Reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-d

Endpoint summary

Description of key information

Data on the components of the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol, 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

Reference

Endpoint:

sub-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: 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

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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 conclusion

Dose descriptor:

NOAEL

1 522 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data are available on TEG and TTEG, components of the reaction mass. The key study is a 13 week study on TEG.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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:

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 toxicity 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 or test system 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.

Critical effects observed:

no

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 conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 036 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Subacute inhalation toxicity study available for TEG, a component of the reaction mass.

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

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

not specified

Qualifier:

according to guideline

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 or test system 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

13 week study available where TTEG, a component of the reaction mass, was used as the vehicle control group.

Additional information

Repeat Dose - Oral

Data on the components of the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol, 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, a 13 week sub-chronic toxicity study (equivalent or similar to OECD 408) by Bushy Run Research Center (UCC, 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 study was considered as 20000 ppm mid dose (or 1522 mg/kg/day in males and 1699 mg/kg/day in females).

In a supporting study by the Mellon Institute of Industrial Research (UCC, 1949), male and female Sherman rats were fed 0.015, 0.062, 0.25, or 1% TEG (equivalent to 11, 47, 190, and 830 mg/kg/day TEG) in the diet for 32 days. Additional groups of rats were fed 0.025% 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 TEG that produced no effect and is considered study NOAEL was the high dose of 830 mg/kg/day.

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 (UCC, 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.

Based on these available data, significant toxicity is not expected for the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol via the oral route of exposure.

Repeat Dose – Inhalation

Overall evidence indicates no inhalation hazard for the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol, based on data for triethylene glycol (TEG).

In a 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 key 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. Based on this finding, no systemic toxicity is expected for the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol.

Justification for classification or non-classification

Based on data on triethylene glycol (TEG) and tetraethylene glycol (TTEG), components of the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol, significant toxicity is not expected for the reaction mass and classification is not warranted.