Bis(hydroxyethyl) terephthalate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

The basis for this read-across approach is that the target substance is expected to undergo transformation into terephthalic acid (202-830-0; 100-21-0) and ethane-1,2-diol (203-473-3; 107-21-1). The toxicity of the metabolites will accurately predict the toxicity of the bis(2-hydroxyethyl)terephthalate (BHET; 959-26-2; 213-497-6). Refer to the JUSTIFICATION FOR READ-ACROSS OF TOXICOLOGICAL INFORMATION in Section 13 of this dossier for further details.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across: supporting information

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

no

Remarks:

pre-dates GLP

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

24 months; with interim sacrifices at 6, 12 and months

Frequency of treatment:

daily in feed

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

142 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Control animals:

yes, concurrent no treatment

Details on study design:

Male and female rats were randomly assigned to treatment groups and fed diets containing 0, 20, 142 or 1000 mg/kg bw/day terephthalic acid for 24 months. At 6 and 12 months, animals of each concentration group were sacrificed. At 18 months 20 rats of each sex were sacrificed. The surviving rats were sacrificed at 24 months.

Positive control:

Not relevant

Details on results:

Terephthalic acid induced bladder stones were seen in 13/126 females in the high dose group. At the scheduled 6 and 12 month sacrifices, no bladder calculi were detected. At the 18 month sacrifice sand like particle or bladder calculi were only seen in two of the high dose females. There were 19/118 females with what was described as transitional cell adenomas by one pathology laboratory and epithelial hyperplasia by another. Two of these were detected in high dose females after 18 months (2/27), 15 were seen in seen in high dose females at the end of the study (15/79), and one was seen in a control female at the end of the study. None were found in the males, nor at any other dose level. It was believed that rats fed high concentrations of terephthalic acid in the diet develop bladder calculi which appear to cause chronic inflammation of the bladder epithelium, bladder hyperplasia and subsequently bladder tumours. An apparent threshold effect exists because animals exposed to lower concentrations for their lifetime do not form bladder calculi or tumors. The high dose group in this study (1000 mg/kg/day) corresponds to an approximate dietary concentration of 2.0% to 2.8% in adult Fisher rats.

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Dose descriptor:

LOAEL

Effect level:

6 other: mmol/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects were seen at the highest dose level

Dose descriptor:

NOAEL

Effect level:

142 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

6 other: mmol/kg bw/day

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

Bladder stones were seen in the high dose (1000 mg/kg bw/day) female group. Bladder calculi cannot occur unless the solubility of the stone components were exceeded. Based on urinary solubility of Ca-terephthalate, normal human urine would become saturated with Ca-terephthalate at a terephthalic acid concertation of approximately 8 to 16 mM. The concentration of terephthalic acid that would need to be absorbed to produce this effect in humans is approximately 2400 mg/kg bw/day.

This is of little relevance to human risk assessment.

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Assessing the potential oncogenicity and chronic toxicity when fed to rats for two years.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Purity: >= 97%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: in suspended stainless steel wire front and bottom cages, 3 males or 5 females per cage
- Diet: Ground diet, ad libitum
- Water: provided by an automatic dispensing system with demand-controlled values in each cage

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Ethylene glycol was added to pilot batches consisting of ground Purina Lab Chow and the pilot batches were mixed for 15 minutes in a Hobart vertical mixer. Aliquots of the pilot batches then were mixed for 5 minutes with basic Purina Lab Chow to prepare diets containing the percentage necessary to attain the dosage levels 1000, 200 or 40 mg/kg bw/day. Group mean body weights were predicted for the middle of the diet consumption period. This procedure was followed every 2 weeks until the body weights stabilized at 580 days on study. Group mean body weights were derived from the biweekly group/sex mean weight and the diet consumed was derived from diet consumption measurements recorded for the first week in each biweekly period. Thus, percentages in diets were adjusted every 2 weeks at which times fresh diets were prepared.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 months

Frequency of treatment:

daily

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

771 males, 783 females

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

During the study all rats were observed daily for mortality, physical condition and for signs of clinical or behavioral effects.
All animals were weighed every two weeks for the first year and monthly thereafter.
Food consumption was determined every other week on the first 16 cages of rats from each sex and dosage group until the body weights stabilized at 580 doses.

