Q3C(R8) Impurities: 
Guidance for Residual 
Solvents 
 
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
December 2021 
ICH 
 
    
Q3C(R8) Impurities: 
Guidance for Residual 
Solvents 
 
Guidance for Industry 
 
 
 
 
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U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
  
December 2021 
ICH
  Contains Nonbinding Recommendations 
 
FOREWORD 
 
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for 
Human Use (ICH) has the mission of achieving greater regulatory harmonization worldwide to 
ensure that safe, effective, and high-quality medicines are developed, registered, and maintained 
in the most resource-efficient manner.  By harmonizing the regulatory expectations in regions 
around the world, ICH guidelines have substantially reduced duplicative clinical studies, 
prevented unnecessary animal studies, standardized safety reporting and marketing application 
submissions, and contributed to many other improvements in the quality of global drug 
development and manufacturing and the products available to patients.  
 
ICH is a consensus-driven process that involves technical experts from regulatory authorities and 
industry parties in detailed technical and science-based harmonization work that results in the 
development of ICH guidelines.  The commitment to consistent adoption of these consensus-
based guidelines by regulators around the globe is critical to realizing the benefits of safe, 
effective, and high-quality medicines for patients as well as for industry.  As a Founding 
Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a major role in the 
development of each of the ICH guidelines, which FDA then adopts and issues as guidance to 
industry.  
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
PART VI: 
 
2-METHYLTETRAHYDROFURAN ......................................................................................... 2 
CYCLOPENTYL METHYL ETHER ......................................................................................... 5 
TERTIARY-BUTYL ALCOHOL ............................................................................................... 8 
  Contains Nonbinding Recommendations 
 
Q3C(R8) Impurities: Guidance for Residual Solvents 
Guidance for Industry1 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
PART VI: 
IMPURITIES : RESIDUAL SOLVENTS (MAINTENANCE) 
PDEs FOR 2-METHYLTETRAHYDROFURAN, CYCLOPENTYL METHYL ETHER, 
AND TERTIARY-BUTYL ALCOHOL 
 
This guidance provides recommendations for permitted daily exposures (PDEs) for three 
additional residual solvents: (1) 2-methyltetrahydrofuran, (2) cyclopentyl methyl ether, and (3) 
tert-butyl alcohol. This guidance is intended to recommend acceptable amounts for these residual 
solvents in pharmaceuticals for the safety of the patient. As part of the maintenance process for 
the ICH guidance for industry Q3C Impurities: Residual Solvents (December 1997),2 the Q3C 
PDE levels are added and revised as new toxicological data for solvents become available.  
 
The contents of this document do not have the force and effect of law and are not meant to bind 
the public in any way, unless specifically incorporated into a contract. This document is intended 
only to provide clarity to the public regarding existing requirements under the law. FDA 
guidance documents, including this guidance, should be viewed only as recommendations, unless 
specific regulatory or statutory requirements are cited. The use of the word should in Agency 
guidance means that something is suggested or recommended, but not required. 
 
1 This guidance was developed within the Expert Working Group (Quality) of the International Council for 
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use and has been subject 
to consultation by the regulatory parties, in accordance with the ICH process. This document has been endorsed by 
the ICH Assembly at Step 4 of the ICH process, April 2021. At Step 4 of the process, the final draft is recommended 
for adoption to the regulatory bodies of the ICH regions.  
2 We update guidances periodically. For the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
1 
  Contains Nonbinding Recommendations 
 
2-METHYLTETRAHYDROFURAN 
 
Introduction 
 
2-Methyltetrahydrofuran (2-MTHF, synonyms: 2-methyloxolane, tetrahydrosylvan; tetrahydro-
2- methylfuran; CAS Number 96-47-9) is a colorless, volatile liquid with ether-like odor. 2-
MTHF is an organic solvent usually synthesized as a racemic mixture consisting of two 
enantiomeric forms ((S)+ and (R)-). Solubility in water is limited and decreases with increasing 
temperature. It has a vapor pressure of 102 millimeters of mercury (mmHg) (20C) (1). For 
practical reasons, 2-MTHF is a racemic mixture when used as a solvent in synthetic processes. 
 
