# Commen **ts on ETBE and**  **tert -**  **But anol**  **(TB A)** 

**Pr eliminary**  **Materials fo**  **r IRIS T ox**  **icological Re** **view** Genotoxicity

, Mut ag enicity

, and Cancer Endpoin ts 

# On **Behalf of**  **Ly ondell**  **Chemical Compan** **y**

**Pr esent**  **ed by**  **Susan Bor ghoff** 

**, PhD , D** **AB T**  **fo r**  **Douglas McGr eg**  **or**

**, PhD** 
**December 12, 2013**

# To **pics for Comments on IRIS**  **Materials**

**for ETBE & TBA** 

**2** 

## ETBE (Ethy **l tert**  **- buty**  **l Ether)** 

• Mutation Assays

• Genetic To xicology Assays

• Cancer Endpoints **TBA (**  **tert -**  **Butanol )** 

• Mutation Assays

• Genetic To xicology Assays

• Cancer Endpoints

### No significant **effects on**  **mutant numbers in any**  **study**
**Strains Used Ac** **tiv ation**  **Highest Ineffectiv** **e Dose**  **Reference**
TA 1535, TA 1537, TA 1538, TA 98 +/- liver S9 (Rat) 500 μg/plate Institut Pasteur de Lille (1992) (Not included) TA 1535, TA 97, TA 98, TA 100 +/- liver S9

(Rat & Syrian Hamster ) 10,000

μg/plate Zeiger et al. (1992) TA 1535, TA 1537, TA 1538, TA 98, TA 100 +/- liver S9

(Rat) 5,000

μg/plate Pharmakon Europe (1994)

(Not included ) 

# ETBE: Mutation **Assay s**  **with Salmonella** 

**typhimurium**

**3**

### No **significant effects on mutation**  **frequency in either**  **study** 

# ETBE: **Mutation Assay** **s with Mammalian**  **Cells**

**In vitro As**  **say Ac** **tiv ation**  **Highest Dose**  **Reference** Gene mutation, CHO - at hprt locus +/- liver S9 (Rat)

Two acceptable independent assays 5,000 μg/mL BBRC (1995c); Ve rgnes & Kubena (1995a)

Chromosomal aberrations - CHO +/- liver S9 (Rat)

Duplicate cultures in a non independent assay 5,000 μg/mL BBRC (1995b); Ve rgnes (1995)

**4**

### No significant **increases in micronucleated**  **c ells; 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);

Ve rgnes & 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 Drinking water 10,000 ppm (w/w),

13 wks Japan Bioassay Research Center (2007c) Male and female Fischer 344/ DuCrj rat Inhalation 5,000 ppm 6h/d, 5d/wk , 13 wks Japan Bioassay Research Center (2007d) 

# ETBE: **In V**

**ivo Micronucleus**  **Assay s**  **with Rodent Bone**  **- Marrow**  **Cells**

**5**Suggested Klimisch Score - 2 (reliable wi th restrictions) 

### We **ng et al**  **., 2012 (Study to**  **add to Section 2.8,**  **Ta ble**  **2 -**  **11 )** 



## ETBE: DNA

**Damage to Liver Cells**  **of C57BL/6**  **Wi ld**  **- ty**  **pe and**  **Aldh2 -**  **/ -**  **Mice**

**6** 

### Fold **- increases**  **over the**  **control proportions**  **of comets** 
**As say**  **Dose (ppm)**  **Statistically significant**  **–**

**fold increase in comets Male**  **Female +/+**  **- /**  **- +/+**  **- /**  **- Comet**  **assay**

**i n**  **liv er**  **cells** 500 1.76 1750 1.83 5000 1.93 1.99 1.84 **net hOGG1 Comet**  **assay in liv** **er cells**  500 2.71 1750 2.51 5000 2.99 3.44 3.15 **8 -**  **OH -**  **dG residues**  **in liv** **er DNA**  500 1.81 1750 1.74 5000 1.31 1.93 1.61

## ETBE: Comet **Assay on**  **Peripheral Leucocy tes**  **of C57BL/6**  **Wi ld**  **- ty**  **pe and**  **Aldh2 -**  **/ -**  **M ice** 

**7** 

### Statistically

**significant fold**  **- increases**  **in comets (ov** **er control) in**  **peripheral leukocy** **tes** 

### We **ng et al., 201**  **1; 2013 ( Studies**  **to add**  **to Section 2.8, T able 2**  **- 11**  **)**

**As say**  **Klimisch Score**  **Dose**

**(ppm) Statistically**  **significant**

**fold increase in comets**  **Male Female**  **+/+ -** **/ -**  **+/+ -** **/ -**  **Comet assay**  **( Weng**  **et al. 201**  **1)** 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 r** **eference w ith**  **suggested Klimisch**  **Score of 3 (not reliable)  Borderline significant (p=0.052)

