Comments on ETBE and tert-Butanol (TBA) 

Preliminary Materials 

for IRIS Toxicological Review

Genotoxicity, Mutagenicity, and Cancer Endpoints 

On Behalf of Lyondell Chemical Company

Presented by 

Susan Borghoff, PhD, DABT

for 

Douglas McGregor, PhD

December 12, 2013
Topics for Comments on IRIS Materials 

for ETBE & TBA

2

ETBE (Ethyl tert-butyl Ether)

• Mutation Assays

• Genetic Toxicology Assays

• Cancer Endpoints

TBA (tert-Butanol)

• Mutation Assays

• Genetic Toxicology Assays

• Cancer Endpoints
No significant effects on mutant numbers in any study

Strains Used Activation

Highest 

Ineffective 

Dose

Reference

TA1535, TA1537,

TA1538, TA98

+/- liver S9 

(Rat) 500 μg/plate Institut Pasteur de Lille (1992)(Not included)

TA1535, TA97, 

TA98, TA100

+/- liver S9 

(Rat & Syrian 

Hamster)

10,000 

μg/plate Zeiger et al. (1992)

TA1535, TA1537, 

TA1538, TA98, 

TA100

+/- liver S9 

(Rat)

5,000 

μg/plate

Pharmakon Europe (1994)

(Not included)

ETBE: Mutation Assays with Salmonella 

typhimurium

3
No significant effects on mutation frequency in either study

ETBE: Mutation Assays with Mammalian Cells

In vitro Assay Activation Highest Dose Reference

Gene mutation, 

CHO - at hprt

locus

+/- liver S9 (Rat)

Two acceptable 

independent 

assays

5,000 μg/mL BBRC (1995c);

Vergnes & Kubena (1995a)

Chromosomal 

aberrations - CHO

+/- liver S9 (Rat)

Duplicate 

cultures in a nonindependent 

assay

5,000 μg/mL BBRC (1995b);

Vergnes (1995)

4
No significant increases in micronucleated cells; total of 6 studies

Species Route Highest Dose Reference

Male and female OF-1 

mouse

Oral gavage 5,000 mg/kg-bw x 1

Institut Pasteur de Lille (1992c)

(Not included)

Male and female CD-1 

mouse

Inhalation 5,000 ppm

6h/d x 5 days

BBRC (1995a);

Vergnes & Kubena (1995b)

Male and female 

Fischer 344/DuCrj rat i.p. 1,000 mg/kg-bw x 2

Japan Bioassay Research 

Center (2007b)

Male and female 

Fischer 344/DuCrj rat Oral gavage 2,000 mg/kg-bw x 2

Japan Bioassay Research 

Center (2007a)

Male and female 

Fischer 344/DuCrj rat

Drinkingwater10,000 ppm (w/w), 

13 wks

Japan Bioassay Research 

Center (2007c)

Male and female 

Fischer 344/DuCrj rat Inhalation 5,000 ppm6h/d, 5d/wk, 13 wks

Japan Bioassay Research 

Center (2007d)

ETBE: In Vivo Micronucleus Assays with Rodent 

Bone-Marrow Cells

5
Suggested Klimisch Score - 2 (reliable with restrictions) 

Weng et al., 2012 (Study to add to Section 2.8, Table 2-11)

ETBE: DNA Damage to Liver Cells

of C57BL/6 Wild-type and Aldh2 -/- Mice

6

Fold-increases over the control proportions of comets

Assay

Dose 

(ppm)

Statistically significant –

fold increase in comets

Male Female

+/+ -/- +/+ -/-

Comet assay in liver 

cells

500 1.76

1750 1.83

5000 1.93 1.99 1.84

net hOGG1 Comet assay

in liver cells

500 2.71

1750 2.51

5000 2.99 3.44 3.15

8-OH-dG residues in 

liver DNA

500 1.81

1750 1.74

5000 1.31 1.93 1.61
ETBE: Comet Assay on Peripheral Leucocytes of 

C57BL/6 Wild-type and Aldh2 -/- Mice

7

Statistically significant fold-increases in comets (over control) in 

peripheral leukocytes

Weng et al., 2011; 2013 (Studies to add to Section 2.8, Table 2-11) 