2-MTHF is increasingly used as a catalytic solvent in exchange of tetrahydrofuran and is much 
less miscible with water compared to tetrahydrofuran. 
 
Genotoxicity 
 
2-MTHF was not mutagenic in the Ames bacterial reverse mutation assay with Salmonella 
typhimurium (Reference (Ref.) 3) and Escherichia coli WP2 uvrA (Ref. 2). 2-MTHF was also 
tested in vitro in a L5178Y mouse lymphoma cell TK+/- assay (Ref. 3), in a chromosome 
aberration assay in human peripheral blood lymphocytes (Ref. 2), and in vivo in a bone marrow 
micronucleus test integrated into a 3-month oral repeated-dose toxicity study in rats (Ref. 2). All 
test results were negative except for the mouse lymphoma assay in the presence of S9, which 
was considered inconclusive without further explanation (Ref. 3). In conclusion, there is no 
evidence that 2-MTHF is genotoxic. 
 
Carcinogenicity 
 
No data for 2-MTHF are available. 
 
Reproductive and developmental toxicity 
 
2-MTHF was tested in a good laboratory practice-compliant prenatal developmental toxicity 
study according to Organization for Economic Co-operation and Development (OECD) TG414 
in rats with doses of 100, 300, and 1,000 milligrams per kilogram per day (mg/kg/day) (Ref. 4). 
At 1,000 mg/kg/day, 2-MTHF caused slightly reduced maternal weight gain, slightly lower 
gravid uterus weight, and marginally reduced fetal body weight. Only slight effects on fetal 
growth were observed, and overall fetal survival and development were considered unaffected at 
the highest dose. The no-observed-adverse-effect level (NOAEL) was considered 1,000 
mg/kg/day. However, because detailed toxicity information is not available, this study was not 
used to support the calculation of a PDE. In an acute embryo toxicity and teratogenicity test in 
zebrafish, 2-MTHF was tested at concentrations ranging from 860  8,600 milligrams/liter 
(mg/L) (Ref. 5). Acute embryo toxicity was observed for 2-MTHF at a nominal lethal 
concentration 50% (LC50) value of 2,980 mg/L. Sublethal effects were also observed, such as an 
increase in edema at nominal concentrations  1,720 mg/L, as well as an increased number of 
embryos without detectable blood circulation and insufficient pigmentation at a nominal 
concentration of 2,580 mg/L. Teratogenic effects were not observed with 2-MTHF in this assay. 
2 
  Contains Nonbinding Recommendations 
 
Repeated-dose toxicity 
 
Two 3-month oral repeated-dose toxicity studies in Crl:CD (SD) rats have been described with 2- 
MTHF racemate  one without a recovery period (Ref. 2) and one with a 1-month recovery 
period (Ref. 6). The top dose in the first study was 26 mg/kg/day (Ref. 2), and in the second 
study 1,000 mg/kg/day (Ref. 6). 2-MTHF treatment-related observations were not seen in the 
first study (Ref. 2). In the second study, groups of 10 male and 10 female rats per dose group 
were treated with doses of 80, 250, 500, and 1,000 mg/kg/day (Ref. 6). The 1-month treatment-
free recovery period included five animals/sex for the control and the high-dose groups. 
Treatment-related observations were generally seen at doses  500 mg/kg/day. Besides slight 
effects on kidney weights (increased at  500 mg/kg/day), blood cholesterol (increased at 1,000 
mg/kg/day), and prothrombin time (decreased at  500 mg/kg/day), the only test article-related 
microscopic observation was hepatocellular centrilobular hypertrophy at 1,000 mg/kg/day. 
However, no effects were observed in the recovery group and the observed effects can therefore 
be regarded as completely reversible. The no-observed-effect level (NOEL) in the second study 
was considered 250 mg/kg/day. 
 
