**I U C L I D**

**D a t a S e t**

**Existing Chemical** ID: 637-92-3

**CAS No.** 637-92-3

**EINECS Name** 2-ethoxy-2-methylpropane

**EC No.** 211-309-7

**Molecular Formula** C6H14O

**Producer Related Part**

**Company:** Lyondell Chemical Europe Inc.

**Creation date:** 22-MAR-2006

**Substance Related Part**

**Company:** Lyondell Chemical Europe Inc.

**Creation date:** 22-MAR-2006

**Printing date:** 22-MAR-2006

**Revision date:**

**Date of last Update: 09-MAR-2006**

**Number of Pages: 156**

**Chapter (profile):** Chapter: 1, 2, 3, 4, 5, 6, 7, 8, 10

**Reliability (profile):** Reliability: without reliability, 1, 2, 3, 4

**Flags (profile):** Flags: without flag, confidential, non
confidential, WGK

(DE), TA-Luft (DE), Material Safety Dataset, Risk

Assessment, Directive 67/548/EEC, SIDS

date: 22-MAR-2006

1\. General Information Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[1.0.1 Applicant and Company Information]{.underline}**

**-**

**[1.0.2 Location of Production Site, Importer or
Formulator]{.underline}**

**-**

**[1.0.3 Identity of Recipients]{.underline}**

**-**

**[1.0.4 Details on Category/Template]{.underline}**

**-**

**[1.1.0 Substance Identification]{.underline}**

**-**

**[1.1.1 General Substance Information]{.underline}**

**Substance type:** organic

**Physical status:** liquid

**Source:** EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

11-FEB-2000

**[1.1.2 Spectra]{.underline}**

**-**

**[1.2 Synonyms and Tradenames]{.underline}**

Synomyms and tradenames

**Remark:** 1,1-dimethylethyl-ethyl-ether

tert-butyl-ethyl-ether

ethyl-tert-butyl-oxide

2-ethoxy-2-methylpropane

ethyl-1,1-dimethylethyl-ether

30-NOV-2003

**[1.3 Impurities]{.underline}**

**-**

**[1.4 Additives]{.underline}**

**-**

\- 1/156 -

date: 22-MAR-2006

1\. General Information Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[1.5 Total Quantity]{.underline}**

**-**

**[1.6.1 Labelling]{.underline}**

**-**

**[1.6.2 Classification]{.underline}**

**Remark:** F, R11

30-NOV-2003

**[1.6.3 Packaging]{.underline}**

**-**

**[1.7 Use Pattern]{.underline}**

**Type:** industrial

**Category:** Fuel industry

**Remark:** Information on the physico-chemical properties of ETBE and

relative performance characteristics of ETBE-gasoline blends

is presented.

Phys-chem properties are reported in IUCLID sections 2.2,

2.3, 2.4 and 2.6.

Performance characteristics for ETBE include:

RON (ASTM2699) = 111.9

MON (ASTM D2700) = 97.2

Oxygen content = 15.66 w/w%

Key conclusions include:

\* The physico-chemical properties (low RVP, low solubility

in water, high boiling point) of ETBE make it suitable for

blending into gasoline.

\* Its Blending Octane Numbers equal or exceed those of MTBE.

\* It is stable and does not oxidise in gasoline blends,

however longer-term storage of neat ETBE requires the use of

antioxidants to avoid possible peroxide formation.

\* Gasoline containing 15% ETBE does not adversely influence

lubricant performance or piston and engine cleanliness.

23-JAN-2002 (22)

\- 2/156 -

date: 22-MAR-2006

1\. General Information Substance ID: 637-92-3

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**Type:** industrial

**Category:** Fuel industry

**Remark:** This paper from the European Commission (Excise, Duties and

Transport, Environment and Energy Taxes) reviews the potential

socio-economic and environmental (automotive emissions) impact

of the use of ETBE in gasoline in Europe. It notes that

Directive 85/536/EEC identifies a role for biofuels in

reducing Member States\' dependence upon oil imports, and

authorises the use of up to 15% v/v ETBE in gasoline.

02-JAN-2004 (11)

**[1.7.1 Detailed Use Pattern]{.underline}**

**-**

**[1.7.2 Methods of Manufacture]{.underline}**

**-**

**[1.8 Regulatory Measures]{.underline}**

**-**

**[1.8.1 Occupational Exposure Limit Values]{.underline}**

**Remark:** ACGIH TLV-TWA: 5 ppm

30-NOV-2003 (1)

**[1.8.2 Acceptable Residues Levels]{.underline}**

**-**

**[1.8.3 Water Pollution]{.underline}**

**-**

**[1.8.4 Major Accident Hazards]{.underline}**

**-**

**[1.8.5 Air Pollution]{.underline}**

**-**

**[1.8.6 Listings e.g. Chemical Inventories]{.underline}**

**-**

**[1.9.1 Degradation/Transformation Products]{.underline}**

**-**

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date: 22-MAR-2006

1\. General Information Substance ID: 637-92-3

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**[1.9.2 Components]{.underline}**

**-**

**[1.10 Source of Exposure]{.underline}**

**Remark:** This product is used in closed systems.

Exposure may occur during sampling.

**Source:** From ECB IUCLID dataset for 637-92-3

23-DEC-2003

**Source of exposure:** Human: exposure of the operator by intended use

**Remark:** This study examined occupational exposure of service station

refuelling attendents and automotive mechanics to gasoline

oxygenates in four regions of the USA (New York City area,

Minnesota, Arizona, Oregon) during the winter of 1994.

The report introduction states that ETBE was included in

these analyses, however no ETBE was reported after analysis

of \'bulk\' gasoline samples nor were any data or results

presented for inhalational exposure to this substance.

15-JAN-2004 (4)

**[1.11 Additional Remarks]{.underline}**

**-**

**[1.12 Last Literature Search]{.underline}**

**-**

**[1.13 Reviews]{.underline}**

**-**

\- 4/156 -

date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

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**[2.1 Melting Point]{.underline}**

**Value:** = -94 degree C

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

28-OCT-2003 (78)

**[2.2 Boiling Point]{.underline}**

**Value:** = 73.1 degree C

**Year:** 1994

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (22)

**Value:** = 72.8 degree C

**Year:** 1990

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (30)

**Value:** = 70 degree C at 1013 hPa

**Decomposition:** no

**Method:** other: ASTM D 86

**GLP:** no data

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003 (2)

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date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[2.3 Density]{.underline}**

**Type:** relative density

**Value:** = .7455

**Year:** 1994

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (22)

**Type:** density

**Value:** = .74 g/cm³

**Year:** 1990

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (30)

**Type:** density

**Value:** = .75 g/cm³ at 20 degree C

**Method:** other: ASTM D 1298

**GLP:** no data

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003 (2)

**[2.3.1 Granulometry]{.underline}**

**-**

**[2.4 Vapour Pressure]{.underline}**

**Method:** other (measured): ASTM D323

**Year:** 1994

**Result:** Reid Vapour Pressure for ETBE = 0.298 bar

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (22)

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date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

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**Method:** other (measured): comparable to ASTM D323

**Year:** 1990

**Remark:** Evaporative emissions for 3 ETBE-gasoline blends were

determined by heating the samples from 40.0 to 75.6 degrees

C in a model carburettor under laboratory conditions.

Vapour production decreased as the concentration of ETBE in

the fuel increased ie

Baseline gasoline = 9.16 psi

10% ETBE blend = 8.57 psi

20% ETBE blend = 8.32 psi

**Result:** Reid Vapour Pressure = 4.3 - 4.5 psi

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (30)

**Value:** = 128 hPa at 20 degree C

**Method:** other (measured)

**GLP:** no data

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

**[2.5 Partition Coefficient]{.underline}**

**Partition Coeff.:** octanol-water

**log Pow:** = 1.48 at 25 degree C

**Method:** other (measured): FDA Technical assistance Handbook document

3.02

**Year:** 1994

**GLP:** yes

**Remark:** ETBE partitioned between n-octanol saturated with MilliQ

purified water, and MilliQ purified water saturated with

n-octanol

Phase ratios in preliminary test 50:50 and 25:75

n-octanol/water showed no difference in percent partitioning.

Shaking times in preliminary test (0, 0.5,1.0, 2.0 hr) showed

equilibration complete in 0.5 hr.

ETBE concentrations: 1 x 10-2 M and 1 x 10-3 M in

\- 7/156 -

date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

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water-saturated n-octanol.

Volume of octanol: 50ml

Phase ratio in definitive test 50:50

Equilibration time 0.5hr (3 samples) plus two samples taken at

59 min (high concentration) and 51 min (low concentration) to

show equilibrium reached.

Temperature 25 +/-1 degree C

ANALYSIS: Concentrations in each phase were determined by

GC-FID (4 replicates)

Comment: Two different sets of test conditions (in this case

low and high concentrations of test substance) were used in

this study whereas OECD guideline would require three.

**Result:** Test concentration 1 x 10-2 M, Kow = 28.3

Test concentration 1 x 10-3 M, Kow = 31.4

**Test substance:** ETBE, 95.31% pure, supplied by ARCO Chemical Co.

**Reliability:** (2) valid with restrictions

Study available for review. GLP compliant near-guideline

investigation, clearly reported, acceptable for assessment.

28-OCT-2003 (62)

**Partition Coeff.:** octanol-water

**log Pow:** = 1.56

**Year:** 1972

**GLP:** no

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (34)

**log Pow:** = 1.28

**Method:** other (calculated)

**Year:** 1985

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

\- 8/156 -

date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

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**Partition Coeff.:** water - air

**Year:** 1995

**GLP:** no data

**Remark:** Liquid/air partition coefficients were determined using the

closed vial technique (Falk et al., 1990, Br J Ind Med, 47,

62-64). In brief, 0.5 ml olive oil or 2 ml pooled human blood

or physiological saline was added to a 22 ml gas tight vial.

After addition of 2 ul of aqueous ETBE (concentration not

stated) the vial was allowed to equilibrate at 37 degrees C

for 40 min. The gas phase was analysed for ETBE content by

head-space gas chromatography. Partition coefficients were

obtained by comparing peak areas between the sample vial and a

reference vial without liquid phase.

The following partition coefficients were obtained:

blood/air: 11.7

water/air: 8.3

oil/water: 190

oil/blood: 16.2 (calculated from \[oil/air\] / \[blood/air\])

**Test substance:** No details.

**Conclusion:** The blood air partition coefficient indicates efficient
uptake

of ETBE from, and elimination via, inhaled air. The greater

oil/air partition coefficient is consistent with preferential

movement of ETBE from blood to fatty tissue.

**Reliability:** (2) valid with restrictions

Study available for review. Briefly reported but acceptable

for assessment.

01-NOV-2003 (47) (67)

**Year:** 2000

**Remark:** Vial-equilibration method of Sato et al (1979, Br J Ind Med

36, 231) as used.

Water-air partition coeffcient 10.0 +/- 0.8

Rat blood-air partition coefficient 11.6 +/- 1.5

Olive oil-air partition coefficient 189 +/- 29

Olive oil-water partition coefficient 17.2

**Test substance:** No details available.

**Reliability:** (2) valid with restrictions

Study available for review. Briefly reported experimental

investigation, acceptable for assessment.

28-OCT-2003 (48)

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date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[2.6.1 Solubility in different media]{.underline}**

**Solubility in:** Water

**Value:** = 6.23 g/l at 25 degree C

**Remark:** The solubility of ETBE in water was investigated over the

temperature range 279.05 to 348.15 degrees K (+/- 0.05 degree

K\) by visually following the disappearance or reappearance of

cloudiness as the two liquids were heated or cooled,

respectively.

The results were reported as the molar solubility ratio of

ETBE in water (0.0011 at 298.15 degrees K).

**Test substance:** Described as ethyl tert-butyl ether, density 0.7356
at 298.15

degrees K.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

methods and results clearly reported, acceptable for

assessment.

24-FEB-2006 (26)

**Solubility in:** Water

**Value:** = 11 g/l at 22 degree C

**Remark:** Excess ETBE was added to a vial containing distilled water
and

the two liquids mixed. The phases were allowed to separate,

and the concentration of ETBE in the water phase determined

using gas chromatography.

**Test substance:** ETBE

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

briefly reported methods and results, acceptable for

assessment.

24-FEB-2006 (68)

**Value:** = .5 other: % Weight

**Remark:** Determination of pH according to ASTM D 1087 is not

applicable

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

\- 10/156 -

date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Solubility in:** Water

**Value:** = 2.37 other: w/w% at 20 degree C

**Year:** 1994

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

02-NOV-2003 (22)

**Solubility in:** Water

**Value:** = 12 g/l at 20 degree C

**Test substance:** Described as ethyl tert-butyl ether, density 0.7456,
boiling

point 73 degrees C.

**Reliability:** (4) not assignable

Study available for review. Early research paper with briefly

decribed methods and results.

09-MAR-2006 (28)

**Solubility in:** Water

**Value:** ca. 8 g/l

**Test substance:** ETBE, CAS No. 637-92-3

**Reliability:** (4) not assignable

Secondary literature (data compendium, review article or

meeting proceedings).

24-FEB-2006 (74)

**Solubility in:** Water

**Value:** = 11.2 g/l at 22 degree C

**Remark:** Analysis of ETBE in water performed using gas chromatograhy.

**Test substance:** ETBE

**Reliability:** (4) not assignable

Study available for review. Briefly reported, limited

information on methods use, acceptable for assessment.