Assay

Klimisch

Score

Dose 

(ppm)

Statistically significant 

fold increase in comets

Male Female

+/+ -/- +/+ -/-

Comet assay

(Weng et al. 2011)

2 (reliable with 

restrictions)

500 1.4

1750 1.6

5000 1.6 1.7 1.6

net hOGG1 Comet 

assay

(Weng et al., 2013)

3 (not reliable)

500 1.35

1750 1.61

5000 1.61 1.74 1.56
*New reference with suggested Klimisch Score of 3 (not reliable)

** Borderline significant (p=0.052) 

8

ETBE: Micronucleus Assay on Circulating 

Reticulocytes in C57BL/6 wild-type and Aldh2 -/- mice

Assay

Dose 

(ppm)

Statistically significant fold increase 

in micronucleated cells

Male Female

+/+ -/- +/+ -/-

Micronucleus assay in 

C57BL/6 mice

(Weng et al., 2013*)

500

1750 1.18

5000 1.42 1.24 1.25**

Statistically significant fold-increases in micronucleated cells

ETBE Inhalation (6hday, 5 days/wk, 13 weeks)
Aldh2 -/- as fold-increase over Aldh2+/+

ETBE Metabolites in Mouse Blood (Weng et al., 2013)

9

Acetaldehyde TBA 2-Methyl-1,2-

Propanediol

Male Female Male Female Male Female

+/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/-

Cmax (μM) 81 495 107 677 270 1426 777 2206 72 108 67 356

Fold increase in 

AUC

(Aldh2 -/- over +/+)

2.69-fold 2.52-fold 5.78-fold 3.01-fold 1.76-fold 4.95-fold
Metabolic Degradation of Alkyl Ethers to TBA

CH3 

H3C C OCnH2n+1 

CH3 

P450

-RCHO 

CH3 

H3C C OH 

CH3 

CH3 

H3C C O-Glucuronide

CH3 

CH2OH 

H3C C OH 

CH3

CHO 

H3C C OH 

CH3

TBA TBA - Glucuronide

COO ̄

H3C C OH 

CH3

2-Methyl -1,2-propanediol 2-Hydroxyisobutyrate

Acetone

MTBE or ETBE

10
Parameter Default Value Reference

A: Alveolar ventilation rate (minute 

volume) 0.17 L/min Amberg et al. (2000)

B: Exposure time 360 min/day, 5 days/wk JBRC (2010b)

C: ETBE concentration 0, 2.12, 6.36, or 21.20 mg/L JBRC (2010b)

D: Net respiratory uptake of ETBE 0.26 rounded to 0.3 Nihlén et al. (1998)

E: Body weight (0-104 wks)

Males: 0.41, 0.40, or 0.35 Kg 

Females: 0.22, 0.22, or 0.20 Kg JBRC (2010b)

Formula A × B × 5/7 × C × D ÷ E = mg/Kg bw/7-day week

Calculated net ETBE uptake rate Males: 0, 97, 292 or 973 mg/Kg bw/day 

Females: 0, 126, 379 or 1390 mg/Kg bw/day

Calculation of ETBE Uptake by Rats (Amberg et al., 2000 & Nihlén et al., 1998)

11

F344/DuCrlCrl Rats Exposed to ETBE Vapour 

for 104 Weeks (JBRC, 2010b)
Calculation of ETBE Uptake by Rats 

Minute volume based on power law (allometric) relationship with body weight formula

of Bide et al. (2000) J. Appl. Toxicol. 20, 273–290

F344/DuCrlCrl Rats Exposed to ETBE Vapour 

for 104 Weeks (JBRC, 2010b)

12

Parameter Default Value Reference

A: Alveolar ventilation rate (minute 

volume)

Vm = 0.499.BW0.809 

Males: 0.25; 0.24; 0.21 L/min

Females: 0.15, 0.15, 0.14 L/min

Bide et al (2000)

E: Body weight (0-104 wks)

Males: 0.41, 0.40, or 0.35 Kg 

Females: 0.22, 0.22, or 0.20 Kg JBRC (2010b)

Formula A × B × 5/7 × C × D ÷ E = mg/Kg bw/7-day week

Calculated net ETBE uptake rate Males: 981 mg/Kg bw/day 

Females: 1145 mg/Kg bw/day
Exposure concentration (ppm) 0 500 1500 5000 Stats. Analysis

No. animals/group 50 50 50 50

Peto Coch/A

Organ Tumour diagnosis r

Pituitary

Thyroid

Mammary gl.