The NOEL of 250 mg/kg/day was used in the PDE calculation: 
 
 
PDE =        25   x   50             = 50 mg/day 
0 
5 x 10 x 5 x 1 x 1 
F1 = 5 to account for extrapolation from rats to humans 
F2 = 10 to account for differences between individual humans  
F3 = 5 for a 3-month study in rodents 
F4 = 1 because no severe effects were observed  
F5 = 1 because a NOEL was established 
 
Limit = (50 x 1,000)/10 = 5,000 ppm 
 
Conclusion 
 
The calculated PDE for 2-MTHF is 50 milligrams per day (mg/day) based upon the NOEL of the 
rat sub-chronic oral study. Since the PDE is 50 mg/day, it is recommended that 2-MTHF be 
placed into class 3 Solvents With Low Toxic Potential in Table 3 in the ICH Q3C guidance. 
 
References 
 
1.  Aycock DF. Solvent applications of 2-methyltetrahydrofuran in organometallic and 
biphasic reactions. Org Process Res Dev. 2007;11:156-159. 
2.  Antonucci V, Coleman J, Ferry JB, Johnson N, Mathe M, Scott JP, et al. Toxicological 
assessment of 2-methyltetrahydrofuran and cyclopentyl methyl ether in support of their 
use in pharmaceutical chemical process development. Org Process Res Dev. 
2011;15:939-941. 
3 
  Contains Nonbinding Recommendations 
 
3.  Seifried HE, Seifried RM, Clarke JJ, Junghans TB, Sanet RH. A compilation of two 
decades of mutagenicity test results with the Ames Salmonella typhimurium and 
L5178Y mouse lymphoma cell mutation assays. Chem Res Toxicol. 2006;19(5):627-
644. 
4.  ECHA 2020. Tetrahydro-2-methylfuran. URL:  
https://www.echa.europa.eu/de/web/guest/registration-dossier/-/registered-  
dossier/13699/7/9/1. (last accessed 5 November 2020) 
5.  Bluhm K, Seiler TB, Anders N, Klankermayer J, Schaeffer A, Hollert H. Acute embryo 
toxicity and teratogenicity of three potential biofuels also used as flavor or solvent. Sci 
Total Environ. 2016;566-567:786-795. 
6.  Parris P, Duncan JN, Fleetwood A, Beierschmitt WP. Calculation of a permitted daily 
exposure value for the solvent 2-methyltetrahydrofuran. Regul Toxicol Pharmacol. 
2017;87:54-63.   
4 
  Contains Nonbinding Recommendations 
 
CYCLOPENTYL METHYL ETHER 
 
Introduction 
 
Cyclopentyl methyl ether (CPME: CAS Number 5614-37-9) is used in pharmaceutical 
chemical  development as an alternative to its more common analogues, such as 
tetrahydrofuran and tert- butyl methyl ether (Refs. 1 and 2). 
 
The vapor pressure of CPME is 44.9 mmHg at 25C, the Log Pow is 1.59, and the water 
solubility is 1.1 grams per 100 grams (23 C) (Refs. 3 and 4). 
 
CPME is classified as an irritant to skin (H315) and eye (H319) in accordance with EC 
No. 1272/2008, in the Globally Harmonized System of Classification and Labelling of 
Chemicals.  CPME did not show the potential to induce skin sensitization in the local lymph 
node assay. In rats, LD50 (lethal dose for 50 percent of animals) for acute oral exposure is 
1,0002,000 milligrams/kilogram (mg/kg), for dermal exposure it is greater than 2,000 
mg/kg, and for inhalation exposure it is greater than 21.5 mg/L. No human toxicity data have 
been reported (Ref. 2). 
 
