyes of all animals were examined by the laboratory veterinarian prior to the start of the study utilizing penlight illumination. Eyes were also examined upon termination of the study at terminal necropsy (see Pathology section).

In-Life Observations. A careful clinical examination was conducted on all animals prior to the start of the study and at least weekly thereafter for the duration of the study. This examination included thorough evaluations of the skin, fur, mucous membranes, respiration, nervous system function (e.g., tremors, convulsions) and behavior pattern. An additional observation for morbidity, mortality and the availability of feed and water was made each day of the work-week as well as twice daily on weekends and holidays.

Functional Observational Battery. A functional observational battery (FOB) was conducted by a trained observer on all surviving rats during the week prior to necropsy. The observer (blind to the identity of the animal) assessed the following parameters upon examination of each individual rat: eyes (pupil size, palpebral fissure, lacrimation, etc.), respiration (increased, wheezing, etc.), movement/behavior (bizarre behavior, vocalization, tremors, convulsions, muscle tone, etc.), skin and fur (rough haircoat, perineal soiling, piloerection, bluish, etc.), mucous membranes, general appearance (salivation, nasal discharge, obese, etc.), and feces and urine (decreased, dark, mucoid, reddish, etc.). The rat was then placed into an observation box (a clear plastic box with approximately 50 x 50 cm floor and 25 cm high walls) for approximately 20 seconds. Parameters evaluated during this observation period included: movement/behavior (gait, pattern, activity level, coordination, etc.) and sensory responsiveness (touch, auditory, nociceptive).

Evaluation of Dermal Application Site.
The condition of the skin at the application site was subjectively evaluated weekly using this laboratory's modification of the acute dermal irritation scoring system recommended by the Organization for Economic Co-operation and Development (OECD, 1981b):

Erythema and Eschar:
Within normal limits ...............................................................0
Very slight erythema (barely perceptible) ...............................1
Well-defined erythema ...........................................................2
Moderate-to-severe erythema ..................................................3
Severe erythema to slight eschar formation ..............................4
Edema:
Within normal limits ...................................................................0
Very slight (barely perceptible) ................................................1
Well-defined (edges raised) ................................................ .....2
Moderate (raised -1 mm) ...................................................... ...3
Severe (raised >1 mm) ..............................................................4
Scaling and Fissuring:
Within normal limits ................................................................. ..0
Slight scaling ................................................................................1
Moderate-to-severe scaling ......................................................2
Slight fissuring.. ...........................................................................3
Moderate-to-severe fissuring ...................................................4
Necrosis, scabs/crusts and scars:
- = condition not present
+ = condition present

Necrosis, scabs and/or scars were noted if present; however, they were not graded.
Necrosis, scabs and/or scars were noted if present; however, they were not graded.
Sacrifice and pathology:
Hematoloy Blood samples were collected from the orbital sinus of fasted rats lightly anesthetized with methoxyflurane approximately one week prior to necropsy. The following hematologic parameters were evaluated for each animal: hematocrit (HCT), hemoglobin concentration (HGB), erythrocyte count (RBC), total leukocyte count (WBC), and platelet counts (PLAT). Blood smears were prepared and stained with Wright's stain for all animals from which blood samples were collected. Complete blood smear examinations were conducted manually on high dose and both controls (sham and vehicle control) which included differential leukocyte counts (the number of leukocytes counted was specified if other than 100 cells were counted) and an assessment of erythrocyte, leukocyte and platelet morphology. Hematologic determinations were made on an ELT-8, Ortho Instruments, Westwood, MA.