Uterus

Spleen

Adenoma

C-cell adenoma

Fibroadenoma

Endometrial stromal polyp

Endometrial stromal sarcoma

Adenocarcinoma

Mononuclear cell leukaemia

18

7

8

8

2

2

8

14

5

10

15

2

3

7

11

1*

4

9

3

1

6

13

3

2*

8

2

4

4

p < 

0.05

Japanese Bioassay Research Center (2010b)

Number of female rats with specified tumors

13

ETBE: Chronic Inhalation Bioassay (104 weeks) 

Female F344/DuCrlCrl Rats

Fisher’s exact test * p < 0.05 ** p < 0.01
Fisher’s exact test * p< 0.05 ** p < 0.01

Japanese Bioassay Research Center (2010b)

ETBE: Chronic Inhalation Bioassay (104 weeks) 

Male F344/DuCrlCrl Rats 

14

Exposure concentration (ppm) 0 500 1500 5000 Stats. Analysis

No. animals/group 50 50 49 50 Peto Coch/A

Organ Tumour diagnosis r

Subcutis

Liver

Pancreas

Pituitary

Thyroid

Adrenal

Testis

Preputial gl.

Lung

Spleen

Peritoneum

Fibroma

Hepatocellular adenoma

Hepatocellular carcinoma

Islet cell adenoma

Adenoma

C-cell adenoma

C-cell carcinoma

Pheochromocytoma

Leydig cell tumour

Adenoma

Bronchiolar-alveolar carcinoma

Mononuclear cell leukaemia

Mesothelioma

8

0

0

7

13

7

3

9

42

1

1

6

0

7

2

0

7

10

4

4

8

44

1

3

8

2

3

1

0

6

18

9

1

3

45

1

1

11

3

2*

9**

1

2

6

5

2

4

46

3

0

8

0

p<0.01

p < 

0.05

p < 

0.01

Number of male rats with specified tumors
Grades: 1, Slight; 2, Moderate; 3, Marked; 4, Severe

Japanese Bioassay Research Center (2010b)

15

ETBE: Incidences and Grades of Selected Liver Pathology

2-year Inhalation with F344 Rats

Exposure concentration (ppm) 0 500 1500 5000

No. animals/group 50 50 49 50

Sex Liver lesion

Male

Clear foci 5 (1) 0 0 4 (1)

Acidophil foci 31 (1) 28 (1) 36 (1) 30 (1)

9 (2)

Basophil foci 18 (1) 10 (1) 13 (1) 31 (1)

2 (2)

Female

Clear foci 2 (1) 0 0 3 (1)

Acidophil foci 2 (1) 1 (1) 4 (1) 2 (1)

Basophil foci 32 (1)

4 (2)

24 (1)

7 (2)

27 (1)

5 (2)

23 (1)

5 (2)

Number of rats with specified liver lesions
ETBE: Chronic Oral Gavage Bioassay, Female SpragueDawley Rats (2-year dose administration; held till death)

Ramazzini Foundation; Maltoni et al. (1999)

16

Dose (mg/Kg bw/day, 4 d/wk) 0 250 1000

Dose (mg/Kg bw/day, 7 d/wk basis) 0 143 571

No. animals/group 60 60 60

Organ Tumour diagnosis

Mammary gl.