24-FEB-2006 (77)

**[2.6.2 Surface Tension]{.underline}**

**-**

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date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

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**[2.7 Flash Point]{.underline}**

**Value:** \< 21 degree C

**Type:** closed cup

**Method:** other: ASTM D 56

**GLP:** no data

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003 (2)

**[2.8 Auto Flammability]{.underline}**

**Value:** = 375 degree C

**Method:** other: DIN 51794

**GLP:** no data

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

**[2.9 Flammability]{.underline}**

**-**

**[2.10 Explosive Properties]{.underline}**

**Remark:** Lower Explosive Limit : 1.23 % Vol

Upper Explosive Limit : 7.7 % Vol

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

**[2.11 Oxidizing Properties]{.underline}**

**-**

\- 12/156 -

date: 22-MAR-2006

2\. Physico-chemical Data Substance ID: 637-92-3

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**[2.12 Dissociation Constant]{.underline}**

**-**

**[2.13 Viscosity]{.underline}**

**Value:** at 40 degree C

**Result:** viscosity = 0.528 mm2/s (static) at 40 degrees C

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

28-OCT-2003 (30)

**[2.14 Additional Remarks]{.underline}**

**-**

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date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

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**[3.1.1 Photodegradation]{.underline}**

**Type:** air

**Light source:** Xenon lamp

**Light spect.:** = 330 - 370 nm

**INDIRECT PHOTOLYSIS**

**Sensitizer:** OH

**Year:** 1990

**GLP:** no data

**Remark:** Light source = High intensity xenon arc lamp

Flow system with hydroxyl radicals from photolysis of methyl

nitrate in air containing nitric oxide.

Concentrations of test substances were measured by gas

chromatograph.

Rate constants determined relative to diethyl ether.

A values of 4.8 x 10\^12 cm3/(molecules\*sec) was obtained.

**Test substance:** No details available.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

08-JAN-2004 (6)

**Type:** air

**Light source:** other: fluorescent backlights

**Year:** 1991

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Experiments were performed using a 140 l pyrex reactor

surrounded by 22 fluorescent backlights (GE F15T8-BL). Fourier

transform infrared spectroscopy was used to monitor loss of

reactants and formation of products in presence of NO, Cl and

NO and Cl.

**Remark:** This research was designed to investigate the mechanism of
the

oxidation of ETBE, and did not include the determination of

rate constants.

**Result:** Tert-butyl formate (76 +/- 6% yield), formaldehyde and

2-ethoxy-2-methyl propanal were detected as degradation

products.

**Test substance:** Ethyl tert-butyl ether, \>99.9% purity (confirmed by
GC-MS),

source not stated.

**Conclusion:** The authors conclude that attack by OH radicals on ethyl
group

yields the corresponding formate; interaction with the

tert-butyl group yields 2-ethoxy-2-methyl propanal, or a

variety of compounds formed by intramolecular hydrogen

abstraction or fragmentation. Formaldehyde can be produced as

\- 14/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

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a by-product.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (85)

**Type:** air

**Year:** 1991

**GLP:** no data

**Remark:** The reactivity of ETBE with different NMOC/NOx ratios was

estimated using a previously-derived OH rate constant

(Wallington et al. (1989) Int J Chem Kinet 21, 993).

A rate constant of 1.4x10\^12 cm3 /(molecule\*sec) was obtained.

The following was proposed as a mechanism for OH-initiated

oxidation of ETBE:

ETBE + OH + 1.8NO -\> 0.8HCOOC(CH3)3 + 0.2C2H5OC(CH3)2CHO + HO2

\+ 0.8HCHO + 1.8NO2

(From:

Chang and Rudy (1990) Proceedings of the international

conference on tropospheric ozone and the environment, Air and

Waste Management Association, Los Angeles, CA, March 1990;

Wallington et al. Int.J. Chem Kinet 20, 541).

Authors conclude that use of ETBE in fuel may have a

beneficial impact on urban ozone levels.

01-NOV-2003 (46)

**Type:** air

**Reliability:** (4) not assignable

German language report, not reviewed.

02-NOV-2003 (5)

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date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

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**Year:** 1990

**GLP:** no data

**Remark:** Results summarised from other publications:

Wallington et al. (1988) Environ Sci Technol 22, 842:

8.12 cm3/(molecules\*sec) at 298 degrees K

Bennett and Kerr (1989) J Atmos Chem 7, 87:

5.63 cm3/(molecules\*sec) at 294 degrees K

Wallington et al (1989) Int J Chem Kinet 21, 993:

8.83 cm3/(molecules\*sec) at 295 degrees K

**Reliability:** (2) valid with restrictions

Secondary literature (data compendium, review article or

meeting proceedings).

08-JAN-2004 (6)

**[3.1.2 Stability in Water]{.underline}**

**-**

**[3.1.3 Stability in Soil]{.underline}**

**-**

**[3.2.1 Monitoring Data (Environment)]{.underline}**

**-**

**[3.2.2 Field Studies]{.underline}**

**-**

**[3.3.1 Transport between Environmental Compartments]{.underline}**

**Type:** fugacity model level I

**Year:** 2003

**Air:** 96.2 **% (Fugacity Model Level I)**

**Water:** 3.658 **% (Fugacity Model Level I)**

**Soil:** .098 **% (Fugacity Model Level I)**

**Remark:** INPUT DATA SET

The following physico-chemical properties of ETBE were used:

Molecular weight = 102

Data temperature = 20 degrees C

Log Kow = 1.48 (at 25 deg C; Montgomery, 1994)

Water solubility = 23700 mg/l (2.37% w/w; 20 degrees C;

Chatin et al., 1994)

Vapour pressure = 29800 Pa (temperature not known; IUCLID

database)

Melting point = -94 degrees C (EPIWIN database)

\- 16/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

The reaction half-life was set at negligible reaction in all

compartments.

LEVEL I RESULTS

The environmental distribution, as calculated from the above

parameters using the Mackay Level I model (EQC program) is as

follows:

To Air 96.2%

To Soil 0.098%

To Water 3.658%

To Fish 5.52E-06%

To Sediment 2.17E-03%

**Reliability:** (2) valid with restrictions

Recognised modelling method.

08-JAN-2004 (53) (54) (88)

**Type:** fugacity model level III

**Year:** 2003

**Remark:** INPUT DATA SET

The following physico-chemical properties of ETBE were used:

Molecular weight = 102

Data temperature = 20 degrees C

Log Kow = 1.48 (at 25 deg C; Montgomery, 1994)

Water solubility = 23700 mg/l (2.37% w/w; 20 degrees C;

Chatin et al., 1994)

Vapour pressure = 29800 Pa (temperature not known; IUCLID

database)

Melting point = -94 degrees C (EPIWIN database)

The reaction half-life was set at negligible reaction in all

compartments.

LEVEL III RESULTS

The Level III program was used, with the \'default\' model and

used to calculate the ultimate fate of the substance, taking

into account the levels of release into specific compartments.

The distribution between environmental compartments obtained

by this method is as follows:

To Air To Water To Soil To all three

\% in air 99.31% 0.42% 2.76% 1.34%

\% in soil 0.10% 4.29E-04% 76.82% 13.80%

\% in water 0.58% 99.53% 20.40% 84.82%

\% in sediment 3.11E-04% 0.05% 0.01% 0.05%

**Reliability:** (2) valid with restrictions

Recognised modelling method.

08-JAN-2004 (19) (53) (88)

**[3.3.2 Distribution]{.underline}**

**-**

\- 17/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[3.4 Mode of Degradation in Actual Use]{.underline}**

**-**

**[3.5 Biodegradation]{.underline}**

**Type:** aerobic

**Inoculum:** activated sludge, domestic

**Concentration:** 150 mg/l related to Test substance

**Result:** other: inherently biodegradable by adapted STP organisms

**Method:** ISO DIS 9408 \"Ultimate aerobic biodegradability - Method by

determining the oxygen demand in a closed Respirometer\"

**Year:** 1998

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Studies were performed in a degradation respirometer (ISO
9408

standard) containing either 30 or 380 mg (dry wt)/l activated

sludge. The inoculum was obtained from an urban STP in the

Paris area. Degradation was followed by electrolytic

respirometry using a D-12 Sapromat apparatus (Voith, Germany).

Respiration was measured in the presence of 150 mg/l ETBE was

corrected for endogenous respiration (oxygen uptake in the

absence of ETBE).

**Remark:** Gordona terrae and Rhodococcus equi, isolated from cultures

used in these studies, were able to grow on ETBA as sole

carbon source.

**Result:** No degradation of ETBE was observed at an inoculum

concentration of 30 mg/l.

At 380 mg/l, oxygen consumption measurements showed that

degradation of ETBE commenced after a lag of approx. 22 hr.

The amount of oxygen consumed at 360 hr was consistent with

total removal of ETBE.

Addition of a second amount of ETBE resulted in degradation

with no lag, and a degradation rate of 2.8 mg ETBE/l/hr.

Similar results were obtained in triplicate samples of

activated sludge over a period of several weeks (no further

details).

**Test substance:** ETBE, Aldrich Chemical Co. (no further details).

**Conclusion:** Results from these studies demonstrate degradation of
ETBE

(and TBA) by a concentrated, adapted inocculum of

microorganisms from an urban wastewater treatment plant.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (29)

\- 18/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** aerobic

**Inoculum:** activated sludge, adapted

**Concentration:** 89 mg/l related to Test substance

**Degradation:** = 100 % after 80 hour(s)

**Result:** other: inherently biodegradable by adapted WWTP organisms

**Year:** 1996

**GLP:** no data

**Method:** Acclimated culture from a petroleum refinery WWTP (adapted
to

degrading MTBE) was used as inoculum. The test substance

concentration was89 mg/l, and incubations were performed in

replicate at 30 degrees C in a 16 place Comput-Ox respirometer

(N-CON Systems Inc, GA). Oxygen uptake, substrate removal and

intermediate accumulation measured. ETBE concentration was

determined by GC. Based on the results, degradation kinetics

were modeled using standard procedures and models.

**Result:** Graphical data indicate that oxygen uptake by the cultures

increased from 0 to 140 mg/l in 80 hr while the concentration

of ETBE decreased from 80 to 0 mg/l over the same period.

Small amounts of TBA (approx. \<1 mg/l) were detected between

50-100 hr.

Degradation rate for ETBE = 0.027/h

Comment: additional studies on TBA included in this

experimental series showed complete degradation of 82 mg/l TBA

over 80 hr, with a calculated degradation rate of 0.029/hr.

**Test substance:** No details available.

**Conclusion:** High levels of ETBE degradation were achieved with
sludge from

a WWTP acclimated to MTBE.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (24)

\- 19/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** aerobic

**Inoculum:** other: mixed bacterial population isolated from

gasoline-polluted soil

**Year:** 1998

**GLP:** no data

**Method:** A mixed bacterial population capable of degrading ETBE were

isolated from gasoline-polluted soil (Kharoune et al. (1997)

Int Symp Environ Technol Oostende, Belgium, 381-384).

Degradation studies were conducted at 28 degrees C in a BSB

digi V 1.01 respirometer (Buhler) at an agitation speed of 125

rpm. Ten respirometer flasks containing ETBE (200 mg/l) were

inoculated with defined amounts of biomass (see results), with

two endogenous respiration control flasks (no ETBE). Oxygen

consumption, total cell protein, pH, oxygenate- and COD

concentrations measured.

ETBE and TBA were analysed by GD-FID.

Mineralisation of ETBE was assessed by measuring COD.

**Result:** At a concentration of 243 mg/l total cell protein,

mineralisation of 200 mg/l ETBE was complete within 21 hr (as

demonstrated by disappearance of substrate, zero carbon mass

balance (COD) and cessation of oxygen uptake).

Rate of ETBE degradation increased with inoculum size

(sub-linear relationship):

Inoculum Oxygen ETBE Removal rate

uptake (mg ETBE/hr/g

(mg/l/hr) (mg/l/hr) cell protein)

12.9 mg/l 5 1.1 1666

19.5 5 1.86 1706

120 15 5.60 1750

243 30 9.75 1666

Accumulation of TBA was reported (but not quantified) in

systems containing 120 mg total cell protein/l and above.

The rate of ETBE degradation (9.3-9.9 mg/l/hr) was

approximately constant over a substrate concentration range of

200-1200 mg/l, however this decreased to 3.2 or 2.4 mg/l/hr at

ETBE concentrations of 2000 or 3000 mg/l, respectively.

(Equivalent removal rates were 1800-1830 mg/hr/g total protein

at 200-1200 mg ETBE/l, and 1570 or 1400 mg/hr/g at higher

substrate concentrations.)

**Test substance:** ETBE, purity not stated, Elf Aquitaine, France or
Fluka France

(no further details).

**Conclusion:** A decrease in ETBE concentration, removal of COD along
with

substrate-dependent oxygen uptake were observed when ETBE by

was incubated with a microbial consortium obtained from a

gasoline-polluted site. Degradation rates of 1800-1900 mg

\- 20/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

ETBE/g cell/hr were achieved at substrate concentrations of

200-1200 mg/l, however higher concentrations of ETBE were

toxic.

**Reliability:** (2) valid with restrictions

Study available for review. Briefly reported experimental

investigation, acceptable for assessment.

