1

Data Selection for Calculating

the Acceptable Intake of

Ethylene Oxide (CAS# 75-21-8)

Zhanna Sobol

May 2, 2018

Genetic Toxicology Association Annual Meeting – PDE Workshop

2

Acknowledgments

• Pamela Heard

• Michelle Kenyon

• Krista Dobo

• Will Drewe

• Georgia Clarke

• Liz Lepley

3

Outline

• Background on Ethylene Oxide

• Review study selection guidance in ICH M7 Addendum

• Describe Available carcinogenicity studies for Ethylene Oxide

• Discussion Question 1 – What study would you choose?

• Describe Pfizer’s selection of best study for AI calculation

• Additional considerations for AI

• Discussion Question 2 – Should additional considerations be

factored into AI calculation?

4

Ethylene Oxide is a Flammable Gas

• Potential for Human Exposure

– major industrial chemical used for synthesis of numerous

products such as ethylene glycol (antifreeze and polyester)

– Used as cold sterilizing agent for food and medical equipment

– Fumigant and fungicide

– Humans may be exposed through environmental contamination

of air/water or in the workplace where ethylene oxide is utilized

– Amounts in food are low as ethylene oxide evaporates or breaks

down shortly after fumigation

• Regulatory and/or Published Limits

– OSHA PEL (permissible exposure limit) = 1 ppm (averaged over

an 8-hour workshift) = 1.8 mg/m3 = 18 mg/day

5

Ethylene Oxide is Genotoxic

• Mechanism of Genotoxicity – direct-acting alkylating agent (epoxide)

• In vitro

– positive for induction of reverse mutations in strains TA98, TA100, TA102, TA1535, TA1537 and in E. coli with and without metabolic activation

– sister chromatid exchanges in human lymphocytes

– unscheduled DNA synthesis in mouse germ cells

– forward mutations (HPRT assay) in Chinese Hamster Ovary cells

• In vivo

– translocations in mice

– micronuclei in mouse bone marrow cells (NTP, 1987)

– chromosomal aberrations in bone marrow of rats (NTP, 1987)

– In workers caused chromosomal aberrations and sister chromatid exchange in peripheral lymphocytes and micronuclei in bone-marrow cells (ATSDR, 1990)

6

Ethylene Oxide is Carcinogenic

• Linear extrapolation from a

carcinogenicity study is the

appropriate approach for

calculating a compound-

specific limit

• Which study to choose?

7

ICH M7 Addendum Guidance for Study Selection

• Carcinogenicity studies are not considered robust when:

– < 50 animals per dose per sex

– < 3 dose levels

– Lack of concurrent controls

– Intermittent dosing (< 5 days per week)

– Dosing for less than lifetime

• Exception

–

8

Additional Considerations

• Selection of Tumor and Site

– Lowest TD50 for organ site, animal species and sex from the most robust study

– Do not use data compiled from “all Tumor Bearing Animals” (TBA)

– mixed tumor types (e.g.,adenomas and carcinomas) in one tissue (e.g., liver) can be used

– lowest TD50 is considered a more conservative estimate than harmonic mean

• Route of administration

– If tumor sites did not appear to be route-specific, the TD50 from the route with the lowest TD50 value should be used

– If tumors are site-specific without distal tumors and TD50 is lower than for other routes, warrants separate AI

• Human relevance of rodent carcinogenicity study findings

– Forestomach tumors, tumors at site of contact only without distal tumors

9

Which TD50 Should I choose?

10

Carcinogenicity Studies with Ethylene Oxide in CPDB

++Taken from the CPDB. The TD50 values represent the TD50 from the most sensitive tumor site, species, and sex for each study.

TD50 (mg/kg/day)

Rat Mouse

21.3 63.7

Study Animals/

dose group

Duration/

Exposure

Controls Doses

(adjusted doses)+Most sensitive tumor

site/sex

TD50(mg/kg/d)++

NTP 1987

(TR326)

50/sex/group

B6C3F1 mice

6h/day, 5

days/wk,

102 weeks,

inhalation

50 50 and 100 ppm

(24.7 and 49.4

mg/kg/day)

Lung multiple tumor

type/female

61.8

Lynch et al.,

1984

80/male/

Fischer 344

rats, pooled

controls*

7h/day, 5

days/wk,

104 weeks,

inhalation

80 50 and 100 ppm

(12.8 and 25.6

mg/kg/day)

Peritoneum

Mesothelioma/male

30.8

Snellings et

al., 1984

120/male

and female/

Fischer 344

rats**

6h/day, 5

days/wk,

104 weeks,

inhalation

120 10, 33, 100 ppm

(2.2, 7.2, 21.9

mg/kg/day)

Brain multiple tumor

type/male

70.7

Dunkelberg

1982

50/female/Sp

rague-

Dawley rats

2 days/wk,

150 weeks,

gavage

50 7.5 and 30 mg/kg

(2.14 and 8.57

mg/kg/day)

primarily

forestomach tumors

without distal site

tumors

7.43

11

Study Animals/

dose group

Duration/

Exposure

Controls Doses

(adjusted doses)+Most sensitive tumor

site/sex

TD50(mg/kg/d)++

NTP 1987

(TR326)