Clinical Chemistry. Blood samples were collected from the orbital sinus of fasted rats lightly anesthetized with methoxyflurane approximately one week prior to necropsy. The following parameters were evaluated for each animal: alkaline phosphatase activity (AP), alanine arninotransferase activity (ALT), aspartate aminotransferase activity (AST), total protein (TI?), albumin (ALB), globulin (GLOB - calculated), total bilirubin (TBILI), glucose (GLU), urea itrogen (UN), cholesterol (CHOL), triglycerides (TRIG), creatinine (CREAT), phosphorus (I?), calcium (CA), sodium (NA), potassium (K) and chloride (CL). All analyses were conducted with a Monarch 2000 automated chemistry analyzer (Instrumentation Laboratory Inc., Lexington, MA.). Globulin values were calculated as the difference between total protein and albumin levels and a Beckman E4A flame photometer (Beckman Instruments Inc., Brea, CA) was used to determine Na, K and Cl levels.

Urinalysis. Urine samples were obtained from all nonfasted rats approximately one week prior to necr0psy.b~m anual compression of the urinary bladder. The following parameters were evaluated for each urine sample: specific gravity (T. S. Meter, American Optical, Keene, NH) and a semiquantitative estimate of bilirubin, glucose, ketones, blood, pH, protein and urobilinogen (Urotron test strip analyzer, BioDynamics, Indianapolis, IN). In addition, the color and appearance, as well as the presence of microsediment in a pooled sample from each dose group was determined.

Pathology. All animals were fasted overnight prior to necropsy. Each animal was weighed, anesthetized with methoxyflurane and euthanatized. To
prevent terminal aspiration of blood, the trachea was exposed and clamped prior to decapitation. Eyes were examined by visual inspection of the cornea, lens and other internal components via placement of a moistened glass slide on the corneal surface using a fluorescent light. All observations made on the eyes were recorded with the gross necropsy observations. A complete gross necropsy of all rats was performed by a veterinary pathologist.

Weights of the liver, kidneys, testes, and brain were recorded and the organ weight to final body weight ratios were calculated for each animal. A complete set of tissues plus any additional gross lesions were collected from each animal and preserved in neutral, phosphate-buffered 10% formalin. Lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin solution by tracheal instillation using a hand-operated syringe. The nasal cavity was flushed by a retrograde infusion of the formalin solution via the pharyngeal duct. Sections of the integument were collected from the dermal test site as well as immediately adjacent to the dermal test site. These sections of the integument were supplemental to the section of skin and subcutis routinely collected from the abdominal / inguinal region.

A histologic examination via light microscopy by a veterinary pathologist included a complete set of tissues and organs, including lesions from all ten male and female rats from the sham control group, the vehicle control group and the high dose group. Histopathologic examination was extended to the intermediate and low dose animals and included 1) integument from the dermal test site, 2) integument immediately adjacent to the test site, and 3) all gross lesions noted in any organ or tissue at necropsy. Tissues were prepared for light microscopic evaluation by standard processing procedures, sectioned at approximately 6 p an d stained with hematoxylin and eosin
Other examinations:
No additional information available.
Statistics:
Descriptive statistics (means and standard deviations) were reported for differential leukocyte counts and feed consumption. Body weights, absolute and relative organ weights, urine specific gravity, clinical chemistry data, and appropriate hematology data were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett'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. Statistical outliers were identified by a sequential test, but were only excluded from analyses for documented, scientifically sound reasons unrelated to treatment with the test material. A separate comparison of sham control to vehicle control was conducted using the same statistical methods. If statistical significance was noted between the two control groups during the comparison a succeeding comparison of all treatment groups to the sham control and all treatment groups to the vehicle control was conducted.

The nominal alpha levels used and test references are as follows:
Bartlett's test (Winer, 1971) ...............................................................a = 0.01
Parametric ANOVA (Steel and Torrie, 1960) ...................................a = 0.10
Non-parametric ANOVA (Hollander and Wolfe, 1973). ................ a = 0.10
Dunnett's test (Winer, 1971).............................................................. a = 0.05, two-sided
Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) ..................a = 0.05, two-sided
Bonferroni correction (Miller, 1966)
Outlier test (Grubbs, 1969)................................................................ a = 0.02, two-sided