02-JAN-2004 (50)

**Type:** aerobic

**Inoculum:** other: mixed bacterial population isolated from

gasoline-polluted soil

**Year:** 1998

**GLP:** no data

**Method:** Degradation of ETBE was investigated at 28 degrees C in a 2
l

continuous fixed-bed recycling biofilm reactor (60x8 cm

internal diameter) containing a sintered SIRAN glass ring as

biomass carrier (55-60% pore volume, 0.15 m2/g surface area).

A recycle rate of 650 l/d and airflow of 4.5 l/d were

employed, along with a feed rate was 2 l/d (salts solution and

ETBE at 50-345 mg/l in distilled water).

The reactor was inoculated with ETBE-mineralising bacteria

isolated from gasoline polluted soil (Kharoune et al. (1997)

Int Symp Environ Technol Oostende, Belgium, 381-384). Cell

growth was monitored at 600 nm, while the amount of biomass

(protein dry wt) present on the sintered glass carrier was

quantified after sonication.

Degradation of ETBE, together with identification of its major

breakdown products, was quantified by GC-FID while

mineralisation was followed using COD and oxygen uptake.

**Result:** The concentration of ETBE in the reactor was increased from
50

mg/l to 345 mg/l between days 15 and 86.

The operation of the reactor was defined by three states:

\- State 1: between days 15-40, a removal rate of 299 mg/l/d

was achieved at an ETBE loading rate of 327 mg/l/d. Efficiency

of conversion was approx. 92% with a COD removal rate of 801

mg/l/d.

\- State 2: between days 40-67, the reactor destabilised, the

concentration of residual ETBE in effluent increased and a

removal rate of 280 mg/l/d was achieved at a loading rate of

345 mg/l/d (80.9% yield).

\- State 3: the ETBE loading rate was reduced and reactor

performance returned to high performance by day 69, with 99%

removal efficiency at a loading rate of 280 mg/l/d.

Graphical data demonstrate good correlation (R2=0.8984)

between removal of ETBE and removal of COD.

**Test substance:** ETBE, purity not stated, Elf Aquitaine, France or
Fluka France

(no further details).

**Reliability:** (2) valid with restrictions

\- 21/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Study available for review. Briefly reported experimental

investigation, acceptable for assessment.

02-JAN-2004 (51)

**Type:** aerobic

**Inoculum:** other: soil microcosms

**Result:** other: biodegradable by soil microcosms under specialised

conditions

**Year:** 1995

**GLP:** no data

**Method:** Degradation was evaluated in soil microcosms containing 5 g

dry weight soil and water in 13x100 mm septum-capped tubes

containing 100 mg/l ETBE (saturating concentration). The tubes

were filled to the top (to prevent loss by evaporation) and

incubated at 20 degrees C in the dark.

Hydrogen peroxide added to the incubations every 7-10 d to a

concentration of 0.01% as oxygen source.

ETBE concentrations were measured by gas chromatography.

Direct chemical oxidation of ETBE to TBA by hydrogen peroxide

was also assessed.

**Result:** Soil with no additional hydrogen peroxide, or soil
pretreated

with hydrogen peroxide (to oxidise and precipitate soil iron

and prevent chemical oxidation), was also used to assess

biodegradation in some studies. Control incubations contained

soil that had been subject to repeated cycles of autoclaving.

After 90 d incubation, no biodegradation of ETBE was seen in

aerobic systems containing soil, nutrient solution and ETBE.

Hydrogen peroxide catalysed the very rapid chemical oxidation

of ETBE by soil samples (and also in simple solution), with

the appearance of TBA in minutes.

Soils pretreated with hydrogen peroxide prior to addition of

ETBE showed steady degradation over 30 d, with a calculated

degradation rate of 2.5 mg/l.d per g soil. The authors suggest

that hydrogen peroxide provided a source of oxygen for the

microorganisms and also removed ferrous iron form the system

(ie preventing competitive chemical oxidation of ETBE).

**Test substance:** No details available.

**Conclusion:** Biological oxidation of ETBE to TBA was demonstrated in
soil

microcosms supplemented with hydrogen peroxide.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (90)

\- 22/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** aerobic

**Inoculum:** other: two bacterial strains isolated from
gasoline-polluted

soil

**Year:** 2001

**GLP:** no data

**Method:** Aerobic degradation of ETBE by two bacterial strains (E1 and

E2), isolated from gasoline-contaminate soil, was

investigated.

Incubations contained 200 mg/l ETBE (sole carbon source) and

mineral salts and were performed in triplicate at 28 degrees

C. Residual amounts of ETBE and presence of TBA was quantified

by GC. Oxidation of ETBE was also followed using COD. Abiotic

control incubations contained heated-killed cells, while

others included metyrapone (inhibitor of cytochrome P-450) or

methimazole (inhibitor of flavin-containing monooxygenase).

Bacterial cytochrome P-450 content was quantified from the

CO-difference spectrum of post-30,000 g cell-free

hommogenates.

**Result:** Strain E1 (tentatively identified as Comamonas
testosteroni):

Complete degradation in 10 d, no lag

Degradation rate 80 mg/g cell protein/day

Protein concentration increased x2.5

Stain E2 (unidentified):

Complete degradation in 15 d.

Degradation rate 58 mg/g cell protein/day

Protein concentration increased x 3

COD analyses indicate complete degradation of ETBE

Abiotic controls indicated 3-4% of ETBE lost by

volatilisation.

There was no accumulation of TBA with either strain.

Degradation of ETBE was strongly inhibited by 5 mM metyrapone

(both strains, 13-18% decrease) but not methimazole. Reduced

CO-difference spectra contained a major absorbance peak at 450

nm (consistent with presence of cytochrome P-450; specific

content not reported).

**Test substance:** ETBE, purity not stated, Elf Aquitaine, France or
Fluka France

(no further details).

**Conclusion:** The results indicate that ETBE is utilised as sole
carbon

source (complete degradation) by the two bacterial strains

isolated from a gasoline contaminated site, possibly by a

cytochrome P-450-dependent pathway.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

02-JAN-2004 (49)

\- 23/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** aerobic

**Year:** 1997

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Remark:** Several propane-oxidizing bacteria were examined for their

ability to degrade ETBE.

A pure culture of Mycobacterium vaccae JOB5 (ATCC 292678) was

capable of degrading 80-100% ETBE (30 mg/l; analysis by GC) in

24 hr after aerobic incubation at 28 degrees C; degradation by

other microorganisms was highly variable (0-30%).

**Test substance:** ETBE, 99%, Aldrich Chemical Co., Milwaukee, WI.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (79)

**Type:** anaerobic

**Inoculum:** other: from methanogenic anoxic aquifer

**Concentration:** 70 mg/l related to Test substance

**Result:** under test conditions no biodegradation observed

**Year:** 1993

**GLP:** no data

**Remark:** ETBE was incubated with sediment and groundwater collected

from a methanogenic portion of a shallow anoxic aquifer

polluted by municipal landfill leachate.

Slurries of 50 g sediment and 75 ml groundwater were placed in

sterile 160ml bottles together 70mg/l ETBE. Bottles

(triplicate incubations) were sealed, incubated at room

temperature, in the dark.

EBTE concentration was measured by GC.

No degradation was detected following 168 d.

**Test substance:** No details available.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (80)

\- 24/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** anaerobic

**Inoculum:** other: sediment and water from landfill leachate impacted

aquifer

**Result:** under test conditions no biodegradation observed

**Year:** 1994

**GLP:** no data

**Remark:** Sediment and groundwater were collected from a landfill

leachate impacted aquifer and from a gasoline polluted

aquifer.

Anaerobic slurries containing 50g sediment in 75 ml water were

incubated under sulphate-reducing conditions for 244 d,

nitrate-reducing conditions for 85 d or methanogenic

conditions for 90 d at degrees C, dark. The initial

concentration of ETBE was 70 mg/l.

GC was used to following depletion of ETBE, HPLC for sulphate

and nitrate depletion.

Under these condition, no anaerobic biodegradation was

observed.

**Test substance:** No details available.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (63)

**Type:** anaerobic

**Inoculum:** other: soil

**Result:** other: biodegradable by soil microcosms

**Year:** 1994

**GLP:** no data

**Remark:** Soil from various depths from 3 sites used in static

soil/water microcosms in sealed test tubes (in triplicate,

autoclaved control) at 20 degrees C, in the dark. The soils

were characterised as to their anaerobic microbial

populations, soil anions, moisture content, organic content,

nitrogen availability, ammonium fixation rate and soil pH.

ETBE concentration approx. 80 mg/L, and degradation followed

under denitrifying and methanogenic conditions by GC-FID for

\>250 days.

Graphical data indicated that ETBE degradation occurred with a

lag of about 120 d under anoxic conditions in soil of low

organic content and low pH (5.5):

0 d 100 d 250 d

Site 1 80 60 10 mg/l ETBE

Site 2 70 60 40

The amount of TBA formed once degradation commenced was quite

\- 25/156 -

date: 22-MAR-2006

3\. Environmental Fate and Pathways Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

variable, possibly indicating degradation of TBA.

No degradation was seen in organic rich soils.

Addition of nitrate and nutrients did not significantly

increase degradation under denitrifying conditions.

Graphical data show that under methanogenic conditions,

degradation of ETBE occurred only in oligotrophic soils with

low organic matter and was maximal at approx. pH 5.5:

0 d 100 d 250 d

Site 1 80 70 50 mg/l ETBE

**Test substance:** No details available.

**Conclusion:** ETBE was degraded slowly under anaerobic conditions by
some

soil microcosms included in this study.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental investigation,

acceptable for assessment.

01-NOV-2003 (89)

**Remark:** The product is highly volatile and has a very low

biodegradability. Ecological data are not available, owing

to the high volatility of the product which does not allow

to carry out the test procedures.

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

**[3.6 BOD5, COD or BOD5/COD Ratio]{.underline}**

**-**

**[3.7 Bioaccumulation]{.underline}**

**-**

**[3.8 Additional Remarks]{.underline}**

**-**

\- 26/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[AQUATIC ORGANISMS]{.underline}**

**[4.1 Acute/Prolonged Toxicity to Fish]{.underline}**

**Type:** semistatic

**Species:** Cyprinodon variegatus (Fish, estuary, marine)

**Exposure period:** 96 hour(s)

**Unit:** mg/l **Analytical monitoring:**

**NOEC:** = 2500 - measured/nominal

**LC50:** \> 2500 - calculated

**Method:** EPA OTS 797.1400

**Year:** 1994

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** TEST ORGANISM

Juvenile sheepshead minnows were obtained from Aquatic

Biosystems Inc., Co, and maintained under flow-through

conditions in a 40 l glass tank prior to testing. At the

conclusion of the test, the control fish had a mean wet weight

of 0.34 g (loading rate 0.23 g/l) and a mean length of 28 mm.

DILUTION WATER

Natural seawater (pH 8.0) was adjusted with dechlorinated tap

water to a salinity of 15-17 ppt during a 14 d acclimation

period, and to 20 ppt during the test.

Other characteristics (measured):

Organochlorine pesticides \<2 ug/l (limit of detection)

Organophosphorus pesticides \<0.5 ug/l (limit of detection)

PCBs \<1.0 ug/l (limit of detection)

ACCLIMATION PERIOD

The following conditions were recorded during a 14 d

acclimation period:

Temperature = 21.0-22.3 degrees C

Salinity = 15-17 ppt

pH = 7.2-7.9

Dissolved oxygen = at least 7.1 mg/l

Fish were fed live brine shrimp and/or a commercial dry diet

during acclimation.

SCREENING TEST

The screening test used exposure concentrations of 0, 1.0, 10

or 100 mg/l. It was terminated after 24 hr due to 100%

survival at all tested concentrations.

DEFINITIVE TEST

Thirty sheepshead minnows were distributed between three

replicate test vessels (20 l glass aquaria containing 15 l of

test solution, depth approx. 18 cm) and exposed to a nominal

concentration of 2500 mg/l ETBE. Twenty additional fish were

assigned to two control tanks. The test medium was replaced

with fresh solution after 24, 48 or 72 hr. A 16 hr light (8 hr

\- 27/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

dark) cycle was maintained (15 minute transition between light

and dark).

Dissolved oxygen, pH, salinity and temperature were recorded

in each vessel before and after renewal of the medium, and

also at 0 hr and 96 hr. Aeration was not required.

The mean number of survivors and occurrence of sub-lethal

effects were assessed in the control and test vessels every 24

hr.

Reviewer\'s comment: Unlike the acute mysid study performed by

this laboratory (see Section 4.2), the report does not state

whether or not the vessels were covered to prevent evaporative

loss of ETBE.

STATISTICAL METHODS

Since no mortality was recorded, no EC50 could be derived form

the data. The NOEC was defined as the concentration of ETBE

that did not cause any sublethal effects and allowed at least

90% survival of the test organisms.

**Result:** ENVIRONMENTAL CONDITIONS (range for all vessels)

Salinity = 20 ppt

pH = 7.5-8.0

Dissolved oxygen = 5.6-8.4 mg/l

Temperature = 21.3-22.5 degrees C

SURVIVAL

There was no mortality in the control or test vessels.

SUBLETHAL EFFECTS

There were no sublethal effects in the control or test

vessels.