Genotoxicity 
 
The results of genotoxicity tests have been reported (Refs. 1 and 2). CPME was not mutagenic 
in the Ames bacterial reverse mutation assays in S. typhimurium test strains TA98, TA100, 
TA1535, TA1537, and E. coli WP2 uvrA with and without metabolic activation at 
concentrations up to 5,710 micrograms per plate (Ref. 1) and 5,000 micrograms per plate (Ref. 
2). Negative results were also obtained in in vitro mammalian chromosome aberration tests in 
human lymphocytes at concentrations up to 1.1 milligrams per milliliter (mg/mL) and in 
Chinese hamster lung cells at concentrations up to 1.0 mg/mL (Ref. 2). An in vivo rat 
micronucleus test integrated in a 3-month oral repeated-dose study up to a dose of 31 mg/kg/day 
(Ref. 1) and an in vivo mammalian erythrocyte micronucleus test in CD-1 mice at single oral 
doses up to 2,000 mg/kg (Ref. 2) also did not indicate any genotoxic potential. In conclusion, 
there is no evidence that CPME is genotoxic. 
 
Carcinogenicity 
 
No data are available. 
 
Reproductive and developmental toxicity 
 
In a two-generation reproductive toxicity study, CPME was administered to rats in drinking 
water at doses of 313, 1,250, or 5,000 mg/mL (Ref. 5). Other than decreased body weights of 
pups in the F1 generation and F2 generation that were observed at the highest dose, no other 
significant changes in reproductive parameters were reported. The NOAEL of this study was 
estimated to be 193.45 mg/kg/day (1,250 mg/L in drinking water). However, because detailed 
toxicity information from this study is not available, this study was not used to support the 
calculation of a PDE. 
5 
  Contains Nonbinding Recommendations 
 
Repeated-dose toxicity 
 
CPME was studied in two oral and one inhalation repeated-dose studies in rats. 
 
In a 28-day study with a 14-day recovery period, Crj: Crl:CD(SD) rats were administered 
CPME by oral gavage at 15, 150, or 700 mg/kg/day in corn oil (Refs. 2 and 6). Six 
unscheduled deaths occurred in males at 700 mg/kg/day between days 12 and 15 of treatment 
and were attributed to poor clinical conditions. Salivation was commonly observed in males 
and females at 700 mg/kg/day. Salivation occurred twice in one male at 150 mg/kg/day; 
however, this finding was not considered adverse. Decreased motor activity, piloerection, 
abnormal gait, tremors, convulsion, hunched posture, fast  respiration, and thin appearance 
were observed in males at 700 mg/kg/day. Decreased body weight gain was observed in 
females at 700 mg/kg/day. All clinical findings and changes in bodyweight gains resolved 
after the recovery period. There were no other toxicological effects of CPME in this study. The 
NOEL of this study was determined to be 150 mg/kg/day. 
 
In a 90-day study, Sprague Dawley Crl:CD(SD) rats were administered up to 31 mg/kg/day 
CPME by oral gavage in corn oil (Ref. 1). There were no CPME-related ante-mortem or post-
mortem findings. Detailed information on the experimental design and study results, such as 
clinical signs, hematology, and blood chemistry findings, were not publicly available, 
although the authors considered the NOEL of this study to be 31 mg/kg/day. In another 90-
day study, Sprague Dawley rats were administered up to 500 mg/kg/day CPME by oral 
gavage in water (Ref. 7). The NOAEL of this study was estimated to be 32 mg/kg/day. 
However, because detailed toxicity information from this study is not publicly available and 
this study was not conducted under Good Laboratory Practice, this study was not used to 
support the calculation of a PDE. 
 
In a 90-day study with a 28-day recovery period, Crj: CD (SD) IGS rats were exposed to 
gaseous CPME up to 4 mg/L (6 hours/day, 5 days/week) by whole-body inhalation 
exposure (Ref. 2). Toxic effects occurred at 4 mg/L and included clinical findings of salivation 
and nasal discharge, decreased body weights, increased levels of alanine aminotransferase 
and potassium (in males),  increased absolute and body weight-relative kidney weight (in 
males), hyaline droplets in the proximal tubular epithelium of the kidney, and simple 
hyperplasia of the mucosal epithelium of the urinary bladder. All adverse effects were 
reversible following the recovery period. The NOEL of this study was determined to be 0.84 
mg/L. 
 