Sacrifice and pathology:

Interim sacrifices were performed at 6, 12 and 18 months. 10 rats per sex per treatment group were sacrificed at 6 and 12 months, and 20 per sex per group at 18 months. Prior to each sacrifice, clinical status was determined by examination of selected haematology, urinalysis and clinical chemistry parameters. Criteria of effect also included mortality, clinical signs, diet and water consumption, body weight change, organ weights, and the incidence of tumors and other pathologic findings.
The following clinical chemistry parameters were measured at approximately 6-month intervals on the animals that were to be sacrificed: total bilirubin, serum urea nitrogen, blood glucose, alkaline phosphatase, serum glutamic oxoacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum albumin, creatinine and calcium.
The following hematologic parameters were also measured at approximately 6-month intervals on the animals selected for sacrifice: red blood cell count, haematocrit, haemoglobin mean cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell and differential counts.
Blood samples for clinical chemistry and haematology were collected by retro-orbital sinus puncture from rats under methoxyflurane anaesthesia.
Urinalysis, performed on the same group of animals as above, included measurements of urine volume, pH, specific gravity, protein, glucose, ketones, bilirubin, occult blood and nitrate. Urine samples were also examined for colour, turbidity, presence of phosphate, calcium oxalate, uric acid, amorphous crystals, BBC's, WBC's, urinary epithelial cells, spermatozoa, bacteria and yeasts.
Post-mortem examination: With the exception of a few animals where autolytic change or cannibalization precluded the evaluation of certain organs, rats that died or were sacrificed when moribund were examined for gross anatomic and histologic alterations. The rats selected for sacrifice were anesthetized with methoxyflurane and killed by severing the brachial vessels to permit exsanguination. A complete necropsy was performed on each rat. Weights of the liver, kidneys, spleen, heart, brain, lung and testes were recorded. Tissues were preserved in 10% neutral buffered formalin and processed for histologic examination.

Statistics:

To evaluate the statistical significance of possible changes in continuous data, the analysis of variance validated by Bartlett's test for homogeneity of variance was used. Individual mean differences were identified by Duncan's multiple range test when indicated by a significant F value. In the case of heterogeneous variances, as indicated by Bartlett's test, an F test and Cochran or Student's t-test were used to identify significant differences. Enumerative data were evaluated statistically by use of the NXR Chi-square test, the 2 x 2 Chi-square test correct for continuity or Fisher's Exact Test, where appropriate. Non-parametric data were evaluated statistically by the multiple sum of ranks test. Mortality and tumor incidence were evaluated by life table techniques. The fiducial limit of 0.05 was employed as the critical level of differences not attributable to chance.

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Among the female rats of both sexes, the mortality rate was equivalent for all dose groups. However, the high-dose male rats has a statistically significant (p<0.001) increase in mortality rate from the 9th through the 16th month of the study. The last high-dose male rat died after 474 days of study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weight gain of male rats was normal for the first 6 months of the study. However, by the end of the first year the high-dose males had gained substantially less weight than either control group. This difference continued until the death of the last surviving high-dose male in the 15th month of the study.

The average weight gain among the high-dose female rats was statistically higher than that of the controls at six and 12 months into the study. However, the relatively small difference from the control means and the absence of a clear dose-response relationship suggest that the difference observed was adventitious.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The dosages attained closely approximated the dosage goals. None of the 6 -month diet consumption means of the dosage groups differed significantly from those of the controls. Therefore, diet consumption was not altered by the ingestion of ethylene glycol.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

There was no significant difference between the water intake of the treated female rats and their controls at any time. However, at the first measurement (at 12 months), the high-dose male rats drank approximately twice as much as the controls. All high-dose males had died by the 18-month measurement period. The water consumption of mid- and low-dose male rats was not affected by ethylene glycol treatment at any time.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology data for female rats revealed no changes of physiological importance. Among the males, statistically significant changes in RBC count (p < 0.01), hematocrit (p < 0.05), hemoglobin concentration (p < 0.05), and neutrophil count (p < 0.05) occurred in the high-dose group at 12 months. No effects were seen at the lower dose levels or in any other hematologic parameter at any dose level at any time.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No changes occurred in female rats, but in high-dose males, urea nitrogen and creatinine showed fourfold mean increases compared to controls after 12 months of treatment. No changes were observed at the lower dose levels or in any other clinical chemistry measurement at any dose level at any time.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes occurred at 6 months, but at 12 months there were statistically significant increases in urine volume (p < 0.001) and reduction in urine specific gravity (p < 0.001) and pH (p < 0.01) in the high-dose male rats. The only change seen in female rats at 12 months was a reduction in mean pH at the highest dose level (; < 0.05). No treatment re-related effects were recorded at 18 months, but at 24 months, high-dose female rats had significantly lower mean urine volume (p < 0.01) and higher specific gravity (p < 0.05) than controls. The toxicologic significance of the 24-month female rat data is questionable in that the control means were much higher than the means of those parameters at earlier time intervals, including 18 months. No treatment-related changes were seen in these parameters at lower dosages or in any other parameter except for a slight reduction (p < 0.05) in urine specific gravity in males rats at the mid-dose level.

