Comments on ETBE and tert-Butanol (TBA) 

Preliminary Materials 

for IRIS Toxicological Review

General and Non-Cancer Endpoints 

On Behalf of 

Lyondell Chemical Company

Susan Borghoff, PhD, DABT

Senior Managing Scientist

December 12, 2013
General Comments 

ETBE/TBA Preliminary Materials

2

• Draft literature strategy; processes for identifying and screening 

scientific literature and literature results

• Preliminary evidence tables, and

• Preliminary exposure-response arrays 

Both summarizing key characteristics and findings from studies 

that EPA proposes to consider in identifying hazards and 

characterizing exposure response relationships.

The stated purpose of these materials is to document EPA’s systematic review 

of the scientific literature, demonstrate a standardized approach to presenting 

key study data, and foster early public involvement and transparency. 
General Comments 

ETBE/TBA Preliminary Materials

Lyondell has reviewed the preliminary materials and offers for 

consideration our comments on:

the clarity and transparency of the materials;

the approach for identifying pertinent studies;

the selection of studies for data extraction to preliminary 

evidence tables and exposure-response arrays;

methodological considerations that could affect the 

interpretation of, or confidence in, study results; and

additional studies published or nearing publication that may 

provide data for the evaluation of human health hazard or 

exposure-response relationships.

3
Draft Literature Search Strategy (ETBE/TBA) 

• Well documented database search and strategy; > 600 citations for 

ETBE, > 2900 for TBA identified; European Chemical Agency (ECHAs) 

database for registered 

substances:http://echa.europa.eu/web/guest/information-on-chemicals/registeredsubstances

• For complete clarity and transparency, further text is recommended to 

support the outline (Figure 1.1) of the literature search approach.

• Specific criteria for selection of primary vs. not primary sources of 

data; Request EPA provide their worksheet that details the basis for selecting 

and/or excluding citations.

• Citations identified as “Not Primary Source of Health Effects Data”

• What is the decision process for including specific citations?

• Will they be reviewed for quality prior to incorporation?

• Will there be an opportunity for public comment prior to finalizing?

4
Preliminary Evidence Tables (ETBE/TBA) 

• Decision process for data extraction; unclear why certain endpoints 

are considered individually and others grouped in evidence tables and 

exposure response arrays.

• Recommendation that study quality be considered prior to 

including data; consider similar approach used by OECD (Klimisch et al., 

1997). 

• Request clarity on data normalization strategy used; although 

helps to make studies comparable, it is not the same as extracting the 

specific data. 

• Clarify why some reproductive endpoints were presented as 

incidence and endpoints as percentage change. 

• Statistical analysis of data; Not clear what data was statistical 

analyzed. 

5
Preliminary Evidence Tables (ETBE/TBA)

Selection of Studies and Endpoints for Data Extraction

• Borghoff et al., (2001) was only considered for mechanism data and 

not other endpoints of toxicity because it was a 10-day study.

• Inconsistency between reviews for selected endpoints extracted; 

Serum enzymes for ETBE but not for TBA. 

• Certain studies were selected for body weight changes and other 

studies not used (section 2.6 of TBA review).

• The approach used does not allow for consideration of how changes 

in one endpoint influences a change in another; data selected in 

isolation.

Organ weight changes without histology

Body weight changes without food and water consumption

Target organ effects without systemic toxicity measures

Overall clarity in the decision process for selecting endpoints 

would provide transparency.

6
Preliminary Evidence Tables(ETBE/TBA)

Selection of Studies and Endpoints for Data Extraction

Request further explanation for not extracting data from the 

following studies;

• Dermal exposure studies,

• Acute or short-term exposures (less than 90 days); except in 

the context of immunotoxicity, neurotoxicity, 

developmental or reproductive toxicity, and

• Endpoints related to possible mechanisms of toxicity

7
Preliminary Exposure Arrays (ETBE/TBA)

• The presentation of results in the exposure response arrays is 

questionable; Statistical findings alone may not be meaningful and 

often severity of changes should be considered.

• Recommend consistent presentation of lesions; Easier to compare 

across studies.

• In the ETBE review; hepatocellular adenomas (benign 

neoplasms) have been combined with hepatocellular 

carcinomas (malignant neoplasms). Recommendation: presentation 

of malignant neoplasms should be separated from benign neoplasms with 

pre-neoplastic lesions having a separate category.

• There were many related endpoints not mentioned in the 

tables/figures for the very comprehensive DART studies. 

Recommendation: Clarify criteria for endpoint selection for inclusion in 

summary tables and arrays. A narrative for each subsection would be 

useful.