Pancreas

Pituitary

Thyroid

Adrenal

Spleen

Fibroma and fibroadenoma

Islet cell adenoma

Adenoma

C-cell adenoma

Pheochromocytoma

Mononuclear cell leukaemia

33

0

24

2

29

3

30

1

24

6

27

6

22

1

22

3

26

5

Number of female rats with specified tumors
Ramazzini Foundation; Maltoni et al. (1999)

ETBE: Chronic Oral Gavage Bioassay, Male Sprague-Dawley

Rats (2 year dose administration; held till death)

17

Number of rats with specified tumors

Dose (mg/Kg bw/day, 4 d/wk) 0 250 1000

Dose (mg/Kg bw/day, 7 d/wk basis) 0 143 571

No. animals/group 60 60 60

Organ Tumour diagnosis

Subcutis

Pancreas

Pituitary

Thyroid

Adrenal

Testis

Spleen

Fibroma

Islet cell adenoma

Adenoma

C-cell adenoma

Pheochromocytoma

Leydig cell tumour

Mononuclear cell leukaemia

0

1

28

4

15

0

3

0

3

29

1

15

1

8

1

2

23

1

6

0

6
Incidences of Uterine Proliferating Lesions

Comparison of RI Re-Evaluation and PWG Consensus

18

Maltoni et al., 1999 vs. Malarkey et al., 2011* 

Dose (mg/Kg bw/day, 4 d/wk) 0 250 1000

Dose (mg/Kg bw/day, 7 d/wk basis) 0 143 571

No. animals/group 60 60 60

Counts reported in ETBE evidence Table 2-10 (derived from Maltoni et al., 1999)

Polyp 14 11 14

Malignancies 2 10* 2

Counts by Ramazzini Institute (SD) & NIEHS Pathology Working Group (PWG)*

SD PWG SD PWG SD PWG

Metaplasia, Squamous 3 3 3 6 4 5

Stromal Polyp 21 21 11 12 14 14

Carcinoma 1 0 1 1 0 0

Leiomyoma 0 0 0 0 2 3

Leiomyosarcoma 1 1 2 0 0 0

Schwannoma, Malignant 0 0 6 7 2 2

Histiocytic Sarcoma 1 1 0 0 1 1
Fisher’s exact test *p < 0.05 in two-tailed test, *a NS in two-tailed test

Japanese Bioassay Research Center (2010a); Suzuki et al (2012)

19

ETBE: Chronic Drinking Water Bioassay (104 weeks) 

Male F344/DuCrlCrl Rats 

Exposure concentration (ppm) 0 625 2500 10000

Calculated average dose (mg/kg bw/day) 0 28 121 542

No. animals/group 50 50 50 50

Organ Tumour diagnosis

Subcutis

Pancreas

Pituitary

Thyroid

Adrenal

Testis

Spleen

Fibroma

Islet cell adenoma

Adenoma

C-cell adenoma

Pheochromocytoma

Leydig cell tumour

Mononuclear cell leukaemia

6

6

25

13

11

29

3

2

7

13*

5*

6

32

4

2

4

15*a

11

11

37

7

2

2

16

9

10

34

7

Number of male rats with specified tumors
Japanese Bioassay Research Center (2010a); Suzuki et al (2012)

ETBE: Chronic Drinking Water Bioassay (104 weeks) 

Female F344/DuCrlCrl Rats 

20

Exposure concentration (ppm) 0 625 2500 10000

Calculated average dose (mg/kg bw/day) 0 46 171 560

No. animals/group 50 50 50 50

Organ Tumour diagnosis

Pituitary

Thyroid

Adrenal

Uterus

Mammary

Spleen

Adenoma

C-cell adenoma

Pheochromocytoma

Endometrial stromal polyp

Fibroadenoma

Mononuclear cell leukaemia

15

8

1

6

6

6

14

8

0

9

4

7

13

6

3

3

12

9

15

13

2

7

7

2

Number of female rats with specified tumors
ETBE: Initiation – Promotion Study in Rats

Hagiwara et al. (2011) & JPEC (2008d)

21

• The carcinogenic promoting ability of ETBE (4-wk DMBDD initiation 

and 23-wk ETBE promotion) was evaluated in male rats; ETBE was 

shown to promote thyroid, colon, and liver tumors in DMBDD initiated rats.

• IARC (1999); consensus statement on the use of multiple- and 

single- organ models; “When data are available only on promoting 

activity, the evidence is suggestive of carcinogenicity in rodents, but the 

information should be evaluated in conjunction with other data on 

biological effects of the agent, such as genotoxicity, initiating activity, and 

cell proliferation.” 