Microscopic examination of rat urine samples for crystals yielded the following results. After 12 months of treatment, urine samples from high-dose male rats contained no triple phosphate crystals in contrast to the samples from all other dose groups which had man such crystals. Calcium oxalate crystals were found in urine samples from all but one of the high-dose animals of both sexes. Oxalate crystals were not found in samples from most of the other animals on study. At 18 and 24 months, all but one of the high-dose female specimens contained calcium oxalate crystals. There was also an increased incidence and amount of uric acid crystals in high-dose females at those times.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Rat kidney and liver weights were affected by treatment at various times during the study. At the 6-month sacrifice, male and female rat absolute and relative (to body weight) kidney weights in the high-dose group were significantly higher than those of the controls. At the 12-month sacrifice, the high-dose males were still affected but the females were not. In addition, absolute and relative liver weights were reduced in the high-dose males compared to controls. At the 18-month sacrifice, there were no effects in the low- and mid-dose males, but the high-dose female kidney weights were high than those of their controls. Although the organs were weighed at the 24-month sacrifice, no statistical comparisons were made because of the relatively large numbers of tumors present. There were no biologically significant changes in the weights of the other organs at any time.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nonneoplastic histopathologic lesions are present. This is a summary of the most important lesions observed in the rats that were sacrificed at 6, 12, 18, and 24 months plus those that died or were sacrificed moribund during the study. F

At the 6-month sacrifice, the incidence of the following renal lesions was significantly increased in the high-dose males: tubular hyperplasia, tubular dilation, peritubular nephritis, and calcium oxalate crystalluria. Not statistically significant but interesting because of their rarity were two cases of granulomatous nephritis; also, in two high-dose males calcium oxalate crystals were present in the urinary bladder. These conditions were absent in the other male dose groups and in females. Among females there was an increased incidence of granulomatous myelitis of the femoral marrow at the highest dose level. Special staining indicated the absence of collagen, reticulin, or calcium oxalate. Finely granular periodic acid-Schiff positive deposits were observed in many of the lesions. This condition was not present in treated animals sacrificed later in the study.

At the 12-month sacrifice all high-dose males had chronic nephritis with calcium oxalate crystalluria and 50% had oxalate crystals in the urinary bladder. The term “chronic nephritis” is used to indicate the presence of multiple severe histopathologic changes including tubular dilation and proteinosis, glomerular shrinkage, tubular cell hyperplasia, and chronic interstitial nephritis. These findings were not present in males at the lower dose levels or in females.

By the time of the 18-month sacrifice, all of the high-dose males had died or were sacrificed moribund. In most of these animals, oxalate nephrosis was the primary cause of death. Kidneys from these animals had tubular obstruction by large birefringent crystals with secondary tubular dilation and degeneration. Calculi were sometimes found within the renal pelvic space, ureters, and urinary bladder, often with an associated (secondary) hydronephrosis. Associated extrarenal lesions included cellular hyperplasia of the parathyroid glands and metastatic calcification within numerous tissues, especially the gastric glandular mucosa, vasculature, heart, smooth muscle, and renal tubular and glomerular basement membranes. Among the high-dose males, there was also a significant increase in the incidence of splenic hemosiderosis, a commonly occurring lesion in the rat.
Among the females sacrificed at 2 years, the incidences of hemosiderosis of the mesenteric lymph node (1.0 g/kg/day), and mild fatty metamorphosis of the liver (1 .O and 0.2 g/kg/day) were significantly increased over the control incidence. No biologically significant lesions were noticed in the male rats sacrificed at 2 years.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The only primary neoplasm tumor type for which there was a statistically significant (p < 0.05) difference was fibroadenoma of the female mammary gland at the 0.04 g/kg/day dose level. Because of the absence of an effect at the two higher dose levels, it is most probable that the difference was the result of chance and unrelated to ethylene glycol treatment.

Other effects:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

16.1 other: mmol/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Critical effects observed:

yes

Lowest effective dose / conc.:

16.1 other: mmol/kg bw/day

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Conclusions:

Urinary calcium oxalate crystals and increased kidney weight were seen in all high-dose rats. The LOAEL of 1000 mg/kg bw/day and the NOAEL of 200 mg/kg bw/day for repeated oral toxicity were reported.

Cross-reference 4

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

SIDS reports: Reliability: Klimisch Code = 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

yes

Remarks:

Laboratory Project Study ID 031079. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI.