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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:
no effects observed
Details on results:
NOTE (It is difficult to separate the DBNPA data from the vehicle only data without rewriting the report. Therefore some but not all of the DBNPA is included here with emphasis placed on the vehicle only to control (animals wrapped only)
In-Life Observations
There were no in-life signs of systemic toxicity associated with repetitive dermal application of DBNPA or the vehicle control, TEG, even under conditions maximizing the amount of each material that could be reasonably applied. There were sporadic observations of lacerations on the chest which were self-inflicted by the animals chewing on the wraps used to hold the test material in place. These self-inflicted lacerations occurred in one male sham control, two highdose males, one female sham control, one female vehicle control and three high-dose females. These lesions appeared to be self-limiting and were treated topically when the severity of the wound deemed it necessary in the judgement of study personnel. The lesions were resolved by study termination. On test day 85 one female control was found at the end of the day as having been wrapped too tightly which resulted in rapid respiration in this rat; the wrap was removed, the animal regained a normal respiration pattern, and was dosed and wrapped the following day. There was no further in-life involvem ent with this animal. However, all hematology values obtained from the blood sample taken at necropsy from this animal were subsequently eliminated from the control group mean; elimination of these values was attributed to the wrapping incident on test day 85.

The test site of the sham control and vehicle control groups was within normal limits throughout the observation period.

A prestudy ophthalmic exam conducted on all male and female rats showed that the eyes of all rats had either no visible lesions or small corneal opacities which are common to rats of this age and strain.

No treatment-related observations were noted in the functional observational battery during this examination.

Bodv Weights and Feed Consumption
Mean body weights and feed consumption from all male and female rats of all dose groups were comparable to concurrent controls. Some statistical
differences were identified for the female rat vehicle control group and the low-dose females versus the female sham controls, and for high-dose female rats versus the female vehicle controls. These differences were not dose related nor were they observed in the males. They were considered representative of normal biological variation and considered not toxicologically significant.

Clinical Pathology
There were no treatment-related effects observed in hematologic parameters, urinalysis parameters or clinical chemistry determinations of male and female rats of any dose level. Statistically significant differences were identified in various clinical chemistry parameters of the vehicle control group compared to the sham control group and the DBNPA treated groups compared to sham control and vehicle control groups. These statistically identified differences were not considered toxicologically significant since there was no doseresponse relationship, the differences were not demonstrated in the other sex of a comparable dose level, the differences between either control group and DBNPA treated groups were very slight (i.e., ALB for males given 309 mg DBNPA/kg/day was 3.8 g/dl identified as statistically significant versus ALB for male sham controls of 3.7 g/dl) or the differences were not consistently identified versus sham controls and vehicle controls.

Organ and Organ/Bodv Weight Ratio.
The mean terminal body weights and absolute and relative liver, kidney, brain and testes weights of rats given DBNPA were comparable to those of the controls and demonstrated no treatment-related effect. There were statistically identified differences in relative kidney and liver weights of the vehicle control males versus the male sham controls, relative brain weights of low-dose male rats versus the male sham controls, absolute kidney and liver weights of female vehicle controls versus female sham controls, absolute kidney weights of high-dose females versus female vehicle controls, absolute liver weights of low- and intermediate-dose females versus female sham controls, relative brain weights of female vehicle controls versus female sham controls, relative brain weights of high-dose females versus the female vehicle controls, and relative liver weights of intermediate- and high-dose females versus female sham controls. These differences were considered of no toxicological significance since there was no dose-response relationship, the differences were not identified in the other sex of a comparable dose level or differences identified in absolute organ weights versus relative organ weights did not correspond and they occurred in the absence of any histopathologic alterations.

Pathology
The dermal test site was unremarkable for all male and female rats treated with the sham (water) control, the vehicle (TEG-solvent) control or the intermediate and lower dose groups (103,309 mg DBNPA/kg/day) of the test material. The other observations made at the time of gross necropsy consisted of a low incidence of diverse observations with no relationship to treatment or dosage. All these other observations (excluding dermal test site) were considered spontaneous in origin and unrelated to treatment with the test material or the vehicle solvent.