• Two-stage models of carcinogenesis in rats demonstrated promoting 

activity of ETBE in liver only; These models have utility in the absence of 

full carcinogenicity tests, and can provide support for results from twoyear bioassays; (DIMS, 2008a; DIMS, 2008b; DIMS, 2010a; DIMS, 2010b).

Therefore, the use of these data on the Exposure-Response Array is 

questioned.
ETBE: MOA for Hepatotumorigenicity in Rats

22

• Recent publication to be included:

Kakehashi et al., 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.

• Oral gavage of ETBE (0, 150 or 1000 mg/kg) for one or twoweeks compared to phenobarbital (500 ppm in diet).

• Conclusion: ETBE MOA of hepatotumorigenicity is similar to 

that of PB except for activation of PPARs. The proposed 

mechanism is related to activation of CAR and PXR through 

oxidative stress, which leads to a sequence of events resulting 

in regenerative cell proliferation. It is unlikely that this MOA has 

human relevance.
Topics for Comments on IRIS Materials for TBA

23

TBA (tert-Butanol)

• Mutation Assays

• Genetic Toxicology Assays

• Cancer Endpoints
TBA: Mutation Assays with Salmonella typhimurium

24

Strains used Activation Dose Reference

S. typhimurium TA 1535, 

TA98, TA100

+/- liver S9 

(Rat)

7800 μg/plate

No response

ARCO (1994) 

Not included by IRIS [or same as 

EG&G Mason, 1981? Included]

S. typhimurium TA1535, 

TA1537, TA98, TA100

+/- liver S9 

(Rat and Syrian

Hamster)

10000 μg/plate

No response

Zeiger et al. (1987) 

Included

S. typhimurium TA102 + liver S9 

(Rat) 

2250 μg/plate

Significant

Williams-Hill et al. (1999) 

Included. Single test 

S. typhimurium TA102 

and 

+/- liver S9 

(Rat) 

5000 μg/plate

No response

McGregor et al. (2005) 

Requires editing 

Two laboratories. Water and 

DMSO solvents. Independent 

repetition
Target cells Activation Dose/Respons

e

Reference

Neurospora crassa ade-3A

locus, reverse mutation

None, but cells 

competent

84% mortality

No response Dickey et al. (1949)

Saccharomyces cerevisiae, 

“petite” mitochondrial 

mutations

None, but cells 

competent

4% TBA, 3 days 

Significant Jimenez et al. (1988)

Human leukaemia (HL-60)

Comet assay

None, but cells 

competent

1 mM, 1hr

Significant Tang et al. (1997) 

Rat-1 fibroblasts 

Comet assay None

0.44 mM, 

0.5-12hr 

Significant 

Sgambato et al. (2009)

Oxidative damage not 

listed in IRIS [8OHdGua]

TBA: In vitro Genotoxicity and Mutation Assays with 

Eukaryotic Cells 

25
NR = No response; Sig = statistically significant; Equiv. = Equivocal

TBA: In Vitro Genotoxicity and Mutation Assays with 

Mammalian Cells 

Target cells Activation Dose/Response Reference

Mouse lymphoma L5178Y

cell tk locus assay

+/- liver S9

(Rat)

5000 μg/mL

+S9: 2 NR

-S9: 1 NR 1 wk (Sig)

McGregor et al. 

(1988)

Mouse lymphoma L5178Y

cell tk locus assay

+/- liver S9

(Rat)

17000 μg/mL

NR

EG&G Mason 

(1981);

ARCO (1994)

Sister-chromatid 

exchange, CHO cells

+/- liver S9

(Rat)

+S9: 15600 μg/mL (Sig)

-S9: 7800 μg/mL (Sig) ARCO (1994b)

Sister-chromatid 

exchange, CHO cells

+/- liver S9

(Rat)

5000 μg/mL

NR NTP (1995)

Chromosomal aberrations, 

CHO cells

+/- liver S9

(Rat)

5000 μg/mL

+S9: 1 NR, 1 Equiv.