8
ETBE: Non-Cancer Endpoints

Preliminary Evidence Tables and Exposure-Response Arrays

Kidney Effects – Tables and Figures 2.1 and 2.2

Liver Effects - Tables and Figures 2.3 and 2.4

Reproductive Effects – Tables and Figures 2.5

Body Weight Effects – Tables and Figures 2.6 and 2.7 

Other Systemic Effects- Tables and Figures 2.8 and 2.9

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ETBE: Kidney Effects (Tables 2.1 and 2.2)

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• Consistency in citing primary references (Suzuki et al., 2012 vs. JPEC, 2010a); 

Atypical hyperplasia, papillary necrosis, average severity of CPN, and relative 

kidney weights were extracted from JPEC report and not Suzuki publication.

• Recommend removing category for kidney weights from 2-year study; 

Questionable endpoint to evaluate toxicity at the end of a chronic study.

• Recommend removing category for incidence of hyaline droplets from 

2-year study; Suggests a lack of understanding of the biology underlying α2uglobulin nephropathy.

• Fully utilize the depth/amount of information in Cohen et al., 2011 

study; Currently restricted to data on nephropathy (CPN). 

• EPA states that both quantitative and qualitative data is to be 

considered; Recommend Including granular casts, a lesion characteristic of 2uglobulin (α2u-g) nephropathy. 
ETBE: Kidney Effects (Tables 2.1 and 2.2)

11

• Recommend removing category of urinalysis endpoints (JPEC, 

2008c; JPEC, 2010a; Suzuki el al., 2012); Casts in the urine and granular casts 

at the junction of the outer and inner stripes of outer medulla of kidney are 

totally different entities with different biological implications

• Corrections in Table based on review of study report (JPEC, 2010b) 

EPA states that mineralization in the papilla was not examined in female 

rats, it should be changed to not observed; mineralization in the papilla 

was observed in male rats only, implies females were examined but the 

lesion was absent.

EPA states that atypical tubule hyperplasia was not examined; should be 

stated as not observed.

Hyperplasia of the papilla observed in this study was not included; 

Papillary hyperplasia is a lesion that results from, and accompanies 

advanced CPN, clarifying that CPN is unique from chronic nephropathy.
ETBE: Kidney Effects (Tables 2.1 and 2.2)

12

Recommend reviewing the following publication for inclusion:

Hard G.C., Banton M.I., Bretzlaff R.S., Dekant W., Fowles J.R., 

Mallett A.K., McGregor D.B, Roberts K.M., Sielken Jr. R.L., ValdezFlores C., and Cohen S.M. (2013). Consideration of Rat Chronic 

Progressive Nephropathy (CPN) in Regulatory Evaluations for 

Carcinogenicity. Toxicological Sciences. 132(2):268-275. 

Although there is no quantitative data in this paper, it describes 

the link between CPN and induction of renal tubule tumors.
ETBE: Liver Effects (Tables and Figures 2.3 and 2.4)

13

• Recommend including studies of shorter duration (< 90-days); 

Early changes can be used to interpret longer term responses (i.e. 

liver enzyme induction and histopathology).

• Remove categories evaluated at the end of 2-year studies; Liver 

weight changes, clinical chemistry, and centrilobular hypertrophy.

Sellers et. al., 2007 , “In these long-term studies, normal physiological aging 

changes and inter-current disease may contribute to inter-animal variability, 

which will confound organ weight interpretation (Long et al.,1998a; 1998b 

). 

The Society of Toxicologic Pathology recommends organ-to-body weight 

ratios in toxicology studies ranging from 7 days to 1 year. 

Liver enlargement in response to hepatic enzyme induction is typically 

associated with centrilobular hypertrophy (Maronpot et al., 2010), not 

typically observed following 104 weeks (Sellers et al., 2007).
ETBE: Liver Effects (Tables and Figures 2.3 and 2.4)

14

• Provide rationale for inclusion and exclusion of specific liver 

toxicity endpoints; Clinical chemistry: ALT vs. cholesterol.

• Consistency in citing primary references; Unclear as to where study 

data is extracted (study reports vs. published papers).

• Clarify for how data was extracted, transformed, and statistically 

analyzed for evidence tables.

• Recommend that quality control checks are conducted on data 

presented in evidence tables prior to review; Specific errors are 

identified in Lyondell’s written comments. 

• Recommend that a distinction is made between adaptive vs. 

adverse effects; Liver weight increases accompanied by hepatocellular 

hypertrophy are common events in toxicity studies in rodents.
ETBE: Liver Effects (Tables and Figures 2.3 and 2.4)

15

• Clarify why liver weights are only extracted from F0 generation; 

Gaoua, 2004b; Fujii et al., 2010; JPEC, 2008e.