The most appropriate and well-documented study for CPME toxicity was the 28-day oral rat 
study. The PDE was calculated based on the identified NOEL of 150 mg/kg/day from this study. 
6 
  Contains Nonbinding Recommendations 
 
PDE =  150 x 50   
   = 15 mg/day 
5 x 10 x 10 x1 x 1 
 
 
F1 = 5 to account for extrapolation from rats to humans 
F2 = 10 to account for differences between individual humans  
F3 = 10 because duration of treatment was less than 3 months  
F4 = 1 because no severe effects were observed 
F5 = 1 because a NOEL was established 
 
Limit = (15 x 1,000)/10 = 1,500 ppm 
 
Conclusion 
The calculated PDE for CPME is 15 mg/day based upon the NOEL from the 28-day oral toxicity 
study. Therefore, it is recommended that CPME be placed into class 2 Solvents To Be Limited 
in Table 2 in the ICH Q3C guidance. 
 
References 
1.  Antonucci V, Coleman J, Ferry JB, Johnson N, Mathe M, Scott JP et al. 
Toxicological assessment of 2-methyltetrahydrofuran and cyclopentyl methyl ether in 
support of their use in pharmaceutical chemical process development. Org Process Res 
Dev. 2011;15: 939941. 
2.  Watanabe K. The toxicological assessment of cyclopentyl methyl ether (CPME) as a 
green solvent. Molecules. 2013;18:3183-3194. 
3.  CPME Material Safety Data Sheet: URL:  
https://www.cdhfinechemical.com/images/product/msds/37_916070364_Cyclopentyl
Methy  lEther-CASNO-5614-37-9-MSDS.pdf. (last accessed on 19 November 2019) 
4.  Watanabe K, Yamagiwa N, Torisawa Y. Cyclopentyl methyl ether as a new and 
alternative process solvent. Org Process Res Dev. 2007;11:251-258. 
5.  European Chemicals Agency (ECHA), 2020. Cyclopentyl methyl ether. CASRN 
5614-37-9. URL: https://echa.europa.eu/registration-dossier/-/registered-  
dossier/26626/7/9/2. (last accessed on 15 November 2020) 
6.  Inoue K, Suzuki H, Yamada T. Comprehensive toxicity evaluation of cyclopentyl 
methyl ether (CPME) for establishing a permitted daily exposure level. Fundam 
Toxicol Sci. 2019;6:145-165. 
7.  European Chemicals Agency (ECHA), 2020. Cyclopentyl methyl ether. CASRN 
5614-37-9. URL: https://echa.europa.eu/registration-dossier/-/registered-  
dossier/26626/7/6/2. (last accessed on 15 November 2020) 
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  Contains Nonbinding Recommendations 
 
TERTIARY-BUTYL ALCOHOL 
 
Introduction 
 
Tertiary-butyl alcohol (t-Butyl alcohol, tert-butanol; TBA: CAS Number 75-65-0) is a 
tertiary aliphatic alcohol and is used for a variety of purposes, including as an alcohol 
denaturant, a dehydration agent, and a solvent (Ref. 1). TBA is soluble in water and has a vapor 
pressure of 31 mmHg (20C). TBA is rapidly absorbed following inhalation or ingestion, but 
poorly absorbed through skin (Ref. 2). 
 