50/sex/group

B6C3F1 mice

6h/day, 5

days/wk,

102 weeks,

inhalation

50 50 and 100 ppm

(24.7 and 49.4

mg/kg/day)

Lung multiple tumor

type/female

61.8

Lynch et al.,

1984

80/male/

Fischer 344

rats, pooled

controls*

7h/day, 5

days/wk,

104 weeks,

inhalation

80 50 and 100 ppm

(12.8 and 25.6

mg/kg/day)

Peritoneum

Mesothelioma/male

30.8

Snellings et

al., 1984

120/male

and female/

Fischer 344

rats**

6h/day, 5

days/wk,

104 weeks,

inhalation

120 10, 33, 100 ppm

(2.2, 7.2, 21.9

mg/kg/day)

Brain multiple tumor

type/male

70.7

Dunkelberg

1982

50/female/Sp

rague-

Dawley rats

2 days/wk,

150 weeks,

gavage

50 7.5 and 30 mg/kg

(2.14 and 8.57

mg/kg/day)

primarily

forestomach tumors

without distal site

tumors

7.43

Workshop Question 1: Which Value would you choose?

++Taken from the CPDB. The TD50 values represent the TD50 from the most sensitive tumor site, species, and sex for each study.

TD50 (mg/kg/day)

Rat Mouse

21.3 63.7A.

B.

C.

D.

E.

12

Our Approach

13

Carcinogenicity Studies with Ethylene Oxide in CPDB

++Taken from the CPDB. The TD50 values represent the TD50 from the most sensitive tumor site, species, and sex for each study.

TD50 (mg/kg/day)

Rat Mouse

21.3 63.7

Study Animals/

dose group

Duration/

Exposure

Controls Doses

(adjusted doses)+Most sensitive tumor

site/sex

TD50(mg/kg/d)++

NTP 1987

(TR326)

50/sex/group

B6C3F1 mice

6h/day, 5

days/wk,

102 weeks,

inhalation

50 50 and 100 ppm

(24.7 and 49.4

mg/kg/day)

Lung multiple tumor

type/female

61.8

Lynch et al.,

1984

80/male/

Fischer 344

rats, pooled

controls*

7h/day, 5

days/wk,

104 weeks,

inhalation

80 50 and 100 ppm

(12.8 and 25.6

mg/kg/day)

Peritoneum

Mesothelioma/male

30.8

Snellings et

al., 1984

120/male

and female/

Fischer 344

rats**

6h/day, 5

days/wk,

104 weeks,

inhalation

120 10, 33, 100 ppm

(2.2, 7.2, 21.9

mg/kg/day)

Brain multiple tumor

type/male

70.7

Dunkelberg

1982

50/female/Sp

rague-

Dawley rats

2 days/wk,

150 weeks,

gavage

50 7.5 and 30 mg/kg

(2.14 and 8.57

mg/kg/day)

primarily

forestomach tumors

without distal site

tumors

7.43

Not relevant

14

Carcinogenicity Studies with Ethylene Oxide in CPDB

++Taken from the CPDB. The TD50 values represent the TD50 from the most sensitive tumor site, species, and sex for each study.

TD50 (mg/kg/day)

Rat Mouse

21.3 63.7

Study Animals/

dose group

Duration/

Exposure

Controls Doses

(adjusted doses)+Most sensitive tumor

site/sex

TD50(mg/kg/d)++

NTP 1987

(TR326)

50/sex/group

B6C3F1 mice

6h/day, 5

days/wk,

102 weeks,

inhalation

50 50 and 100 ppm

(24.7 and 49.4

mg/kg/day)

Lung multiple tumor

type/female

61.8

Lynch et al.,

1984

80/male/

Fischer 344

rats, pooled

controls*

7h/day, 5

days/wk,

104 weeks,

inhalation

80 50 and 100 ppm

(12.8 and 25.6

mg/kg/day)

Peritoneum

Mesothelioma/male

30.8

Snellings et

al., 1984

120/male

and female/

Fischer 344

rats**

6h/day, 5

days/wk,

104 weeks,

inhalation

120 10, 33, 100 ppm

(2.2, 7.2, 21.9

mg/kg/day)

Brain multiple tumor

type/male

70.7

Dunkelberg

1982

50/female/Sp

rague-

Dawley rats

2 days/wk,

150 weeks,

gavage

50 7.5 and 30 mg/kg

(2.14 and 8.57

mg/kg/day)

primarily

forestomach tumors

without distal site

tumors

7.43

Too high

Too high

15

Study Animals/

dose group

Duration/

Exposure

Controls Doses

(adjusted doses)+Most sensitive tumor

site/sex

TD50(mg/kg/d)++

NTP 1987

(TR326)

50/sex/group

B6C3F1 mice

6h/day, 5

days/wk,

102 weeks,

inhalation

50 50 and 100 ppm

(24.7 and 49.4

mg/kg/day)

Lung multiple tumor

type/female

61.8

Lynch et al.,

1984

80/male/

Fischer 344

rats, pooled

controls*

7h/day, 5

days/wk,

104 weeks,

inhalation

80 50 and 100 ppm

(12.8 and 25.6

mg/kg/day)