MEDIAN LETHAL CONCENTRATIONS

The EC50 was \>2500 mg/l at all time points (ie 24, 48, 72 and

96 hr).

NOEC CONCENTRATIONS

The NOEC was 2500 mg/l at all time points.

**Test substance:** ETBE, 95.3% pure, lot no. 12/6/93, supplied by ARCO
Chemical

Company, PA, USA.

**Conclusion:** Under the conditions of this test, the 96 hr EC50 of
ETBE in

the sheepshead minnow (Cyprinodon variegatus) was greater than

2500 mg/l. The NOEC was 2500 mg/l, the highest concentration

tested.

**Reliability:** (2) valid with restrictions

Study available for review. GLP compliant guideline

investigation. Test solution renewed every 24 hr, however

uncertainty over potential evaporative loss limits overall

reliability of result.

02-NOV-2003 (10)

\- 28/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** other: No data available

**Species:** other: No data available

**Exposure period:** 96 hour(s)

**Unit:** mg/l **Analytical monitoring:**

**LC50:** \> 100 -

**GLP:** no data

**Remark:** No toxicity was observed at 100 mg/l.

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Test substance:** ETBE

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

15-JAN-2004 (23)

**[4.2 Acute Toxicity to Aquatic Invertebrates]{.underline}**

**Type:** static

**Species:** Daphnia magna (Crustacea)

**Exposure period:** 48 hour(s)

**Unit:** mg/l **Analytical monitoring:** yes

**NOEC:** = 56 -

**EC50:** = 110 -

**Method:** OECD Guide-line 202

**Year:** 2003

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** TEST ORGANISM

First instar daphnia from a single in-house source were

maintained in 2-3 litre polypropylene beakers at a density of

30-80 daphnids/vessel prior to testing.

DILUTION WATER

Reconstituted water (containing analytical grade CaCl2, MgSo4,

NaHCO3 and KCL), hardness 250 mg/l, pH 7.8, conductivity \<5

uS/cm, aerated to a dissolved oxygen concentration of approx.

half saturation value.

DEFINITIVE TEST

Based on results from a screening test (using concentrations

of 0.1-1000 mg/l), 20 daphnia were divided between two 250 ml

vessels containing ETBE at nominal concentrations of 0

(water), 10, 18, 32, 56, 100, 180, 320, 560 or 1000 mg/l. The

vessels (300 ml conical flasks) were completely filled and

stoppered to minimise evaporative losses of ETBE. In order to

reduce losses due to volatilisation, the test vessels remained

sealed throught the 48 hr of the study. The test was performed

at 21 degrees C, with a 16 hr light / 8 hr dark photoperiod,

\- 29/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

with 20 min dusk/dawn transition periods.

The mean number of survivors and occurrence of sub-lethal

effects were assessed in all vessels every 24 hr.

Temperature was recorded daily in the control vessels.

Dissolved oxygen and pH were measured for each vessel at the

start and the end of the study.

VERIFICATION OF CONCENTRATION

Chemical analysis (GC) was performed over the entire

concentration range (ie all vessels) at 0 hr and 48 hr.

Duplicate samples were stored frozen (-20 degrees C) for

reanalysis, if required.

VALIDATION CRITERIA

The test was considered invalid if 10% or more of the control

organisms were immobilised or stressed after 48 hr, or if the

oxygen concentrtaion of the control vessels fell below 60% of

air saturation values at the end of the test.

STATISTICAL METHODS

The 24 and 48 hr EC50 and 95% confidence limits were

calculated using standard statistical methods (Probit analysis

using ToxCalc computer package). The NOEC was defined as the

concentration of ETBE that did not cause any significant

immobilisation or sublethal effects.

**Result:** ANALYSED CONCENTRATIONS

Analysis at 0 hr and 48 hr showed that measured test

concentrations were generally 81-96% of nominal, with the

exception of the 18 mg/l test concentration which showed

measured concentrations of 71% and 73% at 0 hr and 48 hr,

respectively. As this concentration was below the NOEC (see

below), these findings were not considered by the Study

Directors to have affected the outcome of the study. (A low

measured concentration of 63% nominal was also reported for

the 32 mg/l solution at 0 hr, however reanalysis of a frozen

sample gave a value of 84% nominal.)

ENVIRONMENTAL CONDITIONS

pH = 7.9-8.0

Dissolved oxygen = 8.3 to 8.2-8.4 mg/l (0 hr and 48 hr,

respectively)

Temperature = 21.2-21.6 degrees C

SURVIVAL

There was no mortality or immobilisation in the controls or

test cultures exposed to 10-180 mg/l for 24 hr, or in the

controls or test cultures exposed to 10-56 mg/l for 48 hr.

The percentage immobilisation at study termination (48 hr) was

as follows:

Control: 0/20 (0%)

10 mg/l: 0/20

\- 30/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

18 mg/l: 0/20

32 mg/l: 0/20

56 mg/l: 0/20

100 mg/l: 13/20 (65%)

180 mg/l: 16/20 (80%)

320 mg/l: 19/20 (95%)

560 mg/l: 20/20 (100%)

1000 mg/l: 20/20 (100%)

MEDIAN LETHAL CONCENTRATIONS (nominal)

24 hr: 440 mg/l (95% CI = 370-520)

48 hr: 110 mg/l (95% CI = 91-130)

NOEC CONCENTRATION (nominal)

48 hr: 56 mg/l

**Test substance:** ETBE, CAS No. 637-92-3, 99.97%% pure, Total France.

**Conclusion:** Under the conditions of the test, the 48 hr EC50 for
ETBE in

Daphnia magna was 110 mg/l and the NOEC 56 mg/l.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

23-DEC-2003 (86)

**Type:** semistatic

**Species:** Mysidopsis bahia (Crustacea)

**Exposure period:** 96 hour(s)

**Unit:** mg/l **Analytical monitoring:**

**NOEC:** = 25 - measured/nominal

**EC50:** = 37 - calculated

**Method:** EPA OTS 797.1930

**Year:** 1994

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** TEST ORGANISM

Juvenile mysids from a single in-house source (original

culture from Aquatic Biosystems Inc., Co) were maintained

under flow-through conditions in a 38 l glass tank prior to

testing. At the conclusion of the test, the control mysids had

a mean wet weight of 0.3 mg (loading rate 0.003 g/l).

DILUTION WATER

Natural seawater (pH 8.0-8.1) was adjusted with dechlorinated

tapwater to a salinity of 15-17 ppt during a 14 d acclimation

period, and to 20 ppt during the test.

Other characteristics (measured):

Organochlorine pesticides \<2 ug/l (limit of detection)

Organophosphorus pesticides \<0.5 ug/l (limit of detection)

PCBs \<1.0 ug/l (limit of detection)

ACCLIMATION PERIOD

The following conditions were recorded during a 14 d

\- 31/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

acclimation period:

Temperature = 24.4-25.8 degrees C

Salinity = 15-17 ppt

pH = 7.7-8.0

Dissolved oxygen = at least 7.4 mg/l

SCREENING TESTS

Three screening tests were performed:

1\. Mysids \<24 hr old, 0 or 2500 mg/l ETBE, 24 hr exposure;

2\. Mysids \<24 hr old, 0, 25, 250 or 2500 mg/l ETBE, 24 hr

exposure;

3\. Mysids \<24 hr old or 5 d old, 0, 1, 10, 100 or 1000 mg/l,

96 hr exposure period

DEFINITIVE TEST

Twenty mysids (\<24 hr old) were distributed between two

replicate vessels (3.8 l glass jars containing 1 l of test

solution, depth approx. 6 cm) and exposed to nominal

concentrations of 0, 15, 25, 40, 60 or 100 mg/l ETBE. The test

vessels were tightly covered (method not specified) to prevent

evaporative losses. The test medium was replaced with fresh

solution after 24, 48 or 72 hr. A 16 hr light (8 hr dark)

cycle was maintained.

Dissolved oxygen, pH, salinity and temperature were recorded

in each vessel before and after renewal of the medium, and

also at 0 hr and 96 hr. Aeration was not required.

The mean number of survivors and occurrence of sub-lethal

effects were assessed in all vessels every 24 hr.

STATISTICAL METHODS

The EC50 and slope of the dose-response curve was determined

using standard statistical methods (Probit analysis). The NOEC

was defined as the concentration of ETBE that did not cause

any sublethal effects and allowed at least 90% survival of the

test organisms.

**Result:** ENVIRONMENTAL CONDITIONS (range for all vessels)

Salinity = 20 ppt

pH = 7.9-8.1

Dissolved oxygen = 6.6-8.3 mg/l (mean 7.6 mg/l)

Temperature = 23.1-25.8 degrees C (mean 25.2)

SURVIVAL

There was no mortality in the control cultures. Immobilisation

of a single organism was seen in one replicate from the 15 or

25 mg/l exposures at 72 or 96 hr. Immobilisation was noted at

48 hr in the 40 mg/l vessels, and at 24 hr in the 60 or 100

mg/l vessels. Survival at study termination (96 hr) was as

follows:

Control : 20/20

15 mg/l: 19/20

25 mg/l: 18/20

40 mg/l: 9/20

\- 32/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

60 mg/l: 2/20

100 mg/l: 0/20

SUBLETHAL EFFECTS

No sublethal effects were noted in cultures exposed to 0-40

mg/l ETBE.

Sublethal effects were observed in the 60 mg/l culture

(erratic swimming at 48 and 72 hr; erratic swimming and

lethargy in survivors at 96 hr).

No sublethal effects were noted at 0 or 24 hr in cultures

exposed to 100 mg/l ETBE; full immobilisation thereafter.

MEDIAN LETHAL CONCENTRATIONS

24 hr: 96 mg/l (95% CI = 80-140)

48 hr: 60 mg/l (95% CI = 54-68)

72 hr: 41 mg/l (95% CI = 36-47)

96 hr: 37 mg/l (95% CI = 32-43)

NOEC CONCENTRATIONS

24 hr: 40 mg/l

48 hr: 25 mg/l

72 hr: 25 mg/l

96 hr: 25 mg/l

**Test substance:** ETBE, 95.3% pure, lot no. 12/6/93, supplied by ARCO
Chemical

Company, PA, USA.

**Conclusion:** Under the conditions of this test, the 96 hr EC50 of
ETBE in

the mysid (Mysidopsis bahia) was 37 mg/l and the NOEC 25 mg/l.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

05-OCT-2003 (9)

\- 33/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[4.3 Toxicity to Aquatic Plants e.g. Algae]{.underline}**

**Species:** other algae: Pseudokirchneriella subcapitata

**Endpoint:** growth rate

**Exposure period:** 72 hour(s)

**Unit:** mg/l **Analytical monitoring:** yes

**NOEC:** = 7.5 -

**EC50:** = 1100 -

**Method:** OECD Guide-line 201 \"Algae, Growth Inhibition Test\"

**Year:** 2003

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** TEST ORGANISM

Pseudokirchneriella subcapitata (CCAP 276/20; formerly

Scenedesmus subspicatus) was cultured for 4-5 d at 21 degrees

C (cell density approx. 2x10\^4 to 2x10\^5 cell/ml) prior to

use. (Pre-culture conditions gave an algal suspension in log

phase growth characterised by a cell density of 4.03 x 10\^6

cells per ml, which was diluted to a cell density of 8.66 x

10\^5 cells per ml prior to use.)

TEST MEDIUM

The culture medium (mineral salts) was prepared using reverse

osmosis purified deionised water (Elga Optima 15+), the pH

adjusted to 7.0 ± 0.1 with 0.1N NaOH or HCl and sterilised

using a 0.2 µm membrane filter. Sodium bicarbonate (500 mg/l)

was added after preparation to provide a source of carbon for

algal growth in the sealed test vessels.

DEFINITIVE TEST

Based on results from two screening tests (using ETBE

concentrations of 0, 0.1, 1.0, 10, 100, 100 mg/l and 0, 9.1,

91, 910, 9100 mg/l) the definitive test was conducted using

ETBE concentrations of 0, 9.2, 29, 92, 290, 920, 2900 or 9100

mg/l (3 replicates per concentration). This concentration

range was selected based on recommendations contained in the

draft revised OECD TG201 (July 2002), and was designed to

result in 0-90% inhibition of growth.

Following the recommendations of Mayer et al (2000; Environ.

Toxicol Chem 19, 2551), a nominal cell density of 3 x 10\^3

cells per ml was used at initiation of the test. The flasks

(250 ml, completely filled with test medium) were sealed

(ground glass stoppers) and incubated at 24 degrees C under

continuous illumination (approx. 7000 lux) for 72 hours with

shaking (150 rpm). Samples were removed at 0, 24, 48 and 72

hours for the determination of cell densitity

(haemocytometer).

The pH of each control and test flask was determined at

initiation of the test and after 72 hours exposure. The pH

was measured using a WTW pH 320 pH meter. The temperature

\- 34/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

within the incubator was recorded daily.

Since some loss of test substance may have occurred when the

vessels were openeded for cell sampling, additional flasks

were incubated unopened alongside the definitive test vessels

then analysed for ETBE concentration at 72 hours.

VERIFICATION OF CONCENTRATION

Chemical analysis (GC) was performed over the entire

concentration range (ie all test vessels) at 0 hr and 72 hr,

and also on the unopened duplicate vessels at 72 hr only.

Duplicate samples were stored frozen (-20 degrees C) for

reanalysis, if required.