The rat oral LD50 (combined values for males and females) has been reported to be between 
2,733 and 3,500 mg/kg body weight. The primary acute effects observed in animals are 
signs of alcoholic intoxication. Human clinical test data indicate that TBA is neither an irritant 
nor a sensitizer (Ref. 3). Its potency for intoxication is approximately 1.5 times that of ethanol 
(Ref. 4). Given its wide diversity of use, the potential for human exposure to TBA is high (Ref. 
5). The National Institute for Occupational Safety and Health indicates that TBAs use is 
widespread in the workplace (Ref. 1). A Cosmetic Ingredient Review Expert Panel also 
concluded that TBA is safe as used in cosmetic products with concentrations ranging from 
0.00001 to 0.3 percent (Ref. 3). 
 
Genotoxicity 
 
TBA was not mutagenic in the Ames bacterial reverse mutation assay (Ref. 6). The U.S. 
National Toxicology Program (NTP) studies also showed TBA was not genotoxic in vitro with 
and without metabolic activation (S9) (mouse lymphoma cell mutation assay, chromosome 
aberrations, sister chromatid exchanges). In vivo, no increases in micronucleated 
erythrocytes were observed in  peripheral blood samples from mice administered up to 
40,000 parts per million (ppm) TBA in drinking water for 13 weeks or up to 625 mg/kg 
administered by intraperitoneal injection 3 times at 24-hour intervals (Ref. 6). In conclusion, 
there is no evidence that TBA is genotoxic (Ref. 2). 
 
Carcinogenicity 
 
TBA was investigated by the NTP in two 2-year drinking water studies, one in F344/N rats 
and one in B6C3F1 mice (Refs. 1 and 6). Both studies included 3 treatment groups (60 
animals/sex/group; 50 animals/sex/group completed the study): in rats, doses of 85, 195, and 
420 mg/kg/day in males, and 175, 330, and 650 mg/kg/day in females; and in mice, doses of 535, 
1,035, and 2,065 mg/kg/day in males, and 510, 1,015, and 2,105 mg/kg/day in females) (Ref. 
1). Survival was decreased in high-dose rats and high-dose male mice. Final mean body 
weights were decreased in exposed male and high-dose female rats and high-dose female mice. 
The primary targets of TBA were the kidney (mineralization, hyperplasia, tumors) in male 
rats, and the thyroid gland (follicular cell hyperplasia, tumors) and urinary bladder 
(inflammation and epithelial hyperplasia) in mice. The NTP Technical Report concluded that 
there was some evidence of carcinogenic activity in male rats based on increased incidences of 
renal tubule adenoma or carcinoma (combined) and in female  mice based on increased 
8 
  Contains Nonbinding Recommendations 
 
incidences of follicular cell adenoma of the thyroid gland (Ref. 6). There was no evidence of 
carcinogenicity in female rats and equivocal evidence in male mice. 
 
In mice, the incidence of thyroid follicular cell adenoma was significantly increased in high-
dose females. These tumorigenic effects were associated with an increased incidence and 
severity of  focal follicular cell hyperplasia of the thyroid gland in all TBA-treated groups of 
males and females (Refs. 1 and 6). In contrast, no thyroid tumors were observed in an 18-
month carcinogenicity study of methyl tert-butyl ether by the inhalation route in CD-1 mice 
(Ref. 7). The systemic TBA exposure (as a metabolite of methyl tert-butyl ether) likely exceeded 
the exposure in the NTP study (Ref. 2). However, differences in strain of mice (CD-1 versus 
B6C3F1) or route of administration may be responsible for the differences in response. In the 
absence of evidence suggesting direct thyroid toxicity, it was hypothesized that TBA induced 
thyroid tumors in the drinking water study through increased liver metabolism of thyroid 
hormones, triggering a compensatory increase in thyroid stimulating  hormone production and, 
thus, thyroid follicular cell proliferation and hyperplasia (Ref. 2). Rodents are substantially more 
sensitive than humans to the development of thyroid follicular cell tumors in response to thyroid 
hormone imbalance. Thus, the dose response is nonlinear, and tumors are not expected to occur 
in humans in the absence of altered thyroid hormone homeostasis (Refs. 8 and 9). In partial 
agreement with the above hypothesis, TBA is an inducer of phase 1 and 2 liver enzymes 
following 14 days of oral exposure at doses less than or equal to those used in chronic studies, 
and TBA administration resulted in a small decrease in circulating thyroid hormones in B6C3F1 
mice (Ref. 10). However, no meaningful changes in thyroid stimulating hormone levels were 
observed in this study. A comprehensive review of the mouse carcinogenicity data concluded 
that, in the absence of meaningful effect on thyroid stimulating hormone and toxicity to the 
thyroid, the cause of the increase in either hyperplasia or adenoma incidence remains unclear 
(Ref. 2). TBA administration also resulted in an increased incidence of chronic inflammation and 
hyperplasia of the transitional epithelium of the urinary bladder in high-dose males and females. 
 