Peritoneum

Mesothelioma/male

30.8

Snellings et

al., 1984

120/male

and female/

Fischer 344

rats**

6h/day, 5

days/wk,

104 weeks,

inhalation

120 10, 33, 100 ppm

(2.2, 7.2, 21.9

mg/kg/day)

Brain multiple tumor

type/male

70.7

Dunkelberg

1982

50/female/Sp

rague-

Dawley rats

2 days/wk,

150 weeks,

gavage

50 7.5 and 30 mg/kg

(2.14 and 8.57

mg/kg/day)

primarily

forestomach tumors

without distal site

tumors

7.43

Carcinogenicity Studies with Ethylene Oxide in CPDB

++Taken from the CPDB. The TD50 values represent the TD50 from the most sensitive tumor site, species, and sex for each study.

TD50 (mg/kg/day)

Rat Mouse

21.3 63.7

Included in ICH M7

16

Study Animals/

dose group

Duration/

Exposure

Controls Doses

(adjusted doses)+Most sensitive tumor

site/sex

TD50(mg/kg/d)++

NTP 1987

(TR326)

50/sex/group

B6C3F1 mice

6h/day, 5

days/wk,

102 weeks,

inhalation

50 50 and 100 ppm

(24.7 and 49.4

mg/kg/day)

Lung multiple tumor

type/female

61.8

Lynch et al.,

1984

80/male/

Fischer 344

rats, pooled

controls*

7h/day, 5

days/wk,

104 weeks,

inhalation

80 50 and 100 ppm

(12.8 and 25.6

mg/kg/day)

Peritoneum

Mesothelioma/male

30.8

Snellings et

al., 1984

120/male

and female/

Fischer 344

rats**

6h/day, 5

days/wk,

104 weeks,

inhalation

120 10, 33, 100 ppm

(2.2, 7.2, 21.9

mg/kg/day)

Brain multiple tumor

type/male

70.7

Dunkelberg

1982

50/female/Sp

rague-

Dawley rats

2 days/wk,

150 weeks,

gavage

50 7.5 and 30 mg/kg

(2.14 and 8.57

mg/kg/day)

primarily

forestomach tumors

without distal site

tumors

7.43

Carcinogenicity Studies with Ethylene Oxide in CPDB

++Taken from the CPDB. The TD50 values represent the TD50 from the most sensitive tumor site, species, and sex for each study.

TD50 (mg/kg/day)

Rat Mouse

21.3 63.7

17

AI Calculated based 30.8

• Lifetime AI = TD50/50,000 x 50kg

• Lifetime AI = 30.8 mg/kg ÷ 50,000 × 50 kg× 1000 µg

• Lifetime AI = 30.8 µg/day

18

Additional Consideration

“Higher acceptable intakes may be justified when human exposure to the impurity will be much greater from other sources e.g., food, or endogenous metabolism (e.g., formaldehyde).”

• Ethylene Oxide is generated endogenously from ethylene (Bolt 2000)

– lipid peroxidation

– oxidation of free methionine

– oxidation of hemin in hemoglobin

– and metabolism of intestinal bacteria

• Endogenous levels and risk assessment calculated based on DNA lesions and epidemiological data (Kirman 2004)

• An exposure to ~0.1 ppm ethylene oxide for working lifetime would lead to a theoretical cancer risk of 1 x 10-5 (Valdez-Flores 2011)

– 0.1 ppm = 0.183 mg/m3 = 1.8 mg/day (10 m3 of air breathed in an 8-hour work day)

19

References

• Lynch, D. W., Lewis, T. R., Moorman, W. J., Burg, J. R., Groth, D. H., Khan, A., Ackerman, L. J., and Cockrell, B. Y. Carcinogenic and toxicologic effects of inhaled ethylene oxide and propylene oxide in F344 rats. Toxicol. Appl. Pharmacol. 76: 69-84(1984).

• Snellings, W. M., Weil, C. S., and Maronpot, R. R. A two-year inhalation study of the carcinogenic potential of ethylene oxide in Fischer 344 rats. Toxicol.Appl.Pharmacol. 75: 105-117(1984).

• Dunkelberg, H. Carcinogenicity of ethylene oxide and 1,2-propylene oxide upon intragastric administration to rats. Br. J. Cancer 46: 924-933(1982).

• ICH M7(R2). Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk

• Bolt, H.M. (2000) Carcinogenicity and Genotoxicity of Ethylene Oxide: New Aspects and Recent Advances, Critical Reviews in Toxicology, 30:5, 595-608

• Kirman CR, Sweeney LM, Teta MJ, Sielken RL, Valdez-Flores C, Albertini RJ, Gargas ML (2004) Addressing nonlinearity in the exposure-response relationship for a genotoxic carcinogen: Cancer potency estimates for ethylene oxide. Risk Analysis 24: 1165-1183

• Valdez-Flores C, Sielken RL Jr, Teta MJ (2011). Quantitative cancer risk assessment for ethylene oxide inhalation in occupational settings. Arch Toxicol 85:1189-1193