-S9: 2 NR tests

NTP (1995)

26
TBA: Micronucleus Induction Assay in Peripheral 

Blood Cells

27

Study NTP (1995)

Species Mice (male and female), 10/sex/group

Route Drinking water

Doses

3,000-40,000 ppm

(Highest exposure likely 4,000-6,000 mg/kg 

bw/day)

Duration 13 weeks

Results

Micronucleated normocytes

Males

Control proportion: 0.09

High dose proportion: 0.06

Females

Control proportion: 0.06

High dose proportion: 0.07

Conclusions No response 

No bone marrow toxicity
TBA: In vivo “Adducts” of TBA in Mouse DNA; 

Study Reliability Questioned

28

Reasons for judging the study not reliable

• Study is one of a series from the same laboratory in which Accelerator 

Mass Spectrometry (AMS) is used on DNA from mice dosed with:

– tertiary-single 14C-labelled TBA or

– methoxy14C-labelled MTBE (Yuan et al., 2007); 

– dual tertiary- and methoxy14C-labelled MTBE (Du et al., 2005); and

–

14C-labelled formic acid (Wang et al., 2004). 

• DNA is reduced to elemental carbon, after which the radioactivity is 

measured, but it not known where the radioactivity originated without prior 

analytical steps – which were not investigated.

• Without further analysis, 14C measured could be:

– an adduct;

– metabolically incorporated (e.g., from acetone); or

– a contaminant.

Yuan et al. (2007); if EPA-IRIS agrees, then this study should not 

be included in the review. 
Mauthie et al. (1999), p 541

“AMS provides a measure of total radiocarbon present in the samples which may come from 

MeIQx metabolites, DNA adduct or contaminant. Since AMS measures isotopes only and does 

not differentiate between these sources, data are reported as MeIQx equivalents/sample.”

Phillips et al. (2000), p 224

“AMS is a technique that measures isotope ratios only. It provides no information on the nature 

or chemical form of the isotope and thus has the potential to provide false positives in DNA

binding studies where genomic DNA isolates are measured.”

Himmelstein et al. (2009), p 682

“As all information of the chemical structure of the analyte is consequently lost, comparison of 

the chromatographic properties of the analyte with synthetic adducts is essential to ensure that 

the binding being detected is due to DNA adduct formation. In addition, great care has to be 

taken to avoid contamination and to properly subtract background levels of radioactivity.”

Uncertainties identified in other studies using AMS to measure 

DNA adducts.

29

“Adducts” of TBA with Mouse DNA in vivo
Male mice

TBA Dose (mg/Kg bw/day)* 0 535 1035 2065

Adenoma incidence 1/60 28/50 4/59 1/57

Hyperplasia incidence 

(severe grade)

5/60 (1.2) 18/59* (1.6) 15/59* (1.4) 18/57* (2.1)

Female mice

TBA Dose (mg/Kg bw/day) 0 510 1015 2105

Adenoma incidence 2/58 3/60 2/59 9/59

Hyperplasia incidence 

(severe grade)

19/58 (1.8) 28/60 (1.9) 33/59* (1.7) 47/59* (2.2)

Two-Year TBA Drinking-Water Study (NTP, 1995)

TBA: Thyroid Follicular Cell Adenomas and Hyperplasia

US NTP (1995)

30
Male mice

TBA Dose (mg/Kg bw/day) 0 117 879 2269

Adenoma incidence 0/49 0/11 0/19 0/48

Hyperplasia incidence 0/49 0/11 0/19 0/48

Female mice

TBA Dose (mg/Kg bw/day) 0 120 898 2328

Adenoma incidence 0/50 0/11 0/14 0/49

Hyperplasia incidence 1/35 0/11 0/14 0/29

TBA: Thyroid Follicular Cell Adenomas and Hyperplasia

in CD-1 Mice

31

Inhalation Exposure to MTBE; Doses expressed as TBA

derived via Metabolism (Burleigh-Flayer et al., 1992)
TBA: Thyroid Follicular Cell Adenomas and Hyperplasia

US NTP (1995)

32

• Table 2-3, Including only one category for “Follicular cell 

adenoma and carcinoma” is misleading; The only 

difference between “Follicular cell adenoma” and “Follicular cell 

adenoma and carcinoma” is the presence of one carcinoma in 

male mice at the highest dose level. Diagnoses should be 

recorded separately as has been done for rat data.