** 8** 

## ETBE: Micronucleus **Assay on**  **Circulating Reticulocy** 

**tes in C57BL/6 w**  **ild -** **ty pe**  **and Aldh2**  **- /** **- mice** 

**As say**  **Dose (ppm)**  **Statistically significant fold increase** 

**in micronucleated**  **cells Male**  **Female +/+**  **- /**  **- +/+**  **- /**  **- Micronucleus**  **assay**

**i n**  **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 (**We ng**  **et al., 2013 )** 

**9 Ac**  **etaldehyde TBA**  **2 -**  **Meth yl**  **- 1,2**  **- Pr** **opanediol Male**  **Female Male**  **Female Male**  **Female +/+**  **- /**  **- +/+**  **- /**  **- +/+**  **- /**  **- +/+**  **- /**  **- +/+**  **- /**  **- +/+**  **- /**  **- Cmax**  **( μ**  **M)** 81 495 107 677 270 1426 777 2206 72 108 67 356 **Fold increase in**  **AU C**  **( Aldh2**  **- /**  **- ov** **er +/+)**  2.69 - fold 2.52 - fold 5.78 - fold 3.01 - fold 1.76 - fold 4.95 - fold

# Metabolic Degradation of **Alky l Ethers to TBA** 

CH3 H3C C OCnH2n+1

CH3 P450

- RCHO CH3

H3C C OH

CH3 CH3 H3C C O

- Glucuronide

CH3

CH 2 OH H 3 C C OH

CH 3 CHO H 3 C C OH

CH 3 **TBA TBA**  **- Glucuronide** 

COO  ̄ H 3 C C OH

CH 3 **2 -**  **Methyl -**  **1,2 -** **propanediol 2**  **- Hydroxyisobutyr** **ate**
**Acetone MTBE**  **or ETBE**

**10 Parameter**  **Default Va**  **lue 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**  **we ek**  **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 V**  **apour for 104**  **We eks (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 V**  **apour for 104**  **We eks (JBRC, 2010b**  **)**

**12 Parameter**  **Default Va**  **lue Reference**  A: Alveolar ventilation rate (minute volume) V m = 0.499
. BW 0.809 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**  **we ek**  **Calculated net**  **ETBE uptake rate Males:**  **981 mg/Kg**  **bw /day**  **Females: 11**  **45 mg/Kg bw** **/day Exposure**  **concentration (ppm) 0**  **500 1500**  **5000 Stats.**  **An alysis**  **No. animals/group 50**  **50 50**  **50 Peto**  **Coch /** **A r**  **Organ Tu**  **mour diagnosis** Pituitary Thy roid 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**R esearch**  **Center (2010b)**  **Number of female rats**  **wi th**  **specified tumors** 

**13** 

## ETBE: Chronic Inhalation Bioassay **(104 w eeks)**  **Female F344/ DuCrlCrl**  **Rats**

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



### Japanese Bioassay**R esearch**  **Center (2010b)**  

## ETBE: Chronic Inhalation Bioassay **(104 w eeks)**  **Male F344/ DuCrlCrl**  **Rats**

**14 Exposure**  **concentration (ppm) 0**  **500 1500**  **5000 Stats.**  **An alysis**  **No. animals/group 50**  **50 49**  **50 Peto**  **Coch /** **A r**  **Organ Tu**  **mour diagnosis** Subcutis Liver Pancreas Pituitary Thy roid Adrenal Te stis 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 ** wi** **th specified**  **tumors Grades**  : 1, Slight; 2, Moderate; 3, Marked; 4, Severe



### Japanese Bioassay**R esearch**  **Center (2010b)** 

**15** 

## ETBE: Incidences and Grades of Selected **Liver Pathology**

**2 -**  **ye ar**  **Inhalation w ith F344**  **Rats**

**Exposure concentration (ppm)**  **0 500**  **1500 5000** 

**No. animals/group 50**  **50 49**  **50 Sex**  **Liv er**  **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 **wi th**  **specified liver lesions** 

## ETBE: Chronic Oral Gavage Bioassay **, Female Sprague**   ** Daw** **ley Rats (** **2 -** 

**ye ar**  **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 Tu**  **mour diagnosis** Mammary gl. Pancreas Pituitary Thy roid 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 wi**  **th specified**  **tumors**

### Ramazzini **Foundation; Maltoni**  **et al. (1999 )**  

## ETBE: Chronic Oral Gavage Bioassay **, M ale**  **Sprague -**  **Daw ley**  **Rats (2 y ear dose administration; held till death)** 