Histopathologically, the dermal test site was unremarkable for all males and females treated with the sham control (water), vehicle control (solvent) or the intermediate and low dose groups (309 and 103 mg DBNPA/kg/day). Histopathologic examination of integument away from the dermal test site as well as all internal organs and tissues revealed no observations that were considered related to repetitive treatment with the vehicle control (solvent) or any of the three dose levels of the test material (1031,309 or 103 mg DBNPA/kg/day). The various observations that were made on certain sections of liver, lungs, cornea of eyes, heart, kidneys, nasal tissues, ovaries, stomach, testes, ovaries and thyroid were isolated inflammatory and/or degenerative processes typically encountered as spontaneous occurrences in rats of this age and derivation. These observations were noted in control and treated groups, with no relation to treatment with the vehicle solvent or test material.

The occurrence of interstitial inflammation of the lacrimal glands in some rats of control and treatment groups were considered secondary artifacts due to the previous collection of blood from the orbital venous sinus for determination of hematologic and serum chemistry parameters.
Dose descriptor:
NOEL
Effect level:
3 360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment related effects noted between the sham exposed and vehicle control group (TEG group) which received ~3360 mg/kg/day dermally.
Critical effects observed:
not specified

No additional information available.

Conclusions:
The NOEL in a 13 week rat dermal study is 3360 mg/kg/day of TEG when compared to the sham exposed control group.
Executive summary:

2,2-Dibromo-3-nitrilopropionamide (DBNPA) was evaluated for systemic toxicity following repeated dermal application for six hours/day, five days/week for 13 weeks, excluding holidays. Groups of ten Fischer 344 rats/sex/dose received a 20% solution of DBNPA in tetraethylene glycol (TEG) vehicle at targeted dose levels of 0 (sham control), 103,309 or 1031 mg DBNPA/kg/day. A comparable vehicle control group of male and female rats received only the TEG (needed to solubilize the DBNPA) via dermal application at a dose level of approximately 3360 mg TEG/kg/day. Parameters evaluated included general appearance and demeanor, topical response of skin at the application site, body weights, feed consumption, hematology, clinical chemistry, urinalysis, selected organ weights, gross pathologic and histopathologic observations. In addition, a functional observational battery (FOB) was conducted after twelve weeks of exposure.

There was a topical response of the skin that was dose-dependent and limited to the application site. The dermal irritation was characterized by erythema, edema and scabs/crusts at the test site of most high-dose male and female rats. Transient erythema and/or edema was observed in four male and two female rats given 309 mg DBNPA/kg/day. The test site of the sham control and vehicle control groups was within normal limits throughout the observation period. Gross and histopathologic examination of rats given 1031 mg DBNPA/kg/day showed a localized response at the dermal test site. This localized response did not occur in the rats given 103 or 309 mg DBNPA/kg/day or the controls.

In regard to systemic toxicity, there were no in-life signs of systemic toxicity observed during the study and all groups of rats had comparable rates of weight gain. The FOB revealed no differences among the control and treated groups of rats. There were no treatment-related changes in feed consumption, hematology, clinical chemistry, urinalysis parameters or organ weights. In addition, there were no systemic gross or histopathologic lesions attributed to treatment with DBNPA or the vehicle control.

Additional information

Repeat Dose - Oral

Data on the components of the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade, diethylene glycol (DEG), triethylene glycol (TEG), and tetraethylene glycol (TTEG), were used to assess its repeated dose toxicity via the oral route of exposure.