ANALYSIS OF DATA

The area under the curve was used as a measure of cell growth,

and the percentage inhibition obtained by comparison of the

AUC for the control and test vessels. The average maximum

growth rate for each culture was also calculated from the

linear section of the growth curve.

STATISTICAL METHODS

One way analysis of variance incorporating Bartlett\'s test for

homogeneity and Dunnett\'s multiple comparison procedure was

used applied to the AUC data to determine any statistically

significant differences between the test and control groups.

**Result:** RANGE FINDING TEST

Results from the initial range finding test showed no

significant effect on growth at 1 or 10 mg/l, however growth

was reduced at 100 and 1000 mg/l. In the second range-finder,

growth reduction was observed at all concentrations tested.

DEFINITIVE TEST

The percentage inhibition values for biomass and growth rate

were plotted against test concetration, and the following

results obtained:

EbC50 (biomass, 72 hr) = 37 mg/l; 95% CI 28-49 mg/l;

ErC50 (growth, 0-72 hr) = 1200 mg/l, 95% CI 890-1600 mg/l

NOEC = 9.2 mg/l

Since analytical measurements showed a slight decline in

concentration of ETBE in some of the test vessels over the 72

hr of the study (see below), hence the EC50 values were

recalcalculated based upon the geometric mean of the analysed

concentration. The following results were obtained:

EbC50 (biomass, 72 hr) = 32 mg/l; 95% CI 22-46 mg/l;

ErC50 (growth, 0-72 hr) = 1100 mg/l, 95% CI 810-1500 mg/l

NOEC = 7.5 mg/l

OBSERVATIONS and PHYSICO-CHEMICAL MEASUREMENTS

Cell density in the control cultures increased by a factor of

386 during the definitive test (exceeds Guideline requirement

for a 16-fold increase over 72 hr):

Density at 0 hr: 3.33 X 10\^3 cells/ml

\- 35/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Density at 72 hr: 1.29 x 10\^6 cells/ml

The pH values of the control cultures increased from pH 7.7 at

0 hr to pH 7.9-8.1 at 72 hr. This was less than 1.5 units and

therefore within the limits given in OECD Guideline 201. The

pH of the test vessels was 7.7-7.8 at 0 hr and 7.5-7.9 at 72

hr. In all cases, the pH shift was less than 1.5 units.

VERIFICATION OF TEST CONCENTRATIONS

Analysis of test preparations at 0 hr demonstrated

concentrations were 91-116% of nominal. After 72 hr, there was

a slight decline in measured concentration to 72-94% of

nominal. Analysis of samples from unopened replicate vessels

returned measured concentrations that were 77-103% of nominal

at 72 hr. This slight decline was considered by the Study

Director to reflect possible adsoption of ETBE to algal cells

and/or losses due to volatility when sampling for chemical

analysis.

Given this slight decline in measured test concentration, the

STudy Director considered it was justificable to base the EC50

and NOEC values on geometric mean measured concentrations.

Nominal Analysed 0 hr Analysed 72 hr Geometric mean

(mg/l) (mg/l) (mg/l) (mg/l)

9.2 8.4 6.7 7.5

29 27 23 25

92 91 66 78

290 336 274 303

920 900 748 820

2900 3300 2470 2850

9200 10300 7900 9020

INTERPRETATION

A difference between the EbC50 and ErC50 values is not

uncommon in algal inhibition tests since small changes in

growth rate result in large changes in biomass. Hence the

EbC50 is often lower than the ErC50. Current regulatory advice

(European Chemicals Bureau (2003) Technical Guidance Document

Part II, Chapter 3, Environmental Risk Assessment; OECD (2002)

draft revised Guideline 201, July 2002) notes that for both

risk assessment and classification purposes the ErC50 value

should be used.

**Test substance:** ETBE, CAS No. 637-92-3, 99.97%% pure, Total France.

**Conclusion:** Under the conditions of the test, the 72 hr ErC50 for
ETBE in

Pseudokirchneriella subcapitata was 1200 mg/l (NOEC 9.2 mg/l)

based on nominal concentrations, while calculations based on

geometric mean measured concentrations gave an ErC50 of 1100

mg/l (NOEC 7.5 mg/l).

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

23-DEC-2003 (60)

\- 36/156 -

date: 22-MAR-2006

4\. Ecotoxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[4.4 Toxicity to Microorganisms e.g. Bacteria]{.underline}**

**-**

**[4.5 Chronic Toxicity to Aquatic Organisms]{.underline}**

**[4.5.1 Chronic Toxicity to Fish]{.underline}**

**-**

**[4.5.2 Chronic Toxicity to Aquatic Invertebrates]{.underline}**

**-**

**[TERRESTRIAL ORGANISMS]{.underline}**

**[4.6.1 Toxicity to Sediment Dwelling Organisms]{.underline}**

**-**

**[4.6.2 Toxicity to Terrestrial Plants]{.underline}**

**-**

**[4.6.3 Toxicity to Soil Dwelling Organisms]{.underline}**

**-**

**[4.6.4 Toxicity to other Non-Mamm. Terrestrial Species]{.underline}**

**-**

**[4.7 Biological Effects Monitoring]{.underline}**

**-**

**[4.8 Biotransformation and Kinetics]{.underline}**

**-**

**[4.9 Additional Remarks]{.underline}**

**-**

\- 37/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[5.0 Toxicokinetics, Metabolism and Distribution]{.underline}**

**In Vitro/in vivo:** In vitro

**Type:** Metabolism

**Species:** mouse

**Year:** 1999

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE

Male and female homozygous 2E-/- mice (deficient in cytochrome

P-450 2E1) were obtained from the National Cancer Institute

(Bethesda, MD) and used to establish an in-house breeding

colony.

Female C57BL/6N and female 129/Sv mice were purchased from

Taconic Farms (Germantown, NY). \[Comment: these are the

parental lineage strains of the 2E- knock-out mice, and

contain intact functional 2E1 alleles.\]

Diet: Purina Laboratory Chow, ad libitum

Water: unspecified, ad libitum

PREPARATION OF MICROSOMAL FRACTION

Animals were killed by decapitation, livers removed and stored

frozen at -80 degrees C. After thawing, livers were

homogenised (Ultra-Turrax polytron) in Tris-HCl buffer (50 mM,

1.15% KCl, pH 7.4). The microsomal fraction was isolated by

differential centrifugation. Protein content was determined by

the method of Lowry et al.

NDMA DEMETHYLATION ASSAY

N-demethylation of 14C-N-nitrosodimethylamine (an assay that

is highly specific for cytochrome P-450 2E1) was determined by

following release of 14C-formaldehyde (Hong et al., 1989,

Cancer Res., 49, 2973-2979).

METABOLISM OF ETBE

The incubation mixture (sealed vial, 37 degrees C) contained a

NADPH regenerating system, microsomal protein (1.5 mg/ml) and

ETBE (0.1 mM) in a total volume of 0.4 ml Tris-HCl (50 mM, pH

7.4). The reaction was halted after 30 min by addition of 25%

zinc sulphate (50ul) and saturated barium hydroxide (50 ul).

Formation of tert-butanol (TBA) was determined by headspace GC

analysis.

STATISTICAL METHODS

Values are presented as mean and SD. Data analysed by ANOVA

followed by Newman-Keuls test.

**Result:** NDMA DEMETHYLASE ACTIVITY

No NDMA demethylase activity was detectable in hepatic

\- 38/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

microsomes from 2E-/- knock-out mice. In contrast microsomal

NDMA demethylase activity was present in C57BL/6N (99.8

pmol/min/mg protein) and 129/Sv (100.2 pmol/min/mg protein)

mice.

HEPATIC MICROSOMAL METABOLISM OF ETBE

No metabolism of ETBE was found in hepatic cytosol (data not

presented by Authors).

The following rates of metabolism (conversion of ETBE to TBA)

were recorded for the three strains of mouse:

C57B/6N (female): 0.70 +/- 0.12 nmol/min/mg protein

129/Sv (female): 0.66 +/- 0.14

2E1 -/- (female): 0.51 +/- 0.24

(Mean and SD for 4 or 5 replicate incubations)

Comparable rates of hepatic microsomal conversion of ETBE to

TBA were present in males and females of different ages:

4 month old

\- males: 0.45 +/- 0.11 nmol/min/mg protein

\- females: 0.11 +/- 0.04

8 month old

\- males: 0.68 +/- 0.04

-females: 0.40 +/- 0.19

(Mean and SD for triplicate incubations)

**Test substance:** ETBE, c.99%, Aldrich Chemical Co., Milwaukee, WI.

**Conclusion:** Results from this in vitro investigation indicate that

cytochrome P-450 2E1 plays a minor role in conversion of ETBE

to TBA by mouse hepatic microsomal enzymes.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

27-OCT-2003 (36) (38)

**In Vitro/in vivo:** In vitro

**Type:** Metabolism

**Species:** rat

**Year:** 1997

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE

Male SD rats (9 wk old) were obtained from Taconic Farms

(Germantown, NY) and acclimatised for 1 wk.

Diet: Lab Diet 5012 (PMI Feeds Inc., St Louis, MO), ad libitum

Water: tap water, ad libitum

\- 39/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

PREPARATION OF MICROSOMAL FRACTIONS

Animals were killed by decapitation, and the nasal mucosa

(including olfactory and respiratory epithelium) collected and

stored frozen at -80 degrees C (Hong et al., 1991, Cancer Res,

51, 1509-1514). The liver, lungs and olfactory bulb were also

immediately removed and stored at -80 degrees. After thawing,

tissues were homogenised (Ultra-Turra polytron) in Tris-HCl

buffer (50 mM, 1.15% KCl, pH 7.4). The microsomal fraction was

isolated by differential centrifugation. Protein content was

determined by the method of Lowry et al.

METABOLISM OF ETBE

The incubation mixture (sealed vial, 37 degrees C) contained a

NADPH regenerating system, microsomal protein (1.5 mg/ml

except for nasal mucosa = 0.25 mg/ml) and ETBE (0.25 mM) in a

total volume of 0.4 ml Tris-HCl (50 mM, pH 7.4). The reaction

was halted after 30 min by addition of 25% zinc sulphate

(50ul) and saturated barium hydroxide (50 ul).

Some incubations were conducted in the absence of cofactor

(NADPH) or in presence of carbon monoxide. Enzyme kinetics

were investigated using 0.01-2.5 mM ETBE. The influence of

TAME (tert-amyl methyl ether, a structurally-related

substance) on metabolism of ETBE by olfactory mucosal

microsomes was also investigated.

Formation of tert-butanol (TBA) was determined by headspace GC

analysis.

STATISTICAL METHODS

Values are presented as mean and SD.

**Result:** METABOLISM OF ETBE (0.25 mM) BY MICROSOMAL FRACTIONS FROM

DIFFERENT TISSUES

The following specific activities were obtained:

Nasal mucosa

\- olfactory: 8.78 +/- 0.55 nmol TBA/min/mg protein

\- respiratory: 0.95 +/- 0.18

Liver: 0.24 +/- 0.01

Lung: not detected

Kidney: not detected

Olfactory bulb: not detected

(limit of detection not reported)

Carbon monoxide inhibited conversion of ETBE to TBA by rat

olfactory mucosal microsomes; omission of NAPDH eliminated

metabolism:

Control: 9.5 nmol TBA/min/mg protein

plus CO: 1.2

minus NADPH: no activity detected

KINETIC STUDIES

Metabolism of ETBE by rat olfatory microsomes exhibited

Michaelis-Menton kinetics over the concentration range 0.01 to

\- 40/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

2.5 mM ETBE:

Km = 125 uM

Vmax = 11.7 nmol TBA/min/mg

INFLUENCE OF TAME (0.1-0.5 mM) ON METABOLISM OF ETBE (0.25 mM)

BY RAT OLFACTORY MICROSOMES

TAME inhibited microsomal metabolism of ETBE:

Control: 9.2 nmol TBA/min/mg protein

plus 0.1 mM TAME: 4.8 (48% inhibition)

plus 0.25 mM TAME: 3.7 (60% inhibition)

plus 0.5 mM: 2.2 (76% inhibition)

**Test substance:** ETBE, c.99%, Aldrich Chemical Co., Milwaukee, WI.

**Conclusion:** Results from these studies demonstrate that cytochrome
P-450

dependent metabolism of ETBE was approx. 36 fold greater in

rat nasal olfactory epithelium than in rat liver.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

30-NOV-2003 (37) (38)

**In Vitro/in vivo:** In vitro

**Type:** Metabolism

**Species:** rat

**Year:** 1998

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** MICROSOMES

Liver microsomal fractions were prepared from male

Sprague-Dawley rats (6-8 wk old) pretreated as follows:

\- ETBE in corn oil administered i.p. (dose volume not

specified) at dose levels of 200 and 400 mg/kg for 4 days;

\- ETBE dosed orally at 2 mL/kg in 50% corn oil solution for 2

days;

\- phenobarbitone i.p. at 80 mg/kg in saline for 3 days;

\- B-naphthoflavone i.p. at 40 mg/kg in corn oil for 3 days;

\- dexamethasone at 50 mg/kg in corn oil for 4 days;

\- pyrazole at 200 mg/kg in saline for 4 days;

\- t-butyl alcohol (TBA) 200 or 400 mg/kg for 4 days.