**17** 

### Number of rats **wi th**  **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 Tu**  **mour diagnosis** Subcutis Pancreas Pituitary Thy roid Adrenal Te stis 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., 201**  **1** ***** **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 ev idence**  **Ta ble 2**  **- 10**  **(deriv ed**  **from Maltoni**  **et al., 1999)** Polyp 14 11 14 Malignancies 2 10* 2 **Counts by Ramazzini**  **Institute (SD) & NIEHS Pathology W orking 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 1Fisher ’s e xact test * p < 0.05 in two
- tailed test, * a NS in two
- tailed test 

### Japanese Bioassay**R esearch**  **Center (2010a); Suzuki et al (2012)** 

**19** 

## ETBE: Chronic Drinking W **ater Bioassay**

**(104 w eeks)**  **Male F344/ DuCrlCrl**  **Rats**

**Exposure concentration (ppm)**  **0 625**  **2500 10000**  **Calculated av erage**  **dose (mg/kg**  **bw /** **day) 0**  **28 121**  **542 No. animals/group**  **50 50**  **50 50**  **Organ Tu**  **mour diagnosis** Subcutis Pancreas Pituitary Thy roid Adrenal Te stis 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 **wi th**  **specified tumors** 

### Japanese Bioassay**R esearch**  **Center (2010a); Suzuki et al (2012)**  

## ETBE: Chronic Drinking W **ater Bioassay**

**(104 w eeks)**  **Female F344/ DuCrlCrl**  **Rats**

**20 Exposure**  **concentration (ppm) 0**  **625 2500**  **10000 Calculated av** **erage dose (mg/kg**  **bw /** **day) 0**  **46 171**  **560 No. animals/group**  **50 50**  **50 50**  **Organ Tu**  **mour diagnosis** Pituitary Thy roid 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 wi**  **th specified**  **tumors**

## ETBE: Initiation **– Promotion Study**  **in Rats Hagiw** **ara et al. (201**  **1) & 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 ef**

**fects of the agent, such as genotoxicity , initiating activity** 

**, and cell proliferation.”** 



### • Tw o - 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 two**   ** year 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 PP AR signaling pathways. Toxicol

. Applied Pharmacol. 273 (2013) 390- 400.



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

• Conclusion: ETBE MOA of hepatotumorigenicity is similar to that of PB except for activation of PP ARs. 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.

# To **pics for Comments on IRIS**  **Materials for TBA**

**23** 

## TBA

**( tert**  **- Butanol**  **)**

• Mutation Assays

• Genetic To xicology Assays

• Cancer Endpoints

## TBA: Mutation **Assay s with**  **Salmonella typhimurium** 

**24 Strains**  **used Ac** **tiv ation**  **Dose Reference**  S. typhimurium TA 1535, TA 98, T A100 +/- liver S9 (Rat) 7800 μg/plate

No response ARCO ( 1994)

Not included by IRIS [or same as EG&G Mason, 1981? Included] S. typhimurium TA 1535 , TA 1537, T

A98 , T A100 +/- liver S9

(Rat and Syrian Hamster) 10000 μg/plate

No response Zeiger et al. ( 1987)

Included S. typhimurium TA 102 + liver S9 (Rat) 2250 μg/plate

Significant Williams - Hill et al. (1999)

Included. Single test S. typhimurium TA 102 and +/- liver S9 (Rat) 5000 μg/plate

No response McGregor et al. ( 2005)

Requires editing Two laboratories. Water and DMSO solvents. Independent repetition**Ta rget cells**  **Ac tiv** **ation 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 Ta ng 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 [8OH dGua] 

## TBA: **In vitro Genotoxicity**  **and Mutation Assay** **s with**

**Eukary otic Cells** 

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

## TBA: **In V**

**itro Genotoxicity**  **and Mutation Assay** **s with Mammalian Cells**  **Ta rget cells**  **Ac tiv** **ation 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 wa ter **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

t oxicity

## TBA **: In vivo**  **“Adducts” of TBA in Mouse DNA;**  **Study R** **eliability**

**Q uestioned** 

**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 14 

### C - labelled TBA or

– methoxy 14 

### C - labelled MTBE (Yuan et al., 2007);

– dual tertiary

- and methoxy 14 

### C - labelled MTBE (Du et al., 2005 ); and

– 14 

### C - 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, 14 

### C measured could be:

– an adduct;

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

– a contaminant. 