In rats, an increased incidence of renal tubule adenomas and carcinomas was observed in males 
exposed to TBA, but the increase was not dose dependent. The evidence suggests that these 
tumors are due to an 2-globulin nephropathy-mediated mode of action. 2-Globulin 
nephropathy is a well-recognized sex- and species-specific mechanism of toxicity without 
relevance to humans (Refs. 11 and 12). Foci of linear mineralization in the renal medulla, a 
lesion consistently reported as a long-term consequence of 2-globulin nephropathy, were 
observed in the high-dose male rats (Refs. 1 and 6). Further, TBA was shown to interact with 
2, which explains the accumulation of 2 in the male rat kidney (Ref. 5). Although no 
significant neoplastic findings were observed in female rats, a dose-dependent increase in 
severity of nephropathy was observed at all TBA doses compared to control animals (average 
severity of 1.6, 1.9, 2.3, and 2.9; scale of 04); incidence ranged from 4748 out of 50 animals in 
all groups. An increased incidence of transitional epithelial hyperplasia and suppurative 
inflammation at the two highest doses and renal tubule hyperplasia in a single high-dose animal 
were also observed. The human relevance of the renal findings in female rats is currently 
unclear. 
 
The 2-year carcinogenicity studies were considered the most relevant for calculation of the PDE 
for TBA. From the results of the rat and mouse carcinogenicity studies, PDEs were calculated 
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  Contains Nonbinding Recommendations 
 
based on two different scenarios:  
 
(1)  Renal lesions and tumor findings in male rats are not relevant to humans and, therefore, 
the increased severity in nephropathy observed in female rats at the lowest dose (lowest-
observed- effect level (LOEL) = 175 mg/kg/day is used for the PDE calculation. 
 
or 
 
(2)  Increased incidence of follicular cell hyperplasia in the thyroid of female mice at the 
lowest TBA dose (LOEL = 510 mg/kg/day) is used for the PDE calculation. 
 
Scenario 1 (rat): LOEL (nephropathy) 175 mg/kg/day 
 
175 x 50
PDE =  =35 mg/day 
5x 10 x 1 x 1x 5
F1 = 5 to account for extrapolation from rats to humans 
F2 = 10 to account for differences between individual humans 
F3 = 1 because long duration of treatment (2 years) 
F4 = 1 due to similar severity of effect (nephropathy in females) at the low-dose 
compared to control animals  
F5 = 5 because a NOEL for nephropathy was not established 
 
Limit = (35 x 1,000)/10 = 3,500 ppm 
Scenario 2 (mouse): LOEL (follicular cell hyperplasia) 510 mg/kg/day 
 
510 x 50
PDE  =     =42.5mg/day  
12 x1 0 x 1 x 1x 5
F1 = 12 to account for extrapolation from mice to humans 
F2 = 10 to account for differences between individual humans 
F3 = 1 because long duration of treatment (2 years) 
F4 = 1 because hyperplasia response was of minimal to mild average severity at all 
doses and thyroid tumors were not observed at the low dose 
F5 = 5 because a NOEL for hyperplasia was not established 
 
Limit = (42.5 x 1000)/10 = 4,250 ppm 
 
The ultimate PDE for TBA, calculated based on the identified LOEL of 175 mg/kg/day from the 
2- year rat study, is 35 mg/day. 
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  Contains Nonbinding Recommendations 
 
 
Reproductive and developmental toxicity 
 
TBA has not been associated with induction of skeletal or visceral malformations in rats or mice 
but did induce developmental delays and intrauterine or prenatal mortality at doses of 1,000 
mg/kg/day or greater (Ref. 2). 
 