In the key study by Gaunt et al. (1970) and described by Snellings et al. (2016), NICNAS (2009), and DECOS (2007), fifteen to twenty Wistar rats/sex/dose were fed 0, 0.4, 2.0, or 4.0% DEG (equivalent to 300, 1600, and 3000 mg/kg/day in males and 400, 1800 and 3700 mg/kg/day in females) for 14 weeks (phase 1) or ten male and ten female Wistar rats were fed 0, 0.085, 0.17, 0.4, or 2.0% DEG (equivalent to 50, 100, 230, and 1200 mg/kg/day in males and 60, 130, 290, and 1500 mg/kg/day in females) for 32 weeks (phase 2). Observations and measurements included body weights, food and water intake, urinalysis, hematology, clinical chemistry (14 week), and organ weight and histopathology (kidneys only in 32 week). In the 14 week study, effects included death, decreased body weight, and kidney hydropic degeneration at the high dose of 4% DEG. Oxalic acid observed in the urine in some animals of all dose groups was considered a biomarker and does not indicate toxicity. Since hydropic degeneration of the kidney was observed in one animal exposed to 2% DEG and decreased male and female body weights were observed, the LOAEL is considered 2% for the 14 week exposure. The 14 week NOAEL was therefore 0.4% (300 mg/kg/day). For the 32 week study, reduced body weight was observed at 2%, resulting in a NOAEL of 0.4% (234 mg/kg/day).

In a 4 week feeding study by BASF (1988), five to ten Wistar rats/sex/dose were exposed to 0, 500, 2500, 10000, or 40000 mg/kg DEG in the diet. Body weight, food consumption, clinical chemistry, urinalysis, hematology, gross pathology, organ weights, and organ histology were evaluated. Increased oxalic acid concentration were reported in both sexes and calcium oxalate crystals were observed in the urine of males of the 40000 mg/kg diet dose group. These findings were reversible during a 3 week recovery phase. The NOEL was 10000 mg/kg diet (equivalent to 936 mg/kg bw/day).

In a supporting study by the Mellon Institute of Industrial Research (1949), male and female Sherman rats were fed 0.015, 0.062, 0.25, or 1% DEG or TEG (equivalent to 11, 46, 180, and 850 mg/kg/day DEG and 11, 47, 190, and 830 mg/kg/day TEG) in the diet for 32 days. Additional groups of rats were fed 0.015% DEG, 0.062% DEG, 0.025% TEG, or 0.062% TEG for 90 days. None of the 90-day dosage groups showed any statistically significant deviations from the rats fed the control diet for a similar period. The highest dosage level of DEG that produced no significant effect for 32 days and is the NOAEL for DEG was 180 mg/kg/day, based on increased kidney weight and renal degeneration at the high dose. The highest dosage level of TEG that produced no effect and is considered study NOAEL was 830 mg/kg/day.

In a 13 week sub-chronic toxicity study (equivalent or similar to OECD 408) by Bushy Run Research Center (1990), male and female Fischer rats were exposed to nominal doses of 0, 10000, 20000, or 50000 ppm TEG (equivalent to 0, 748, 1522, and 3849 mg/kg/day for males and 0, 848, 1699, and 4360 mg/kg/day for females) in the diet. Observations or measurements for clinical signs of toxicity, ophthalmologic changes, food consumption, body weight, clinical pathology (interim and final), organ weights, necropsy, and histology were made. Body weight depression compared to controls occurred in males from the high dose group throughout the study and in females during the latter weeks of the study (starting at approximately week 8). Hematology measurements, including decreased erythrocytes and hematocrit in males from the high and mid dose groups and decreased hemoglobin and increased MCV in the high dose group only, were altered at the 13-week measurement period. These changes were considered to be of questionable biological significance based on a lack of similar effect in the females, the small magnitude of the changes, and the lack of corresponding effects in other red cell indexes. Decreases in urine pH at all dose levels in males and mid and high dose levels in females and an increase in urine volume in males from the high dose group were considered to be related to TEG and its metabolites. Observations of small increases in kidney weight (high dose group females) and kidney weight relative to body weight (all groups of males and mid and high dose group females) were also considered to be probably treatment related. Based on the lack of any other significant toxic effects, particularly the lack of histologic evidence of renal injury, hyperplasia, or hypertrophy, the altered urine measurements were considered to be most likely related to excretion of the large amounts of test material (or metabolites) during the course of this study. The study NOAEL for this supporting study was considered as 20000 ppm mid dose (or 1522 mg/kg/day in males and 1699 mg/kg/day in females).