The washed microsomal pellets were resuspended in 100 mM

phosphate buffer, 1 mM EDTA (pH 7.4) and stored at -80°C.

PURIFIED ENZYME STUDIES

CYP P450B1 and NADPH-P450-reductase (following phenobarbitone

pretreatment), P4502E1 (after pyrazole pretreatment) and

P4502C11 from control animals were isolated and purified from

rat liver according to established published methods. The

purified enzymes were reconstituted in a system comprising 0.1

nmol purified P450, 0.3 nmol of P450 reductase, 30 mg of

\- 41/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

dilauroylphosphatidylcholine together with substrate in 1 mL

of 0.1 mM phosphate buffer, pH 7.4. The reaction was

initiated by the addition of NADPH (1 mM).

ANTIBODY STUDIES

Polyclonal antibodies (rabbit anti-rat P450B1 and

anti-P4502E1) were obtained from Oxygene (Dallas, TX, USA).

Goat anti-rabbit IgG were purchased from Dako (Copenhagen,

Denmark). Anti-P4502E1 IgG (shown by Western blotting to

recognize the same antigen as the commercially prepared

P4502E1 polyclonal antibodies purchased from Oxygene) was

prepared from female New Zealand white rabbits.

ENZYME ASSAYS

\- aniline hydroxylase was determined by quantifying formation

of p-aminophenol

\- benzphetamine demethylase (BzD) and erythromycin demethylase

(ErD) activities from formation of formaldehyde

\- ethoxyresorufin-O-deethylase (EROD) and PROD activities were

determined by measuring formation of resorufin

\- para-nitrophenol hydroxylase (pNPH) activity followed

formation of 4-aminocatechol

\- testosterone hydroxylase activity was measured using an HPLC

method

All activities were determined according to published

methods).

METABOLISM AND KINETIC STUDIES

The extent of ETBE deethylation was followed from production

of acetaldehyde using the semicarbazide/semicarbazone method,

and quantified by HPLC (undertaken according to published

methods). Concentrations in the range 0.05 to 20 mM were used

to assess microsomal kinetic constants (Km and Vmax).

INHIBITION STUDIES

Specific chemical P450 inhibitors were added to microsomal

incubations to assess the significance of specific P450

isozymes in ETBE metabolism:

\- triacetyloleandromycin (TAO) for P4503A4

\- 4-methyl pyrazole (4-MP) for P4502E1

\- metapyrone (MET) for P4502B

WESTERN BLOT ANALYSIS

Hepatic microsomes from control and ETBE-treated rats were

analysed by Western blotting using polyclonal antibodies

against P450B1/2 and 2E1.

STATISTICAL METHODS

Students t test was used to compare activity present in

various microsomal fractions etc (no further details).

**Remark:** Reviewers comment: The authors concluded that since ETBE

induced P4502B1 only when dosed orally at high levels (2 ml/kg

bw), it was unlikely that low environmental exposures would

\- 42/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

impact ETBE metabolism in vivo. The reliability of this

conclusion in the absence of appropriate studies (prolonged

low level inhalation exposure) is not certain to this

reviewer.

**Result:** EFFECTS OF ETBE AND TBA ADMINISTRATION ON P450 ACTIVITIES

ETBE at doses of 200 or 400 mg/kg (i.p.) for 4 days had no

effect on total P450, BzD, ErD, pNPH, PROD or EROD. PROD and

pNPH were increased (approx. 7-fold and approx. 2-fold,

respectively) after pretreatment with 2 mL/kg/day for 3 days.

PROD, EROD, ErD, and pNPH activities were used as selective

markers for P4502B1/2, 1A1/2, 3A1/2 and 2E1 respectively.

WESTERN BLOT ANALYSIS

ETBE (dosed orally but no i.p.) at 2mL/kg appeared to induce

both P4502B1 and 2E1.

KINETIC PARAMETERS

ETBE was metabolized in control and induced microsomal enzymes

to actealdehyde. This reaction had an absolute requirement

for molecular oxygen and NADPH, while acetaldehyde production

was inhibited by approx. 80% by carbon monoxide.

Production of acetaldehyde followed simple Michaelis-Menten

kinetics and was linear up to 30 min in the presence of 1 mg

microsomal protein. Only microsomes from PB induced rats

showed enhanced rate of acetaldehyde metabolism.

Purified CYP2B1 catalysed acetaldehyde production in a

reconstituted system, with a high turnover number and a Km

similar for control or induced microsomes. 2E1, 2C11 and 1A1

had modest turnover and the authors suggest that P4502B1 may

be the major contributor (in rat) to ETBE metabolism in vivo.

INHIBITION STUDIES

TAO (P4503A4 inhibitor) did not diminish metabolism of ETBE in

microsomes from PB and PYR induced rats. Met (P4502B

inhibitor) caused \~60-75% inhibition but only in PB induced

microsomes. 4-MP (P4502E1 inhibitor) had little or no effect

on ETBE metabolism.

Immuno-inhibition studies using PYR-induced microsomes and

anti-P4502E1 IgG did not inhibit ETBE metabolism.

Testosterone (0.1 mM) inhibited metabolism of ETBE by 75% in

(untreated) female rat microsomes indicating that P4502A1 may

be important in female rats.

**Test substance:** ETBE, purity not stated, Fluka, Buchs, Switzerland.

**Conclusion:** CYP2B1 and 2E1 were induced in rat hepatic microsomes

following oral (but not i.p.) treatment with ETBE at 2 ml/kg

bw, with an increase in immunodetectable 2B1 and 2E1

apoproteins in these fractions. Following treatment with

other inducing agents, only PB increased metabolic capacity

compared to controls. Reconstituted purified 2B1 showed

\- 43/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

higher turnover than 2E1, 2C11 and 1A1. The authors conclude

that 2B1 has a prominent role in ETBE metabolism and the other

isoforms a secondary role. Purified P4502A1 was not available

for study but investigations in female rat with testosterone

indicated that P450 isoforms belonging to the 2A subfamily

might be important in rat.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

30-NOV-2003 (83)

**In Vitro/in vivo:** In vitro

**Type:** Metabolism

**Species:** human

**Year:** 1997

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** EXPRESSION OF HUMAN CYTOCHROME P-450 ENZYMES

Human CYP2A6 and CYP2E1 were obtained by expressing the

corresponding cDNAs in a baculovirus expression system

(Pattern and Koch, 1995, Arch Biochem Biophys, 317, 504-513).

Sf9 insect cells were infected simultaneously either with the

CYP2A6 and P-450 reductase recombinant viruses or with the

CYP2E1 and reductase recombinant viruses (no further details

provided). A microsomal fraction was prepared by sonication of

the infected cells followed by centrifugations at 40,000 rpm.

P450 content and P450 reductase activity were determined as

described by Patten and Koch (see earlier record).

METABOLISM OF ETBE

The incubation mixture (sealed vial, 37 degrees C) contained a

NADPH regenerating system, microsomal protein (1.5 mg/ml) and

ETBE (0.1 mM) in a total volume of 0.4 ml Tris-HCl (50 mM, pH

7.4). The reaction was halted after 30 min by addition of 25%

zinc sulphate (50ul) and saturated barium hydroxide (50 ul).

Formation of tert-butanol (TBA) was determined by headspace GC

analysis.

STATISTICAL METHODS

Values are presented as mean and SD.

**Result:** Metabolism of ETBE to TBA by human CYP2A6 was approx.
17-fold

greater than metabolism of ETBE by human CYP2E1:

CYP2A6: 13.6 nmol TBA formed/min/nmol P450

CYP2E1: 0.8 nmol TBA formed/min/nmol P450

**Test substance:** No details available.

**Conclusion:** The results demonstrated that cloned human CYP2A6 is
more

active than cloned human CYP2E1 in metabolising ETBE to

tert-butanol in vitro.

\- 44/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

27-OCT-2003 (39)

**In Vitro/in vivo:** In vitro

**Type:** Metabolism

**Species:** human

**Year:** 2001

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** LIVER SAMPLES AND PREPARATION OF MICROSOMAL FRACTION

Liver samples were obtained from 11 adult donors (9 male, 2

female, age 41+/- 11 yr) at onset of brain death, immediately

frozen on dry ice then stored in liquid nitrogen. Microsomal

fractions were prepared (Berthou et al., 1989, Xenobiotica,

19, 401-417) and stored at -80 degrees C until use.

METABOLISM OF ETBE

The incubation mixture (sealed vial, 37 degrees C) contained

NADPH, microsomal protein (1.2 mg/ml) and ETBE (0.5 or 10 mM)

in a total volume of 0.5 ml Tris-HCl (50 mM, pH 7.4). The

reaction was halted after 30 min by addition of 0.3M

perchloric acid (125 ul) for HPLC analysis or zinc sulphate

(200 ul; concentration unspecified) and saturated barium

hydroxide (200 ul) for GC analysis. Blanks were run without

NADPH.

Michaelis-Menten kinetic parameters for ETBE metabolism were

determined by following formaldehyde formation in incubations

containing 0.01-20 mM ETBE and microsomal fractions from 3

human donors.

METABOLISM OF ETBE BY HETEROLOGOUSLY EXPRESSED CYTOCHROME P450

Heterologously-expressed human CYP proteins were purchased

from Gentest Corp. (Woburn, MA, USA). The incubation (0.5 ml)

contained CYP2A6, CYP2B6, CYP2E1 or CYP3A4 (40, 57, 72 or 17

pmol, respectively), NADPH-P450 reductase (concentration

unspecified), NADPH (concentration unspecified) and ETBE (0.5

or 10 mM).

ANALYSIS OF METABOLITES

Formation of tert-butanol (TBA) was quantified by GC, and

deethylation of ETBE to formaldehyde followed by HPLC (after

reaction of formaldehyde with 2,4-dinitrophenylhydrazone

(DNP), crotonaldehyde-DNP internal standard).

OTHER CYTOCHROME P450 DEPENDENT ACTIVITIES

Methoxyresorufin O-demethylase (MROD; marker for CYP1A2

\- 45/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

activity), ethoxyresorufin O-deethylase (EROD; CYP1A1),

4-nitrophenol hydroxylation (4-NP; CYP2E1),nifedipine

dehydrogenation (NIF; CYP3A4) and coumarin 7-hydroxylation

(7-OH; CYP2A6) were determined using standard methods.

INHIBITION STUDIES

Hepatic microsomal metabolism of ETBE (0.5 or 3 mM) was

investigated in the presence of 8-methoxypsoralen (8-MOP; 0.02

mM; selective inhibitor of CYP2A6), diethyldithiocarbamate

(DEDTC; 0.3 mM; CYP2E1) or ketoconazole (5 uM; CYP3A4).

STATISTICAL METHODS

Least squares regression was used to determine correlation

coefficients. t values for the null hypothesis of no linear

association were calculated, and found to be equivalent to r =

0.7 for P\<0.02.

**Result:** METABOLISM OF ETBE

Metabolism of ETBE by human microsomal fractions exhibited

complex kinetics. An Eadie-Hostee plot (v versus v/\[ETBE\])

revealed biphasic kinetics, consistent with a 2-site model,

with a Km1 of 0.11 mM and a Vmax1 of 0.14 nmol/min/mg protein,

and a Km2 of 5.0 mM and a Vmax2 of 1.7 nmol/min/mg protein.

Results obtained for the 11 subjects showed an approx. 4-fold

range in activity when metabolism was assessed using 0.5 or 10

mM ETBE (no quantitative data reported).

ENZYME CORRELATION STUDIES

Enzyme correlation studies showed that demethylation of ETBE

correlated well with NIF and 7-OH activity:

Correlation at 0.5 mM ETBE:

EROD 0.14

MROD 0.25

4-NP 0.58

NIF 0.72\*

7-OH 0.85\*\*

Correlation at 10 mM ETBE:

EROD 0.41

MROD 0.65

4-NP 0.52

NIF 0.68

7-OH 0.88\*

(\* P\<0.02; \*\* P\<0.01)

INHIBITOR STUDIES

Inhibition studies demonstrated that ETBE metabolism was

strongly inhibited by 8-MPO (inhibitor of CYP2A6) and DEDTC

(CYP2E1), and less affected by Ketoconazole (CYP3A4):

Inhibitor = 8-MPO

0.5 mM ETBE: 55.4% inhibition

\- 46/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

3 mM ETBE: 79.1% inhibition

Inhibitor = DEDTC

0.5 mM ETBE: 64.3% inhibition

3 mM ETBE: 83.4%

Inhibitor = Ketoconazole

0.5 mM ETBE: 20.9% inhibition

3 mM ETBE: 7.4% inhibition

METABOLISM OF ETBE BY HETEROLOGOUSLY EXPRESSED CYTOCHROME P450

CYP2A6 exhibited the greatest turnover number, and CYP2B6 the

lowest, for ETBE:

CYP2A6

0.5 mM ETBE: 2.85 min-1

10 mM ETBE: 9.8 min-1

CYP2B6

0.5 mM ETBE: 0.28 min-1

10 mM ETBE: 3.2 min-1

CYP3A4

0.5 mM ETBE: 0.45 min-1

10 mM ETBE: 7.6 min-1

CYP2E1

0.5 mM ETBE: 1.7 min-1

10 mM ETBE: 5.6 min-1

**Test substance:** ETBE, 97%, Aldrich Chemical,
Saint-Quentin-Fallavier, France.

**Conclusion:** Kinetic data, correlation studies, chemical inhibition
and use

of purified heterologously expressed cytochrome P-450

haemoproteins showed that CYP2A6 was the primary enzyme

involved in the metabolism of ETBE. CYP3A4 and CYP2B6 were

also found to be involved, whereas CYP2E1 was found to be less

important.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

30-NOV-2003 (52)

**In Vitro/in vivo:** In vitro

**Type:** Metabolism

**Species:** human

**Year:** 2001

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** METABOLISM OF ETBE BY HUMAN HEPATIC MICROSOMAL FRACTIONS

\- 47/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Liver samples were obtained from consenting liver cancer

patients (age 37-80 yr). The samples were from tissue adjacent

to the tumour, and were snap frozen in liquid nitrogen within

30 minutes of removal. The microsomal fraction was isolated by

differential centrifugation.