In a reproduction/developmental toxicity screening study, TBA was administered to Sprague- 
Dawley rats (12/sex/group) by oral gavage at dose levels of 0, 64, 160, 400, and 1,000 
mg/kg/day for up to 63 days in males and from 4 weeks before mating until postnatal day 20 in 
females (Ref. 13). There were no adverse effects on any reproductive parameters, including 
mating index, fertility index, pregnancy index, or gestation index. For dams receiving 1,000 
mg/kg/day TBA through gestation and lactation, there was a significant reduction in mean litter 
size; a decrease in the number of live born pups per pregnancy; an increase in the number of 
stillborn pups; increased pup mortality up to postnatal day 4; and a decrease in mean pup body 
weight at birth, which continued to weaning. Parental toxicity (transient central nervous system 
effects, reduced body weight and food consumption) was observed at doses of 400 mg/kg or 
greater. The NOAEL for developmental/reproductive effects was identified as 400 mg/kg/day. 
 
At a dose of 1,000 mg/kg/day, mild to moderate transient systemic toxicity was observed in both 
sexes in the parental generation, including reversible central nervous system effects such as 
lethargy and ataxia, and reduced food consumption and weight gain. At 400 mg/kg/day, an 
increased incidence of transient mild lethargy/ataxia in females was observed. The NOEL for 
parental toxicity was 160 mg/kg/day.   
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  Contains Nonbinding Recommendations 
 
Repeated-dose toxicity 
 
In a sub-chronic toxicity study, TBA was administered to F344/N rats (10/sex/dose) ad 
libitum in drinking water at dose levels of 0, 2.5, 5, 10, 20, and 40 mg/mL for 13 weeks 
(equivalent to 176, 353, 706, 1,412, and 2,824 mg/kg/day) (Ref. 6). All high-dose males and 
six high-dose females died during the study. Nephropathy was the most sensitive effect 
observed in the study. An increase in severity of nephropathy was observed in the lower four 
dose groups in males when compared to control animals, as was the accumulation of hyaline 
droplets in the kidney at doses of 353, 706, and 1,412 mg/kg/day. The incidence of 
nephropathy in females at the highest three doses was significantly greater than that in the 
controls. Transitional epithelial hyperplasia and inflammation of the urinary bladder were 
observed at the two highest doses in males and in high-dose females. Based on the 
nephropathy in male rats at the lowest dose, 176 mg/kg/day was considered the LOEL. As 
noted above, 2-globulin nephropathy is a well-recognized sex- and species-specific 
mechanism of toxicity without relevance to humans (Refs. 11 and 12). 
 
TBA was also administered to B6C3F1 mice (10/sex/dose) in drinking water for 13 weeks at 
the same concentrations provided to rats (doses equivalent to 446, 893, 1,786, 3,571, and 
7,143 mg/kg/day) (Ref. 6). Two high-dose males and one high-dose female died. The final 
mean body weights in males at the two highest doses and in females at the high dose were 
significantly lower than that in the control animals. Transitional epithelial hyperplasia and 
inflammation were observed in the urinary bladder of the same groups. A NOEL of 1,786 
mg/kg/day was identified (Ref. 6). 
 
Conclusion 
 
The calculated PDE for TBA is 35 mg/day based upon the LOEL for nephropathy in females 
from the 2-year rat carcinogenicity study. It is recommended that TBA be placed into class 2, 
Solvents To Be Limited in Table 2 in the ICH Q3C guidance. 
 
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