• Insure that data extracted is not misrepresented; Centrilobular

hypertrophy was not evaluated in all the animals (Gaoua, 2004b).

• The exposure-response arrays of liver effects was duplicated; 

Figure 2.4, Exposure to ETBE via inhalation is a copy of Figure 2.3, ETBE oral 

administration. 

• Recommend inclusion of additional endpoints; livers of mice 

exhibited time- and concentration-dependent increases in hepatocyte 

labeling index, quantitative data for response array and mode-of-action 

(Medinsky et al., 1999; Bond et al., 1996a). 
ETBE: Reproductive and Developmental Effects

16

CA OEHHA DART (Nov. 21, 2013 ) Committee voted unanimously 

that ETBE has not been clearly shown to cause developmental 

toxicity, male reproductive toxicity, or female reproductive toxicity.

Therefore ETBE will be removed from the Proposition 65 list.
17

• Provide clarity on data extracted and transformed; Include 

information on sample size based on litter and unit of analysis (litter vs. 

individual pup). 

• Confirm that sample size reflects litters assigned to exposure 

groups and not number of impregnated dams; Litter of origin is 

the appropriate experimental unit of analysis.

• Recommend quality control checks on evidence tables prior to 

review; Specific errors are identified in Lyondell’s written comments. 

• Consistency in citing primary references; Unclear as to where study 

data was extracted, F1 post implantation and total litter losses, (Fuji et 

al.,2010 or JPEC, 2008e).

ETBE: Reproductive Effects(Tables and Figures 2.5
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• Provide criteria for endpoint selection; Suggest inclusion of more 

explanatory text for selecting specific endpoints (i.e. Berger and Horner, 

2003).

• Caution with extracting data from reports translated to English; 

Recommend a complete understanding of how data is collected (Fujii et al., 

2010 ; JPEC, 2008e).

• Clarification of post-implantation loss; Reported differently in the 

original publications vs. in the evidence table (Fujii et al., 2010 ; JPEC, 2008e).

• All data extracted into Evidence Tables should be checked for 

consistency and accuracy; (Gaoua, 2008; Asano et al., 2011; JPEC, 

2008j). 

ETBE: Reproductive Effects - Tables and Figures 2.5
ETBE: Body Weight- Table and Figures 2.6 and 2.7

19

• Consistency in citing primary references; Unclear from what report 

the body weight data was extracted (Hagiwara et al., 2011 vs. JPEC, 

2008d)

• Request clarity as to why EPA only evaluated body weights for 

F0, and not the F1 generation; (Gaoua, 2004b). 

• Recommend that body weight assessments consider (female) 

reproductive stages (i.e., mating, pregnancy, lactation) as well 

as life stages (pups, adults); a single body weight value does not 

representative all possible changes in this study (Fujii et al., 2010). 

• Recommend that body weight changes incorporate changes in 

feed consumption and water intake to provide more 

meaningful data.
ETBE: Other Systemic Effects 

Tables and Figures 2.8 and 2.9

20

• Request explanation as to why responses were combined on one 

exposure array; antibody response, immunological effects, adrenal weight, 

and mortality.

• Consistency in citing primary references; spleen and adrenal weights 

(Suzuki et al., 2012 vs. JPEC, 2010a). 

• Insure data from reports is represented correctly; Evidence table 

notes a statistically significant change in relative adrenal weights for male 

rats whereas the report does not (JPEC, 2010a).

• Include relative adrenal and spleen weights in evidence tables; 

provided in publication the following reports/publications, Medinsky et al., 

1999; Bond et al., 1996.

• Spleen and adrenal weights only presented for F0 and not F1 

generation; (Gaoua, 2004b).
ETBE: Other Systemic Effects 

Tables and Figures 2.8 and 2.9

21

• Rationale for inclusion and exclusion of data; Wild-type and knock-out 

mice evaluated, data presented for only knock-out mice only (Weng et al., 

2011).

• Recommend describing how numerical data is extracted from 

publication figures; Numerical percent change reported in Evidence Table

(Weng et al., 2011).

• Recommend evaluating study quality prior to inclusion of data; 

Study quality concerns including design and methods (Weng et al., 2011).

• Rationale for inclusion and exclusion of specific data; (Li et al., 

2011) 

Only data on one of two strains was included

Not all hematological and immunological markers were included

Extraction numerical data from publication figures
22

• JPEC, 2008h/Aso et al., 2013; Prenatal Developmental toxicity study in 

rats.

