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Type of information:
other: Independent Expert Evaluation
Adequacy of study:
weight of evidence
other: Independent expert evaluation of NTP studies assigned reliability 1
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Reliability ratings are assigned to specific studies or generated data. This report is an expert evaluation based on all available data and, as such, cannot be assigned a reliability rating. The evaluation was of renal effects reported in NTP 13-week and 2-year carcinogenicity studies conducted with tertiary butyl alcohol in rats and mice. The NTP studies were rated as reliability 1.

Data source

Reference Type:
other: Unpublished expert evaluation
Report date:

Materials and methods

Type of study / information:
The author presents a detailed assessment of the urinary system pathology observed in rats and mice following exposure to tertiary butyl alcohol in the NTP subchronic and 2-year carcinogenicity drinking water studies. Hard presents two possible modes of action for the occurrence of renal tumors observed in male rats and relevance for extrapolation to humans.
Test guideline
no guideline required
other: Submission is an expert evaluation of the results observed in a study conducted according to NTP guidelines.
not applicable
GLP compliance:
Submission is an expert evaluation of data for a study that was GLP compliant. Expert evaluation does not require GLP compliance.

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
tertiary butyl alcohol
tertiary butyl alcohol

Results and discussion

Any other information on results incl. tables

Citing studies by Hard et al. (1993) and Swenberg and Lehman-McKeeman (1999), Hard states that one pathway leading to renal tubule tumor formation in male rats only is induction of alpha-2u-globulin (alpha-2u-g) nephropathy. Hard provides the following description of alpha-2u-g nephropathy: “In this syndrome, a xenobiotic binds non-covalently to circulating α2u-g synthesized in the male rat liver. Being a low-molecular-weight protein, α2u-g is freely filtered through the glomerulus, and approximately half of the ultrafiltrate is absorbed into the second segment of proximal convoluted tubule (P2) and catabolized by the hydrolytic enzymes of renal lysosomes. The loose binding of a xenobiotic to α2u-g significantly retards the lysosomal degradation of the protein, resulting in hyaline droplet (and α2u-g) accumulation in tubule cells, secondary cytotoxicity, cell loss and compensatory tubule cell regeneration, sustained during the period of exposure. Prolonged cytotoxicity and regenerative cell proliferation is considered to be the mode of action leading to renal tubule tumor formation. Additional characteristic morphological features of α2u-g nephropathy include granular cast formation at the junction between the outer stripe of outer medulla (OSOM) and inner stripe of outer medulla (ISOM), representing the lodging of sloughed cell debris at the point where tubule lumen narrows from the pars recta into the thin descending limb of Henle. As well, linear mineralization occurs in the papilla after several months of treatment, probably at the pre-bend segment of Henle’s loop.” A second possible pathway involves dose-related enhancement of chronic progressive nephropathy by tertiary butyl alcohol. Chronic progressive nephropathy is itself a risk factor for the development of renal tubule neoplasia.


Reference cited:

Hard et al., 1993. Hazard evaluation of chemicals that cause accumulation of alpha 2u-globulin hyaline droplet nephropathy, and tubule neoplasia in the kidneys of male rats. Environ. Health Perspect. 99: 313-349.


Swenberg JA and Lehman-McKeeman LD, 1999. Alpha 2-Urinary globulin-associated nephropathy as a mechanism of renal tubule carcinogenesis in male rats. In: Species Differences in Thyroid, Kidney and Urinary Bladder Carcinogenesis. CC Capen, E Dybing, JM Rice and JD Wilbourn (eds). International Agency for Research on Cancer, Lyon. IARC Scientific Publications No. 147, pp. 95-118.

Hard cites the following observations from the 13-week and 2-year NTP studies in rats and mice as well as published studies by others to support one or both modes of action as the most likely explanation for renal tumor formation in male rats in the NTP study with tertiary butyl alcohol:


13-Week Studies

- Increase in hyaline droplet accumulation within renal tubule epithelium and tubule lumens in male rats at all dose levels where animals survived to study termination; lesion more prominent but not increased in severity at higher dose levels; protein composition of droplets and angular crystalline shape confirmed with use of Mallory Heidenhain and Lee’s methylene blue basic fuchsin stains; protein droplet accumulation was specific to male rats only; not reported in female rats or mice of either sex.

- Focal tubule mineralization at the “corticomedullary” junction in all female rat groups, including controls; effect was significantly increased in 3 highest male dose groups; no granular cast formation; no effect in mice of either sex.

- Severity of the spontaneous, background lesion, chronic progressive nephropathy (CPN) was exacerbated in treated male rats; dose-related increase in incidence but not severity between female groups; severity classified as minimal in all female groups, including controls; effect not reported in mice of either sex.

- Diffuse or papillary hyperplasia of the urinary bladder transitional cell lining in most male rats but only a few female rats in the high-dose group; hyperplasia occurred in conjunction with grossly visible bladder calculi in the males but not the females; chronic bladder inflammation was present in a few high-dose male and female rats; chronic inflammation and diffuse or focal papillary hyperplasia of the transitional cell lining of the bladder present in most or all male mice in the mid- and high-dose groups; urinary bladder hyperplasia and chronic inflammation present in high-dose group female mice.


2-Year Studies:

- Mineralization within the kidney in all groups of rats, including controls; 2 distinct types of mineralization occurred; “focal tubular mineralization” at the corticomedullary junction occurred with equal incidence in all female groups (slight increase in severity in higher dose groups), in control males, and some exposed males; “linear papillary mineralization” occurred in male rats only and there was a clear dose-response for incidence; no effects in mice of either sex.