In the range-finding 14-day dietary toxicity study for TEG by Bushy Run Research Center (1989), male and female Fischer rats received nominal doses of 10000, 20000, or 50000 ppm in the diet. The corresponding mean ingested doses were 1132, 2311 and 5916 mg/kg/day for the males and 1177, 2411 and 6209 mg/kg/day for the females. No mortality or treatment-related clinical signs were observed. No treatment-related effects were observed in food consumption, absolute body weights, body weight gains, hematology, clinical chemistry, gross pathology, histology, or organ weight data for either sex at any dosage level when compared to controls. The only treatment-related findings in this study were increased urine volume, decreased urine pH, and decreased triple phosphate crystal in the urine of in males and females from the high dose group (study LOEL). The 14 day study NOEL was 20000 ppm (mid dose).

In an OECD 407-equivalent supporting study by Schladt et al. (1998), male and female Wistar rats were exposed daily to TTEG at doses of 0, 220, 660, 2000 mg/kg bw/day via drinking water for 4 weeks. Animals were examined twice a day except on weekends and holidays, when clinical observations were performed once daily. Body weights were measured daily and food and water consumption was determined weekly. At the end of the 4 week period, blood was obtained for hematology and clinical chemistry determinations and urine for urinalysis determinations. Organ weights were obtained at necropsy and gross and histopathologic examination of selected tissues conducted. No treatment-related effects of toxicological significance were noted in any of the animals treated. The NOEL for this key study was 2000 mg/kg/day, the highest dose examined.

In a supporting study, Healing et al. (2016) exposed four male and four female rats to 30% (v/v) (3380 mg/kg/day) and 40% (v/v) (4500 mg/kg/day) TTEG by oral gavage for 28-32 days, high doses which are above current testing limit. While 30% (v/v) TTEG was well tolerated without any treatment related findings, animals given 40% (v/v) TTEG had increases in urinary volume and specific gravity, along with lower urinary pH in the females. As these observed changes may be due to the diuretic properties and expected acidic metabolite formation of TTEG, these changes are not considered adverse and 4500 mg/kg/day represents the study LOEL. The study NOEL was 3380 mg/kg/day.

In a supporting study by Bushy Run Research Center (1991), Fischer 344 male rats (5/group) were dosed with TTEG in drinking water (0, 1000, 5000, 25000, and 50000 ppm, daily, equivalent to 92, 391, 2355, and 6387 mg/kg/day) or by gavage (0, 500, 1000, 2500, and 5000 mg/kg, 5 days/week for 2 weeks), in order to establish the most appropriate mode of administration, and to select doses for a dominant lethal study with TTEG. A dose-related increase in water consumption measurement was observed for the 25000 and 50000 ppm groups likely related to the diuretic properties of this family of substances. A subsequent probe study confirmed that this increase was due to a real increase in water intake (as opposed to increased spillage), which was confirmed by an increase in urine output. Based on the lack of effects on body weights, food consumption, or other measurements, the increased water consumption was not considered to be a result of toxicity of the TTEG but more likely the result of an osmotic diuresis. No effects considered to be treatment-related were observed for any monitor for animals treated via drinking water or gavage. The NOEL was 6387 mg/kg/day for the drinking water study and 5000 mg/kg/day by oral gavage.

Renal toxicity is demonstrated to decrease with increasing molecular weight, as evidenced by the weight of evidence studies examining the effects of DEG, TEG, and TTEG, as well as several supporting studies. Renal toxicity was observed at doses of 230 mg/kg/day in male Wistar rats exposed to DEG, while no effects were observed in male or female Wistar rats exposed to 2000 mg/kg/day TTEG. As such, the endpoint conclusion NOAEL for repeated dose toxicity was based on DEG to provide the most conservative assessment.