• Kakehasi et al., 2013; Mode of action of ethyl tertiary-buty ether 

hepatotumoigenicity in the rat: Evidence for a role of oxidative stess via 

activation of CAR, PXR and PPAR signaling pathways. Toxicol. Applied 

Pharmacol. 273 (2013) 390-400.

• Gaoua, 2003, Male rat pilot study

• Malarkey, 2011 Summary Report sponsored by the NTP and EPA of 

Pathology Materials from Selected Ramazzini Institute Rodent Cancer 

Bioassays, November 29, 2011. 

Additional Studies/Reports - ETBE
TBA: Non-Cancer Endpoints

Preliminary Evidence Tables and 

Exposure-Response Arrays

• Kidney

• Thyroid

• Reproductive

• Development

• Neurodevelopmental

• CNS

• Body weight

• Liver

• Urinary Bladder

23
TBA: Kidney Effects

24

• Insure study results are clearly represented; It is stated “There 

were no changes in kidney histopathology observed” implies that the 

kidneys were evaluated. Kidneys were fixed, but not microscopically 

examined. Lyondell Chemical Company, 2004 -should be cited as HLS, 2004.

• Study quality evaluated; Histopathologic observations not diagnosed 

with accepted terminology (Acharya et al.,1995, 1997). 

• Qualification of lesions; It should be noted that mineralization was 

mainly at the “corticomedullary” junction. This lesion represents an 

imbalance of the Ca/PO4 ratio and is affected by dietary factors, and not 

necessarily an indication of nephrotoxicity. It is distinct from linear 

mineralization seen in the papilla in the 2-year study, which is a “marker” 

of a preceding α2u-globulin nephropathy event the (NTP, 1995; Cirvello et 

al., 1995).
TBA: Kidney Effects

25

• Recommend inclusion of key endpoint; Hyaline droplet 

accumulation is an important pathologic entity that increases in all TBA 

exposed male rats.

• Clarify conflicting text within the preliminary materials; It was 

stated that “no renal histopathological changes reported in mice at 13-

weeks” and also stated that “histopathologic data for the 13-week study 

were not provided.” 

• Recommend clarifying histological lesion with a footnote; On reexamination of kidney slides from the 15-month time point (NTP, 1995), it 

was determined that the mineralization in male rats was linear 

mineralization in the papilla, confirming a chronic end-point associated 

with α2u-g nephropathy occurs at an earlier time point (Hard, 2005).
TBA: Kidney Effects 

26

• Recommend clarifying histological lesions with a footnote; For 

both males and females, transitional epithelium hyperplasia and 

suppurative inflammation were identified by NTP as being part of the 

nephropathy (CPN) complex.

• Extracted data from these publications should be included in 

the Histopathology subsection; Takahashi et al., 1993; Williams and 

Borghoff, 2001; Hard et al., 2001.
27

• Clarify that thyroid weight changes were not evaluated; It would 

be useful to list this endpoint and note that weight changes were not 

evaluated in the 13-week or at the 15-month interim necropsy.

• Clarify the reasoning for the exclusion of selected thyroid 

histological changes; Besides follicular cell hyperplasia, other 

histological changes were noted but not included in the evidence table (Ccell hyperplasia, follicle, cyst, ect). 

• Clarify the timing of when follicular cell hyperplasia was observed in 

evidence table; Observed following chronic exposure to TBA (2-year study). 

• Clarify the lack of data provided in the evidence tables at the high 

TBA exposure concentration; Lethality at this exposure concentration 

should be noted. 

TBA: Thyroid Effects
28

• Clarify the reasoning for including single acute exposure 

study; A single acute exposure of TBA in male mice at dose levels(up 

to 2,000 mg/kg with endpoints measured several days later (Billitti, 

et al., 2005).

• Clarify criteria for data inclusion and exclusion from the 

following reports; 

• Lyondell Chemical Company (2004) OECD 421 

• NTP (1995) B6C3F1 mouse study 

• NTP (1997) F344/N rat inhalation rat study 

• NTP (1997) B6C3F1 mouse inhalation study

TBA: Reproductive, Developmental, and 

Neurodevelopmental Effects
29

• An MTBE rat inhalation study (Bevan et al.,1997) is currently 

included in the “Excluded (not pertinent)” category of the 

Reference List; MTBE is metabolized to TBA which is the primary 

metabolite found in rats following exposure. Since systemic blood levels of a 

chemical are required to cause reproductive toxicity, inclusion of the MTBE 

two-generation study is well-justified within the TBA IRIS process.