### Yu an et al. (2007); if EP A- 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 dif ferentiate 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 D**  **ose (mg/Kg**  **bw /**  **day)*** **0 535**  **1035 2065**  **Ad enoma**  **incidence** 1/60 28/50 4/59 1/57 **Hyperplasia incidence**  **(sev ere**  **grade)** 5/60 (1.2) 18/59* (1.6) 15/59* (1.4) 18/57* (2.1) 

### Female mice **TBA D**  **ose (mg/Kg bw** **/day) 0**  **510 1015**  **2105 Ad**  **enoma incidence**  2/58 3/60 2/59 9/59 **Hyperplasia incidence**  **(sev ere**  **grade)** 19/58 (1.8) 28/60 (1.9) 33/59* (1.7) 47/59* (2.2) 

### Tw **o -** **Ye ar TBA** 

**D rinking**  **- Wa**  **ter Study**  **(NTP , 1995)**  

## TBA: Thy **roid Follicular Cell**  **Adenomas and Hy perplasia** 

**US NTP**  **(1995)**

**30**

### Male mice **TBA D**  **ose (mg/Kg**  **bw /**  **day) 0**  **11 7**  **879 2269**  **Ad enoma**  **incidence** 0/49 0/1 1 0/19 0/48 **Hyperplasia incidence**  0/49 0/1 1 0/19 0/48 

### Female mice **TBA D**  **ose (mg/Kg bw** **/day) 0**  **120 898**  **2328 Ad**  **enoma incidence**  0/50 0/1 1 0/14 0/49 **Hyperplasia incidence**  1/35 0/1 1 0/14 0/29 

## TBA: Thy **roid Follicular Cell**  **Adenomas and Hy perplasia** 

**in CD -**  **1 Mice** 

**31** 

### Inhalation **Exposure to MTBE;**  **Doses expressed as TBA**  **derived via Metabolism**  **( Burleigh**  **- Flayer**  **et al ., 1992)** 

## TBA: Thy **roid Follicular Cell**  **Adenomas and Hy perplasia**  **US NTP (1995)** 

**32** 

## • Ta ble 2 - 3, Including only one category for “ Follicular cell adenoma and carcinoma” is misleading; **The only dif**  **ference between**  **“**

**Follicular cell adenoma”**  **and “Follicular cell**  **adenoma and carcinoma” is**  **the presence of one carcinoma**  **in male mice**  **at the highest**  **dose level.**  **D iagnoses**  **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,**  **suppurativ e**  **)** 0/50 (2/50) 5/50*

(3/50) 24/50*

(13/50*) 46/50*

(15/50*) **Renal tubule hyperplasia**  **(extended ev aluation)**  12/50 16/50 14/50 23/50* **Tr ansitional epithelium**  **hyperplasia** 25/50 (0/50) 32/50

(0/50) 36/50*

(3/50) 40/50*

(17/50*) **Nephropathy sev erity**  3 (1.6) 3.1 (1.9*) 3.1(2.3*) 3.3*(2.9*)



## Tw **o -** **Ye ar TB** **A Drinking -**  **Wa te**  **r Study (NTP , 1995)**  

## TBA: Kidney

**H istopathology** 

**i n Male F344 Rats**  **US NTP**  **(1995)**

**33 Dose (mg/Kg**  **bw /**  **day )**  **0 90**  **200 420**  **Renal Tu**  **bule Ad**  **enoma a**  7/50 7/50 10/50 10/50 **Renal T ubule**  **Ad enoma**  **(multiple) d**  1/50 4/50 9/50* 3/50 **Renal T ubule**  **Ad enoma**  **or Carcinoma**  **d** 8/50 13/50 19/50* 13/50 

# TBA: **Renal Cell**  **Adenomas or Carcinomas in** 

**Male F344 Rats Tw**  **o -** **Ye ar**  **TBA**

**Drinking -** **Wa ter**  **Study (NTP**  **, 1995)**

**34 Dose (mg/Kg**  **bw /**  **day )**  **0 90**  **200 420**  **Renal Tu**  **bule Ad**  **enoma a**  3/50 9/50 9/50 9/50 **Renal T ubule**  **Ad enoma**  **(multiple) d**  1/50 3/50 9/50 3/50 **Renal T ubule**  **Ad enoma**  **or Carcinoma**  **d** 4/50 13/50* 18/50* 12/50* 

### Re **- evaluations**  **(Hard et al., 201**  **1)**

## MOA

**Endpoints for Male Rat Specific Renal T umors;**  **Information to Include and Clarify i**  **n Materials**

**35** 

### • Recommend extracting data from Williams and Borghof f (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., 201 1 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** 

### • **EP A's**  **consideration and application**  **of mechanistic data is**  **unclear and inconsistent.**  EP A 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 EP

A then states " Supporting data that provide mechanistic information for each selected endpoint was also included.”



### • **Generally mechanistic data on key** **h ealth 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 L

**yo ndell Chemical Company** 

• We appreciate the opportunity to provide both written and oral comments on EP

A’ s Preliminary Materials for IRIS To xicological 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 EP A to address these deficiencies before proceeding with preparation of the draft assessment.