- Statistically significant exacerbation of severity (scale of 1 to 4) of CPN in all dosed female rat groups (1.6 in controls versus 2.9 in high-dose group) and high-dose group males (3.0 in controls versus 3.3 in high-dose group); slight increase in severity of CPN also observed at interim sacrifice at higher doses; no effects in mice of either sex.

- Standard and extended evaluations of kidney sections from male rats demonstrated a statistically significant increase in a special type of renal tubule hyperplasia, i.e. atypical tubule hyperplasia (ATH) in the high-dose group; ATH differs from foci of regeneration associated with CPN; ATH is widely accepted as a sequential, preneoplastic stage in the continuum leading to renal adenoma and carcinoma (Hard, 1986; Lipsky and Trump, 1989; Hard, 1990; Dietrich and Swenberg, 1991; Nogueira et al., 1993); while ATH was observed in kidneys of all male rat groups, including controls, only a single ATH lesion was found in a high-dose female rat; no effects in mice of either sex.   


References cited:

Hard GC, 1986. Experimental models for the sequential analysis of chemically-induced renal carcinogenesis. Toxicol. Pathol. 14: 112-122.


Lipsky MM and Trump BF, 1988. Chemically induced renal neoplasia in experimental animals. Int. Rev. Exp. Pathol. 30: 357-383.


Hard GC, 1990. Tumours of the kidney, renal pelvis and ureter. In: Pathology of Tumours in Laboratory Animals. Vol. 1 – Tumours of the Rat, second edition. VS Turusov and U Mohr (eds.). International Agency for Research on Cancer, Lyon. IARC Scientific Publications No. 99, pp 301-344.


Dietrich DR and Swenberg JA, 1991. Preneoplastic lesions in rodent kidney induced spontaneously or by non-genotoxic agents: predictive nature and comparison to lesions induced by genotoxic carcinogens. Mutation Res. 248: 239-260.


Nogueira et al., 1993. Experimental models of renal carcinogenesis. Cancer Res. Clin. Oncol. 119:190-198.

- A non-statistically significant distribution of renal tubule tumors was found in all male rat groups after standard evaluation of the kidney; extended evaluation of the kidneys combined with the standard evaluation resulted in a statistically significant increase in the mid-dose group for adenomas and carcinomas combined and for incidence of multiple adenomas; renal tumor incidence was also statistically significant in high-dose group males when a single renal tubule tumor found on step-sectioning of kidneys from the 15-month interim sacrifice was added to the results from the 2-year evaluation; no renal tubule tumors were found in female rats or mice of either sex.

- Statistically significant increase in hyperplasia of the transitional cell lining of the renal pelvis in mid- and high-dose group male rats and high-dose group females; no progression of transitional epithelial hyperplasia to benign or malignant neoplasms in either sex; no effects in mice of either sex.

- Statistically significant increase in chronic inflammation of urinary bladder with transitional cell hyperplasia in high-dose group male mice; statistically significant increase in chronic inflammation in female mice but only a few had hyperplasia of the transitional cell lining; no evidence of progression of hyperplasia to urinary bladder neoplasia in treated mice; no bladder effects in rats of either sex.


Applicant's summary and conclusion

Following a review of the 13-week and 2-year NTP studies in rats and mice exposed ad libitum to tertiary butyl alcohol in the drinking water, the author concluded:

“There are two scenarios possible with respect to the occurrence of renal tubule tumors in male rats in the NTP carcinogenicity bioassay of TBA. Firstly, TBA satisfies the criteria listed as essential evidence for establishing a role for α2u-g nephropathy in renal tubule carcinogenesis, as accepted by IARC (Rice et al., 1999; Swenberg and Lehman-McKeeman, 1999) and the U.S. Environmental Protection Agency (Baetcke et al., 1991). These criteria, which TBA has been shown to satisfy, are the following:
- renal tubule tumors occurring only in male rats,
- acute exposure inducing hyaline droplet accumulation,
- identification of the accumulating protein as α2u-g,
- chronic histopathologic change including linear papillary mineralization,
- an absence of renal toxicity in female rats and mice,
- negativity for genotoxicity.

In addition, reversible binding of TBA to α2u-g has been demonstrated biochemically. In comparison to some other chemicals producing male rat renal tubule tumors through an α2u-g mechanism, TBA would have to be regarded as a weak renal carcinogen, which is in keeping with its relatively lower level of activity involving α2u-g accumulation (Borghoff et al., 2001).

A second possible scenario is that dose-related enhancement of CPN by TBA is leading to a significant increase in renal tumors evident only upon serial sectioning, because end-stage CPN is itself a risk factor for renal tubule neoplasia. It is also possible that both of these factors, α2u-g nephropathy and enhanced CPN, are playing a role in the observed renal carcinogenesis.”

References cited:
Rice et al., 1999. Rodent tumors of urinary bladder, renal cortex, and thyroid gland in IARC Monographs evaluation of carcinogenic risk to humans. Toxicol. Sciences 49: 166-171.

Swenberg and Lehman-McKeeman, 1999. Previously cited in this endpoint study record.

Baetcke et al., 1991. Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Male Rat. U.S. Environmental Protection Agency, Risk Assessment Forum. Washington, DC.

Boreghoff et al., 2001. Previously cited in this endpoint study record.
Executive summary:

In an expert evaluation conducted by Gordon C. Hard, the author examines the renal tubule tumors observed in male rats exposed ad libitum to tertiary butyl alcohol in the drinking water in an NTP carcinogenicity study and postulates species differences and two modes of action for the observed effects at high doses. It is the author’s opinion that the renal tubule tumors observed in male rats in the NTP carcinogenicity bioassay were most likely related to alpha-2u-g nephropathy (a condition unique to male rats), a dose-related enhancement of chronic progressive nephropathy (end-stage CPN is itself a risk factor for renal tubule neoplasia), or possibly a combination of both factors.