• Reconsider inclusion of the Grant and Samson, 1982 paper based 

on quality; poorly reported and experimentally limited study in which half 

of the pups died due to experimental procedures that were very stressful 

(e.g. cannulation of pups raised away from their dams in a cup). 

TBA: Reproductive Effects
30

• Incomplete and/or exclusion of endpoints without clear 

rationale; the Lyondell Chemical Company (2004) OECD 421 

• Questionable study quality and/or lacking details for inclusion.

• Daniel and Evans (1982). 

• Faulkner, et al., (1989) C57BL/6J mouse study is incomplete.

• Nelson et al., 1989 

TBA: Developmental Effects
31

• Recommend avoiding using catch-all classification of 

"neurodevelopmental”; general categories such as neurobehavioral 

(cognitive, motor), neuropharmacologic, neuropathologic, or 

neurophysiologic findings should be used to avoid conflicting results.

• The selection process for including significant or nonsignificant findings is not transparent; Although cited as a source 

of data, none of the findings from Nelson et al. 1991 paper were 

included in the exposure array. 

• The following citations should be re-reviewed to determine 

quality of the publication and then recommend including 

complete data sets with complete data entry.

–Grant and Sampson, 1981 

–Wood and Laverty, 1979 

–Snell and Harris, 1980 

–Thurman et al., 1980 

–McComb and Goldstein, 1979a and 1979b

TBA: Neurotoxicity and CNS Effects 
32

• Tables recording absolute and relative (body weight) liver weights; 

Consider brain weight as the basis for relative liver weights. It has been 

recognized that brain weight is more stable than body weight.

• Although exposure (to females) occurred during pre-mating, mating, 

gestation, and lactation, the body weight data presented appears to 

be only the lactation body weights; HRL, 2004.

• Numerous transcription errors were identified in the tables; NTP, 

1995.

• Body weight changes following inhalation exposure to TBA should 

have been included in the evidence table; NTP, 1997.

TBA: Body Weight
33

• Not all liver effects reported in this paper were included in the table;

Acharya et al., 1995.

• Clarify the inclusion and exclusion of selected endpoints; in general only 

selected measurements of liver toxicity where included.

• Figure 2-6; Errors identified in the exposure-response array of body 

weight, liver effects, and urinary bladder effects following oral 

exposure to TBA; Data reported in this array needs to be corrected or 

alternatively this figure could be deleted. 

• Recommend reviewing Blanck et al., 2010 for inclusion; Relevant 

endpoints for mode of action for hepatic enzyme inducer.

• Recommend including reviews by McGregor et al., 2010; Provides a 

detailed evaluation of all endpoints discussed and should be considered in the 

overall evaluation.

TBA: Liver Effects
Summary Comments Pertaining to

ETBE/TBA Preliminary Materials

34

• Preliminary materials are intended to provide opportunity for 

early discussion; however, content presented does not adequately 

support this objective.

• Lack of consideration of study quality; A systematic approach for 

evaluating the quality of experimental data should be considered before 

accepted for any further evaluations (such as evidence tables). 

Recommended approach comparable to Klimisch scores. (Klimisch et al., 

1997)

• Time allotted for public review was limited; Encourage EPA to QC 

on data extracted and provide clear identification of where in 

report/publication the data was extracted from, allowing more 

transparency in the review.
Summary Comments Pertaining to

ETBE/TBA Preliminary Materials

35

• Preliminary materials do not adequately provide the documentation for 

a systematic review of the scientific literature. 

• Approach used for creating evidence tables and exposure-responses 

arrays does not identify the findings that are meaningful and does not 

relate endpoints that need to be evaluated together (e.g. organ weight 

changes and body weight changes, toxicity and cancer endpoints).

• Inconsistent criteria for use of mechanistic data. EPA’s application of 

these conflicting criteria is also inconsistent with some mechanistic 

information included, whereas other data is excluded. 

Recommendation: Data on key health endpoints much be included as 

pertinent to assessment of these endpoints.

These and our other comments identify significant deficiencies in the 

preliminary materials that EPA is encouraged to address before 

proceeding with preparation of the draft assessment.
In Summary

36

• The present preliminary materials, although a good start, at 

this point, do not satisfactorily achieve their purpose.

• There are significant deficiencies in the preliminary materials 

that EPA is encouraged to address before proceeding with 

preparation of the draft assessment.

• Overall, there is concern as to whether there will be another 

opportunity for the public to review the final “preliminary 

materials” once a completed and corrected data set are 

finalized and prior to the final IRIS assessment.