EXPRESSION OF HUMAN CYTOCHROME P-450 ENZYMES

Metabolism of ETBE by human CYP2A6, CYP2E1, CYP2B6 and CYP1A2

expressed in the human AHH-1 TK+/- B-lymphoblastoid line

(Gentest, Woburn, MA) was assessed in vitro. The report states

that the cells contained very low levels of endogenous

cytochrome P450 enzyme activity, and each cell line was

engineered to consistently express a specific human CYP cDNA.

The cells were stored frozen at -80 degrees C until use.

ENZYME CORRELATION STUDIES

The following substrate-specific cytochrome P-450 dependent

enzyme were determined in 15 human hepatic microsomal samples

(HepatoScreen kit, Human Biologics, Phoenix, AZ; no assay

details provided):

7-ethoxy resorufin O-delakylation: marker for CYP1A2

coumarin hydroxylation: CYP2A6

S-mephenytoin N-demethylation: CYP2B6

tolbutamide hydroxylation: CYP2C9

S-mephenytoin hydroxylation: CYP2C19

dextromethorphan O-demethylation: CYP2D6

chlorzoxazone hydroxylation: CYP2E1

dextromethorphan N-demethylation: CYP3A4

testosterone oxidation: CYP3A4/5

Demethylation of ETBE by these 15 samples was also quantified.

METABOLISM OF ETBE

The incubation mixture (sealed vial, 37 degrees C) contained a

NADPH regenerating system, expressed CYP proteins (1.5 mg/ml)

or microsomal protein (250-375 ug/ml) and ETBE (0.1 mM) in a

total volume of 0.4 ml Tris-HCl (50 mM, pH 7.4). The reaction

was halted after 30 min by addition of 25% zinc sulphate

(50ul) and saturated barium hydroxide (50 ul). Formation of

tert-butanol (TBA) was determined by headspace GC analysis

(detection limit 15 ng TBA).

STATISTICAL METHODS

Values are presented as mean and SD. Correlation analysis was

performed using CA-Criketgraph III version 1.01 software

(Computer Associates International, Islandia, NY).

**Result:** METABOLISM OF ETBE BY HEPATIC MICROSOMAL FRACTIONS

Metabolism of ETBE by human hepatic microsomal enzymes was

variable: range 179 to 3,134 pmol formaldehyde formed/min/mg

microsomal protein.

Comment: number of samples not specified; graphical data

suggest that at least 10 were used.

\- 48/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

METABOLISM OF ETBE BY HETEROLOGOUSLY EXPRESSED HUMAN

CYTOCHROME P450 ENZYMES

CYP 2A6 expressed the greatest activity toward ETBE:

CYP2A6: 1.61 +/- 0.29 pmol TBA/min/pmol CYP

CYP2E1: 0.34 +/- 0.0

CYP2B6: 0.18 +/- 0.05

CYP1A2: 0.13 +/- 0.0

Note: the report states that activities for CYP1B1, CYP2C8,

CYP2C9, CYP2C19 and CYP2D6 were not detectable in these

samples. No results are presented.

ENZYME CORRELATION STUDIES

ETBE metabolism correlated most closely with expression of

CYP2A6 and, to a lesser degree, with CYP2B6, CYP3A4 and

CYP3A4/5 present in human hepatic microsomes:

CYP2A6: 0.95 (correlation coefficient)

CYP2E1: 0.19

CYP2B6: 0.69

CYP1A2: 0.01

CYP3A4: 0.71

CYP3A4/5: 0.64

CYP2C9: 0.28

CYP2C19: 0.18

CYP2D6: 0.01

There was no correlation with CYP1A2 or CYP2D6 activities.

**Test substance:** ETBE, \>99%, Aldrich Chemical Co., Milwaukee, WI.

**Conclusion:** Metabolism of ETBE (O-demethylation) by human hepatic

microsomal fractions exhibited a high degree of

interindividual variation. This activity was associated with

the expression of cytochrome P450 2A6 in human liver

microsomes and engineered cell lines expressing human

recombinant cDNA. Other isozymes (including CYP2E1, CYP2B6,

CYP3A4 and CYP3A4/5) appeared of minor importance.

**Reliability:** (2) valid with restrictions

Study available for review. Briefly reported experimental

investigation, acceptable for assessment.

30-NOV-2003 (35) (38)

**In Vitro/in vivo:** In vitro

**Type:** Toxicokinetics

**Species:** other: human PBTK model

**Year:** 1999

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** PHYSIOLOGICALLY BASED TOXICOKINETIC MODEL

Organs were grouped together into compartments according to

blood flow (lung and arterial blood; readily perfused tissues;

working muscles; resting muscles; liver) and fat content; the

model for tert butanol (TBA) also included urine. Two muscle

compartments were included since human TK used to validate the

\- 49/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

model (from Nihlen et al (1998) Tox Sci 46, 1; see entry in

this section) were collected under conditions of light

exercise. First order metabolism was assumed since linear

kinetics had been observed experimentally in humans exposed to

5, 25 or 50 ppm ETBE vapour (Nihlen et al., 1998).

Physiological characteristics of the model were based upon

previously published equations (Droz (1992) Ann Occup Hyg, 36,

295). Partition coefficients for ETBE and TBA were based on

published results obtained by the authors and others.

Information on body composition and physiological parameters

were obtained from Nihlen et al (1998).

A sensitivity analysis was performed (Pierce et al (1996)

Toxicol Appl Pharmacol, 139, 49) to identify which

physical/physiological parameters most strongly influenced the

model.

PREDICTIONS IN HUMANS

The PBTK model was used to predict biomarker levels in blood,

urine and exhaled air at the end of a hypothetical work shift

(8 hr duration) and the next morning (24 hr after the start of

the first exposure, immediately prior to the next work shift).

In other simulations, it was assumed that subjects were

exposed to 50 ppm ETBE from 8am to noon, and from 1 pm to 5 pm

5 d/wk for 2 wk. The impact of fluctuations in ETBE exposure

concentration (mean modelled time weighted average = 50 ppm,

with a SD of 25 ppm in scenario 1 and a SD of 100 ppm in

scenario 2) were investigated, as was the influence of work

load (0, 50, 100 or 150 W) and changes in body composition.

**Result:** Reviewer\'s comment: Results in this report are generally

presented either as descriptive information or graphical

plots. Quantitative data are given, where available.

The model adequately described real-life human exposure to 5,

25 or 50 ppm ETBE vapour for 2 hr.

Simulated exposure over 2 weeks was associated with a minor

predicted accumulation of ETBE and TBA at the end of each work

week, with elimination over the weekend.

Physical exercise markedly increased the update of ETBE, with

an approx. 2-fold increase at 100 W (heavy exercise) compared

to that predicted at rest (0 W).

The predicted concentration of ETBE in blood and exhaled air

at the end of a work shift was sensitive to modelled

fluctuations in external exposure:

\- 4.4 or 44 uM ETBE predicted in blood, and 17 or 85 umol

ETBE/min in exhaled air, after simulated exposure to 50+/-25

ppm or 50+/-100 ppm ETBE respectively for 8 hr;

\- 0.8 or 7.3 uM TBA predicted in blood, 0.7 or 7.3 umol

TBA/min in exhaled air, and 1.0 or 8.0 umol TBA/min in urine

after simulated exposure to 50+/-25 ppm or 50+/-100 ppm ETBE

\- 50/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

respectively for 8 hr.

The predicted concentration of TBA in urine at the end of a

work shift or the following morning was relatively unaffected

by fluctuations in external (inhaled) ETBE concentration.

50+/-25 ppm ETBE:

\- endshift urine concentration = 0.8 uM

\- next morning urine concentration = 1.1 uM

50+/-100 ppm ETBE:

\- endshift urine concentration = 7.9 uM

\- next morning urine concentration = 12 uM

The coefficient of variation (CV) for the predicted end-shift

or next morning mean concentration of ETBE in blood (uM) or

elimination of ETBE in exhaled air (umol/min) for 8 subjects

exposed to 50 ppm ETBE for 5 consecutive days was around 10%

or less. The inter-individual CV for predicted end-shift

exhalation of TBA (umol/min) was 9% while that for urinary

excretion (umol/min) was 19%; a larger inter-individual CV of

29-30% was obtained for the equivalent next morning

predictions.

The sensitivity analysis showed the concentration of ETBE in

blood and exhaled air was most strongly influenced by the

blood/air partition coefficient of ETBE and body fat content,

while body weight, partition coefficients and hepatic

metabolism influenced the fate of TBA.

**Conclusion:** The authors describe a PBTK model for inhalation
exposure to

ETBE in humans. The results indicate that the concentration of

ETBE in blood and exhaled air at the end of a work-shift is

sensitive to fluctuations in the external environment, whereas

TBA levels the next morning were less sensitive. TBA in urine

is proposed as a suitable biomarker for the estimation ETBE

exposure.

**Reliability:** (2) valid with restrictions

Study available for review. Accepted modelling method,

suitable for assessment.

30-NOV-2003 (64)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** mouse

**No. of animals, males:** 3

**No. of animals, females:** 3

**Doses, males:** 0 (control), 500, 750, 1000, 1750, 2500, 5000 ppm

**Doses, females:** 0 (control), 500, 750, 1000, 1750, 2500, 5000 ppm

**Route of administration:** inhalation

**Exposure time:** 6 hour(s)

**Year:** 1995

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

\- 51/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Method:** ANIMALS AND TREATMENTS

Animals: male and female CD-1 mice (6-8 weeks)

Supplier: Charles River, Portage, MI, USA

Group size: n=3

Diet: Agway Prolab Animal Diet Rat, Mouse, Hamster 3000 (Agway

Inc.), ad libitum

Water: unspecified, ad libitum

Treatment: 0, 500, 750, 1000, 1750, 2500, 5000 ppm \[14C\]-

ETBE, 6 hr, nose-only

Exposure chamber: stainless steel (Cannon design) nose-only

unit. (Uniformity of exposure concentration was continuously

monitored by gas chromatography (GC) and LSC during the

experiments.)

Immediately after exposure 1 mouse/sex/group was transferred

to an individual glass metabolism cage while another was

sacrificed by exsanguination and the liver removed. The

remaining animal remained in a metabolism cage (for up to 96

hr).

SAMPLE COLLECTION

The following were collected (the longer collection periods

applied only to the 5000 ppm groups):

\- expired volatiles (charcoal traps) at 1,3, 6, 12, 24, 48,

72, 96 and 118 hr;

\- 14CO2 (KOH trap) at 1, 3, 6, 12, 24, 48, 72, 96, and 118 hr;

\- urine (over dry-ice) at 12, 18 24, 48, 72, 96 and 118 hr;

\- faeces (room temperature) at 12, 18 24, 48, 72, 96 and 118

hr;

\- cage wash at 118 hr.

After the last collection point animals were euthanized by CO2

asphyxiation.

SAMPLE PROCESSING

Radioactivity present in 14CO2 and cage wash was quantified by

direct liquid scintillation counting (LSC), while faeces and

liver homogenates were combusted prior to counting. Exhaled

volatiles were extracted from charcoal traps with methanol and

an aliquot assayed by LSC.

Comment: The total dose of radioactivity was not calculated.

STATISTICAL METHODS

No specific methods or software given.

**Remark:** The study was initially designed to incorporate a repeat

dosing phase but this was cancelled.

**Result:** EXPOSURE

Achieved exposure levels throughout the 6 h period were +/-

10% of target (478, 763, 991, 1695, 2507, 5125 ppm)

ELIMINATION OF \[14C\]-ETBE

Comment: The total absorbed dose was not calculated and

elimination values are expressed as % of total radioactivity

recovered in excreta.

\- 52/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

The majority of absorbed radioactivity was eliminated over 48

hr, and 83-93% of the total excreted in urine or as exhaled

volatile organics at all exposure levels. The majority of

exhaled volatile organics were eliminated by 12 hr with

highest proportions in the 1 to 3 h collection. Minor amounts

were eliminated as 14CO2 or in faeces.

The proportion of radioactivity in urine decreased from 74% to

46% as exposure concentrations increased from 500 ppm to 1750

ppm, with a concomitant increase in volatile organics from 10%

to 42%. The relative proportions remained quite similar as

exposure concentrations increased from 1750 ppm to 5000 ppm.

Although not noted by the authors it appeared that there was

no consistent gender difference in elimination

characteristics.

Blood and liver concentrations (mg equiv. ETBE/g) increased

from 154 to 481 (blood) and 208 to 724 (liver) as exposure

concentration increased from 500 ppm to 1750 ppm. Values then

remained similar from 1750 ppm to 5000 ppm.