Repeat Dose – Inhalation

In a supporting study by Central Toxicology Laboratory (2001), groups of ten male and ten female Alpk:APfSD (Wistar-derived) rats were exposed nose-only for 6 hours per day, 5 days per week, to 0, 0.53, 3.00 or 5.06 mg/l DEG (measured) over a period of 11 days, resulting in 9 exposures. Clinical observations, body weights, and food and water consumption were monitored throughout the study. Blood was taken for hematology, clinical chemistry, and urinalysis at the at the end of the exposure (day 12) or after a 28 day recovery period (day 40) and a full necropsy was performed, including determination of organ weights and histopathological examination of select tissues. Body weight was lower throughout the study in main study males exposed to all concentrations of DEG, and a concomitant reduction in food consumption was seen for all treatment groups. Main study females exposed to 5.06 mg/l DEG lost weight initially, but body weight returned to control values by day 6-7. Minor changes in some hematological and clinical chemistry parameters were seen at 5.06 mg/l. These included decreased reticulocyte counts in males, decreased neutrophil counts in females, increased triglycerides in males, increased total bilirubin in females, increased alkaline phosphatase enzyme activity in females, and increased alanine aminotransferase and aspartate aminotransferase in males. While these would normally be considered of no toxicological significance, the fact that DEG produces toxicologically significant effects in a number of these parameters by other routes and at higher doses suggests that the small changes seen here may progress with longer duration inhalation exposure. Therefore, with the possibility of progression of these changes at 5.06 mg/1, together with a questionable effect on body weight, this concentration is considered to be an LOAEC effect level in this study, and the NOAEC is considered to be 3 mg/l DEG.

In another supporting study reported by Ballantyne et al. (2006), groups of 10 to 15 Sprague-Dawley rats/sex/dose were exposed to 494, 2011, or 4824 mg/m3TEG by whole body inhalation for 6 hours/day for 9 days. The highest concentration produced 100% mortality. At the mid-dose, effects included periocular and perinasal irritation, decreased body weight, increased food and water consumption, and increased serum alkaline phosphatase and alanine aminotransferase. Fluid imbalance was also suggested. A NOEC could not be established, as minimal signs of irritation, increased water consumption, and slightly increased alkaline phosphatase were observed at the lowest exposure level. However, preening of the fur at these high aerosol concentration exposures may have led to a confounding factor from the resultant oral intake.

Ballantyne et al. (2006) also reported the results of a 9 day nose-only inhalation toxicity study. Ten male and ten female Sprague-Dawley rats were exposed to measured concentrations of 102, 517, or 1036 mg/m3TEG for 6 hours/day, 5 days/week, over 11 days (9 exposures). No clinical signs, effects on food or water intake, biochemical changes, or histological evidence of hepatorenal dysfunction were observed at any dose level. Male and female body weights were reduced by the end of the exposure period at the high dose, but this did not reach statistical significance. Therefore, 1036 mg/m3was considered as the study NOAEC for TEG.

 

Repeat Dose – Dermal

In a 13-week dermal repeated dose supporting study (Dow Chemical Company, 1990), where TTEG was used as vehicle, and animals of the vehicle control group received a dose of approximately 3360 mg/kg/day dermally, no treatment related effects were noted between the sham exposed and TTEG vehicle control group. Therefore, the NOEL for TTEG is in excess of the current limit dose of 1000 mg/kg/day and no systemic toxicity is expected from dermal exposure to TTEG.

Justification for classification or non-classification

Diethylene glycol is self-classified for repeated dose toxicity according to 67/548/EEC and Regulation (EC) No 1272/2009 due to effects observed on the kidney after oral exposure in animal studies. Based on the concentration of DEG in the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade, this substance is classified as an STOT-RE category 2.