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on species / strain selection:

Age at study initiation: approximately 6 weeks

Sex:

male

Route of administration:

oral: feed

Duration of treatment / exposure:

1 year

Frequency of treatment:

continuous

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

No. of animals per sex per dose: 10 (main group)

Control animals:

yes

Details on study design:

5 additional rats in control and at each dose for metabolite evaluation and 5 additional for renal clearance determination

Observations and examinations performed and frequency:

Clinical observations performed and frequency: Observed pre-exposure. Daily cage-side observations were made to identify significant clinical abnormalities and monitor the general health of the animals. Clinical observations (includes cage-side, hand-held and open field evaluations) were made for main group animals weekly for the first 12 weeks and every two weeks thereafter. Individual body weights and feed consumption were determined weekly for the first 12 weeks, then biweekly until study day 120, then weekly until study termination. Water consumption data were collected for a 24-hour period during urine collection the week prior to necropsy.

Urinalysis: The color, appearance, specific gravity (refractometer), and volume were noted. Semiquantitative analyses of the following were conducted: pH, bilirubin, glucose, proteins, ketones, blood, urobilinogen, microscopic evaluation for crystal types via micro-sediment analysis on individual animals.

Sacrifice and pathology:

Organs examined at necropsy: A complete necropsy was conducted on all main group animals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The nasal cavity was flushed via the nasopharyngeal duct. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, reexamined, and selected tissues incised. The lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. The liver (excluding the high-dose group) and kidneys were trimmed and weighed immediately. One kidney was cut longitudinally and the other kidney was cut transversely. One-half of each kidney was fixed in formalin for kidney pathology, and the remaining half was weighed and frozen in liquid nitrogen for evaluation of metabolic parameters. In addition, any gross observations at necropsy were recorded, and saved any gross lesions in formalin. The ratios of organ weight to terminal body weight were calculated. The frozen kidneys were wrapped in foil, flash frozen in liquid nitrogen, kept at -80°C, and subsequently shipped frozen for evaluation. The kidneys and urinary bladders fixed in formalin were processed by standard histologic procedures and the slides were shipped to Dr. Gordon Hard for histologic evaluation. Representative samples of tissues listed in below were collected and preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in jars with the tissues. Similar necropsy procedures were followed for all animals (main, metabolite, and oxalate clearance) found dead or moribund, except that body weights, organ weights, and urine samples were not obtained. Sampled tissues: adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow brain (cerebrum, brainstem, cerebellum), cecum, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/harderian glands, larynx, liver, lungs, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues/pharynx, oral tissues, pancreas, parathyroid glands, peripheral nerve – tibial, pituitary, prostate rectum salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, and urinary bladder.

Statistics:

Body weights, feed consumption, organ weights, urine volume, and urine specific gravity were evaluated by Bartlett's test for equality of variances (alpha = 0.01; Winer, 1971). Based on the outcome of Bartlett's test, exploratory data analyses were performed by a parametric (Steel and Torrie, 1960) or nonparametric analysis of variance (ANOVA) (Hollander and Wolfe, 1973). If the ANOVA was significant at alpha = 0.05, it was followed respectively by Dunnett's test (Winer, 1971) or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) with a Bonferroni correction for multiple comparisons to the control (Miller, 1966). The experiment-wise alpha level of 0.05 was reported for Dunnett’s test and Wilcoxon Rank-Sum test. Descriptive statistics only (means and standard deviations) were reported for body weight gains and feed efficiency. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed consumption statistics. Outliers were excluded from other analyses only for documented, scientifically sound reasons. Gross pathologic observations were tabulated and considered in the interpretation of final histopathologic data, but were not evaluated statistically.

Details on results:

At the highest dose (400 mg/kg bw/d), the following effects were seen: mortality/moribundity, occasional treatment-related absent/decreased feces, blood in the cage, red urine, red perioral and perinasal soiling and/or perineal soiling, decreased body weight and body weight gain, increased absolute and relative kidney weights, gross pathological observations in the kidney and urinary bladder with secondary treatment-related observations in the lung.

At the second highest dose (300 mg/kg bw/d), the following effects were seen: mortality/moribundity, kidney lesions, urinary obstruction, occasional treatment-related absent/decreased feces, blood in the cage, red urine, red perioral and perinasal soiling and/or perineal soiling, decreased body weight and body weight gain, increased water consumption, increased absolute and relative kidney weights, gross pathological observations in the kidney and urinary bladder with secondary treatment-related observations in the lung.