• Figure 2-3, The legend is divided into non-cancer and 

cancer; Cancer is a malignant condition, so not clear why 

adenoma is listed in this space. Recommend providing 

descriptions for both hyperplasia and adenoma. 
Incidence in Male Rats (Female Rats)

Dose (mg/Kg bw/day) 0 90 200 420

Mineralization 26/50 28/50 35/50 48/50

Linear Mineralization

(Inflammation, suppurative)

0/50

(2/50)

5/50*

(3/50)

24/50*

(13/50*)

46/50*

(15/50*)

Renal tubule hyperplasia 

(extended evaluation) 12/50 16/50 14/50 23/50*

Transitional epithelium 

hyperplasia

25/50

(0/50)

32/50

(0/50)

36/50*

(3/50)

40/50*

(17/50*)

Nephropathy severity 3 (1.6) 3.1 (1.9*) 3.1(2.3*) 3.3*(2.9*)

Two-Year TBA Drinking-Water Study (NTP, 1995)

TBA: Kidney Histopathology in Male F344 Rats

US NTP (1995)

33
Dose (mg/Kg bw/day) 0 90 200 420

Renal Tubule Adenomaa 7/50 7/50 10/50 10/50

Renal Tubule Adenoma 

(multiple)d

1/50 4/50 9/50* 3/50

Renal Tubule Adenoma or 

Carcinomad

8/50 13/50 19/50* 13/50

TBA: Renal Cell Adenomas or Carcinomas in 

Male F344 Rats

Two-Year TBA Drinking-Water Study (NTP, 1995)

34

Dose (mg/Kg bw/day) 0 90 200 420

Renal Tubule Adenomaa 3/50 9/50 9/50 9/50

Renal Tubule Adenoma 

(multiple)d

1/50 3/50 9/50 3/50

Renal Tubule Adenoma or 

Carcinomad

4/50 13/50* 18/50* 12/50*

Re-evaluations (Hard et al., 2011)
MOA Endpoints for Male Rat Specific Renal Tumors;

Information to Include and Clarify in Materials

35

• Recommend extracting data from Williams and Borghoff (2001) into 

the Histopathology section; The study provides exposure response 

induction of measures of 2u-globulin nephropathy which include 

histology changes.

• Recommend extracting data from Hard et al., 2011 into the 

Histopathology section; 

–Dose-related increase in hyaline droplet formation in male rats

–Confirmed the crystal-like form of the hyaline droplets that is 

characteristic of α2u-globulin nephropathy

–Identified the presence of precursors of granular casts at the 

“corticomedullary” junction 

• Recommend extracting additional data from Cirvello et al. (1995); 

Lindamood et al. (1992); and NTP (1995);

–Quantitation of severity of the crystal-like nature of hyaline droplets

–Quantitative cell proliferation
36

• EPA's consideration and application of mechanistic data is 

unclear and inconsistent.

EPA provides inconsistent criteria for mechanistic data, noting that such 

studies were not extracted for evidence tables if "the data in the study 

only included endpoints related to possible mechanisms of toxicity.”

Whereas EPA then states "Supporting data that provide mechanistic 

information for each selected endpoint was also included.” 

• Generally mechanistic data on key health endpoints must be 

included as pertinent to assessment of these endpoints. For 

example:

ETBE induced liver tumors; induction of liver weights, enzymes, etc.

TBA induced renal tumors; mode of action associated with measures of 

a2u-globulin nephropathy; histopathology, cell proliferation, chemical 

binding.

Incorporation of Mechanistic Data
In Summary

37

On behalf of Lyondell Chemical Company

• We appreciate the opportunity to provide both written and 

oral comments on EPA’s Preliminary Materials for IRIS 

Toxicological review for ETBE and TBA.

• Based on the comments presented today and the detailed 

comments submitted by Lyondell Chemical Company, we 

feel that there are significant deficiencies and 

inconsistencies in these materials and encourage EPA to 

address these deficiencies before proceeding with 

preparation of the draft assessment.