To determine renal effects, the severity of nephropathy in the kidney sections was graded on a scale of 0 to 5, with 0 representing no crystal nephropathy and 5 representing end-stage nephropathy indicative of impending renal failure. The test item related nephropathy was not present in the kidney sections from the control group or the lower doses (50 and 150 mg/kg bw/d). In the 300 mg/kg bw/d group, severity grades ranged from 1 to 4 with the mode of occurring at 1 (minimal). In the 400 mg/kg bw/d group, severity grades ranged from 3 (moderate) to 5 (end-stage) with the mode at 4 (marked).

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

2.4 other: mmol//kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw (total dose)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Bis(hydroxyethyl) terephthalate

EC Number:

213-497-6

EC Name:

Bis(hydroxyethyl) terephthalate

Cas Number:

959-26-2

Molecular formula:

C12H14O6

IUPAC Name:

bis(hydroxyethyl) terephthalate

Test material form:

solid

Specific details on test material used for the study:

Bis(2-hydroxyethyl) terephthalate value is read-across from supporting terephthalic acid (202-830-0; 100-21-0) and ethane-1,2-diol (203-473-3; 107-21-1) data.

Results and discussion

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

307 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

2.4 other: mmol/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

614 mg/kg bw (total dose)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Information on the source substances terephthalic acid (202-830-0; 100-21-0) and ethane-1,2-diol (203-473-3; 107-21-1) are considered to be directly applicable to an equivalent molar amount of the target substance.  The most conservative chronic NOAEL value was 150 mg/kg bw/day for ethane-1,2-diol or 2.4 mmol/kg bw/day. Therefore the concentration for further safety assessment of the target substance will be 2.4 mmol/kg bw/day or 307 mg/kg bw/day.

The European CLP (EC 1272/2008 as amended) classification of ethane-1,2-diol was qualitatively determined to be STOT RE Category 2 (oral, kidney) as the effect concentrations in the available animal studies do not trigger CLP classification, yet kidney effects have been reported in humans following chronic exposure to ethane-1,2-diol. Following a precautionary approach, the target substance will similarly be classified as STOT RE Category 2 (oral, kidney).

Executive summary:

The primary adverse effect of lifetime (2-year) high doses of terephthalic acid to rats was almost completely restricted to the urinary tract. Terephthalic acid induced bladder stones were seen in the high dose (1 000 mg/kg bw/day) female group. Bladder calculi cannot occur unless the solubility of the stone components were exceeded. Based on urinary solubility of Ca-terephthalate, normal human urine would become saturated with Ca-terephthalate at a terephthalic acid concertation of approximately 8 to 16 mM. The concentration of terephthalic acid that would need to be absorbed to produce this effect in humans is approximately 2 400 mg/kg bw/day. The 2-year LOAEL for female rats due to bladder calculi formation was 1 000 mg/kg bw/day (6.0 mmol/kg bw/day).

The chronic toxicity of ethylene glycol was assessed in rats. Groups of Fischer 344 rats were fed diets yielding approximate dosages of 40, 200, or 1 000 mg/kg b/day of ethylene glycol for two years. Urinary calcium oxalate crystals and increased kidney weight were seen in all high-dose rats. The LOAEL of 1 000 mg/kg bw/day and the NOAEL of 200 mg/kg bw/day for repeated oral toxicity were reported. A one-year repeated oral dose study exposed Wistar Han rats to 40, 150, 300, or 400 mg/kg bw/day ethylene glycol in feed. The NOAEL in Wistar rats was determined to be 150 mg/kg bw/day, and the LOAEL was 300 mg/kg day/day for renal toxicity in the Wistar rats for 1 year of exposure in diet. All sub-chronic and chronic oral studies in rats appear to converge in a NOAEL around 150 mg/kg bw/day. The LOAEL and NOAEL values determined for ethylene glycol do not trigger the European CLP classification; however, due to the potential of oxalate nephrosis following oral exposure, ethylene glycol has been classified as Specific Target Organ Toxicity following Repeated Exposure (STOT RE) Category 2.

The European CLP (EC 1272/2008 as amended) classification of ethylene glycol was qualitatively determined to be STOT RE Category 2 (oral, kidney) as the effect concentrations in the available animal studies do not trigger CLP classification, yet kidney effects have been reported in humans following chronic exposure to ethylene glycol. Following a precautionary approach, BHET will similarly be classified as STOT RE Category 2 (oral, kidney).

Information on the source substances is considered to be directly applicable to an equivalent molar amount of the target substance. The most conservative chronic NOAEL value was 150 mg/kg bw/day for ethylene glycol or 2.4 mmol/kg bw/day. Therefore, the concentration for further safety assessment of BHET will be 2.4 mmol/kg bw/day or 307 mg/kg bw/day.