**Test substance:** Non-labeled ETBE (98%) was obtained from Pittsburgh
Applied

Research Corp. (PA, USA). 14C-ETBE was obtained from Wizard

Laboratories (CA, USA), chemical purity 99.8%.

**Conclusion:** The majority (83-93%) of an inhaled dose of 14C-ETBE was

eliminated by the CD-1 mice within 48 hr. The proportion of

radioactivity in urine decreased from 74% to 46% as exposure

concentrations increased from 500 ppm to 1750 ppm, with a

concomitant increase in volatile organics from 10% to 42%.

The relative proportions remained quite similar (plateau) as

exposure concentrations increased from 1750 ppm to 5000 ppm.

The authors conclude that processes responsible for the

absorption and elimination of ETBE in the mouse appear to

become saturated at an exposure concentration of 1750 ppm.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

30-NOV-2003 (81)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** mouse

**No. of animals, males:** 6

**Doses, males:** 5000 ppm

**Route of administration:** inhalation

**Exposure time:** 6 hour(s)

**Year:** 1966

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

\- 53/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Method:** ANIMALS AND TREATMENTS

Six male CD-1 mice (Charles River, NC, USA; bwt 29-33 g) were

exposed to \[14C\]- ETBE (5000 ppm, 6 hr) using a nose-only

exposure tower. Environmental conditions were monitored and

recorded automatically. The 12 h light/dark cycle was

interrupted briefly on study days for collection of samples

during a dark phase. All animals received pelleted Standard

NIH-07 rodent chow (Zeigler Bros., PA, USA) and drinking water

ad libitum prior to and following nose-only exposures.

Each mouse received approx. 0.8 uCi of \[14C\]-ETBE.

Immediately post-exposure, 3 animals were sacrified, carcasses

digested and the total content of radioactivity determined by

liquid scintillation counting (LSC). The remaining animals

were transferred to individual glass metabolism cages for 96

hr, then sacrificed (CO2) and carcasses digested for

quantitation of total radioactivity.

SAMPLE COLLECTION

The following were collected:

\- expired volatiles (charcoal traps) at 1,3, 6, 12, 24, 48, 72

and 96 hr;

\- 14CO2 (KOH trap) at 1, 3, 6, 12, 24, 48, 72 and 96 hr;

\- urine (over dry-ice) at 24, 48, 72 and 96 hr;

\- faeces (room temperature) at 24, 48, 72, and 96 h

cage wash at 96 hr

SAMPLE PROCESSING

Urine, 14CO2, cage wash and nose-only tube wash were assayed

for radioactivity by LSC. Faeces and carcasses were digested

prior to LSC and volatiles removed from charcoal traps with

dimethylformamide (DMF) before direct radioassay by LSC.

Thus, total recovery of radioactivity was determined as a

percentage of the calculated dose.

EXPOSURE APPARATUS

Exposure was conducted using a stainless steel Cannon

nose-only unit (Lab products, Maywood, NJ, USA). Uniformity of

concentration throughout the apparatus was established before

the experiments and was continuously monitored during the

experiments. Radioactivity in the apparatus atmosphere was

measured by LSC and the total ETBE concentration by gas

chromatography (GC).

METABOLISM

Urine was analysed for ETBE, TBA, a-hydroxybutyrate (HBA) and

2-methyl-1,2-propandiol (MPD) by GC-MS. Control urine and

feces were collected from an additional animal for

establishing background concentrations.

DATA ANALYSIS

A Microsoft Excel spreadsheet was used to calculate recoveries

of radioactivity expressed as percentage of total

\- 54/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

radioactivity recovered from the animal or by using the total

radioactivity determined in an animal concurrently exposed and

sacrificed immediately following the 6 hr exposure.

**Result:** EXPOSURE

The mean achieved exposure level was 4927 +/- 209 ppm.

ELIMINATION OF \[14C\]-ETBE

The major pathways of elimination of radioactivity (ETBE

equivalents) were urine (57.32%) and expired air (37.31%, as

volatile organics). Approximately 87% of the absorbed dose

was eliminated within 24 h following the end of exposure and

approximately 98% by 96 h. Only minor proportions were

eliminated as 14CO2 (1.59%) or in feces (1.62%).

BIOTRANSFORMATION OF ETBE

In urine levels of TBA were greater than those of ETBE, the

latter decreasing quickly after exposure. In exhaled air ETBE

was present at higher concentration (44.7 mmoles) than TBA

(16.9 mmoles) at 1 h but ETBE levels decreased quickly and

were lower thereafter. In addition the presence of HBA and

MPD were was detected in urine.

**Test substance:** Non-labeled ETBE (97.5%) was obtained from
Pittsburgh Applied

Research Corp. (PA, USA). 14C-ETBE was obtained from Wizard

Laboratories (CA, USA), chemical purity 99.2%.

**Conclusion:** Following single nose-only exposure to 5000 ppm
\[14C\]-ETBE,

mice eliminated radioactivity (mainly as TBA) in urine and

exhaled air. Amounts of unchanged ETBE were also detected

(greatest at 1 h) but decreased quickly with time. HBA and

MPD, being further products of metabolism of TBA, were also

detected in urine.

**Reliability:** (2) valid with restrictions

Study available for review. GLP compliant method developmnent

study, clearly reported, acceptable for assessment.

02-JAN-2004 (14)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** mouse

**No. of animals, males:** 8

**No. of animals, females:** 8

**Doses, males:** 0, 500, 1750 and 5000 ppm

**Doses, females:** 0, 500, 1750 and 5000 ppm

**Route of administration:** inhalation

**Exposure time:** 6 hour(s)

**Year:** 1996

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND TREATMENTS

Animals: male (20-44 g) and female (15-35 g) CD-1 mice

\- 55/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Supplier: Charles River, NC, USA;

Group size: n=8/sex

Diet: Standard NIH-07 rodent chow (Zeigler Bros., PA, USA), ad

libitum;;

Water: unspecified, ad libitum;

Treatment: 0, 500, 1750 and 5000 ppm \[14C\]- ETBE, 6 hr,

nose-only;

Environmental: conditions were monitored and recorded

automatically; the 12 hr light/dark cycle was interrupted

briefly on study days for collection of samples (during dark

phase);

Exposure chamber: stainless steel Cannon nose-only unit (Lab

products, Maywood, NJ, USA). Uniformity of concentration

throughout the apparatus had been established before the

experiments and was continuously monitored during the

experiments (14C by LSC, total ETBE by GC)

Immediately post-exposure, 4 animals/sex/exposure level were

transferred to individual glass metabolism cages and 4

animals/sex/exposure level were euthanized. The carcasses of

the latter were digested and the total carcass content of

radioactivity determined by liquid scintillation counting

(LSC). The surviving animals remained in the metabolism cages

(air flow rate 200 mL/min) for 48 hr, after which they were

sacrificed by CO2 asphyxiation and carcasses digested for

radioassay.

It was estimated (from exposure levels, breathing rate and

specific activity measurements) that the mice were exposed to

15-18 mCi of \[14C\]-ETBE.

SAMPLE COLLECTION

The following were collected:

\- expired volatiles (charcoal traps) at 1,3, 5, 8, 16, 24 and

48 hr;

\- 14CO2 (KOH trap) at 3, 8, 16, 24 and 48 hr;

\- urine (over dry-ice) at 8, 24 and 48 hr;

\- faeces (room temperature) at 24 and 48 hr;

\- cage wash at 48 hr

At 48 h after the end of the nose-only exposure the animals

were euthanized

SAMPLE PROCESSING

Urine, 14CO2, cage wash and nose-only tube wash were assayed

for radioactivity by LSC. Feces and carcasses were digested

prior to LSC and volatiles removed from charcoal traps with

dimethylformamide (DMF) before direct radioassay by LSC.

Thus, total recovery of radioactivity was determined as a

percentage of the calculated dose.

METABOLISM

Urine was analysed for ETBE, TBA, a-hydroxybutyrate (HBA) and

\- 56/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

2-methyl-1,2-propandiol (MPD) by GC-MS. Concentrations of

ETBE and TBA were quantitated in urine and in DMF extracts

from charcoal traps. Control urine and feces were collected

from an additional animal for establishing background

concentrations.

DATA ANALYSIS

A Microsoft Excel spreadsheet was used to calculate recoveries

of radioactivity expressed as percentage of total

radioactivity recovered from the animal or by using the total

radioactivity determined in an animal concurrently exposed and

sacrificed immediately following the 6 hr exposure.

**Result:** EXPOSURE

The mean achieved exposure levels (activity inhaled) were:

555 ppm (17.98 uCi)

1717 ppm (15.39 uCi)

4957 ppm (17.15 uCi)

ELIMINATION OF \[14C\]-ETBE

The major pathways of elimination of radioactivity (ETBE

equivalents) were urine and expired air. The totals

eliminated increased with increasing exposure but in a less

than dose proportional fashion. At all three exposure levels

\>95% of the absorbed dose was eliminated within 48 h following

the end of exposure.

Males, nose only:

\% absorbed eliminated (48 hr)

500 ppm 1750 ppm 5000 ppm

Exhaled volatiles 12.7 23.3 39.9

Exhaled CO2 1.1 2.2 2.9

Urine 81.3 70.7 52.6

Faeces 3.2 1.1 0.6

Total 98.3 97.4 95.9

Females, nose only:

\% absorbed eliminated (48 hr)

500 ppm 1750 ppm 5000 ppm

Exhaled volatiles 26.7 35.8 47.2

Exhaled CO2 1.2 1.9 3.3

Urine 67.2 60.7 47.4

Faeces 2.2 0.6 0.7

Total 97.4 99.0 98.7

(Values = mean for 4 mice/group)

At the 500 ppm exposure urine accounted for majority of dose

whilst at 5000 ppm exhaled air and urine accounted for

approximately similar proportions. Only minor proportions

were eliminated as 14CO2 or in feces.

RETENTION

Carcass accounted for approx. 2-2.5%, 1-1.5% and 0.6-0.8% of

the absorbed radioactivity in the low, intermediate and high

dose groups, respectively.

\- 57/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

BIOTRANSFORMATION OF ETBE

In urine and exhaled air levels of \[14C\]-TBA were greater than

those of \[14C\]-ETBE, the latter decreasing quickly after

exposure. The period of elimination of TBA in exhaled air was

more extended in the intermediate and high dose groups

(detectable amounts present in both sexes at 16 hr

post-exposure) than in the 500 ppm group (undetectable after 1

hr post-exposure). In contrast detectable amounts of TBA were

present in urine from all three treatment groups at 24 hr

post-dose (both sexes).

In addition the presence of HBA and MPD were confirmed

qualitatively in urine.

**Test substance:** Non-labeled ETBE (97.5%) was obtained from
Pittsburgh Applied

Research Corp. (PA, USA). 14C-ETBE was obtained from Wizard

Laboratories (CA, USA), chemical purity 99.2%.

**Conclusion:** Following single nose-only exposure to \[14C\]-ETBE at
500, 1750

or 5000 ppm, mice eliminated radioactivity mainly in urine and

exhaled air. At 500 ppm the majority of the absorbed dose was

eliminated in urine whilst at 5000ppm urine and exhaled air

accounted for approximately similar proportions. \[14C\]-TBA

was the main elimination product in both urine and exhaled

air, and HBA and MPD were identified qualitatively in urine.

There were no gender differences.

**Reliability:** (2) valid with restrictions

Study available for review. GLP compliant near-guideline

investigation, clearly reported, acceptable for assessment.

30-NOV-2003 (15)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** rat

**No. of animals, males:** 3

**No. of animals, females:** 3

**Doses, males:** 0 (control), 500, 750, 1000, 1750, 2500, 5000 ppm

**Doses, females:** 0 (control), 500, 750, 1000, 1750, 2500, 5000 ppm

**Route of administration:** inhalation

**Exposure time:** 6 hour(s)

**Year:** 1995

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND TREATMENTS

Animals: male and female Fischer 344 rats (10-12 weeks)

Supplier: Harlan-Sprague Dawley, Indianapolis, IN, USA

Group size: n=3

Diet: Agway Prolab Animal Diet Rat, Mouse, Hamster 3000 (Agway

Inc.), ad libitum

Water: unspecified, ad libitum

Treatment: 0, 500, 750, 1000, 1750, 2500, 5000 ppm \[14C\]-

ETBE, 6 hr, nose-only

\- 58/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Exposure chamber: stainless steel (Cannon design) nose-only

unit. (Uniformity of exposure concentration was continuously

monitored by gas chromatography (GC) and LSC during the

experiments.)

Immediately after exposure 1 rat/sex/group was transferred to

an individual glass metabolism cage while another was

sacrificed by exsanguination and the kidneys removed. The

remaining animal remained in a metabolism cage (for up to 96

hr).

SAMPLE COLLECTION

The following were collected (the longer collection periods

applied only to the 5000 ppm groups):

\- expired volatiles (charcoal traps) at 1,3, 6, 12, 24, 48,

72, 96 and 118 hr;

\- 14CO2 (KOH trap) at 1, 3, 6, 12, 24, 48, 72, 96, and 118 hr;

\- urine (over dry-ice) at 12, 18 24, 48, 72, 96 and 118 hr;

\- faeces (room temperature) at 12, 18 24, 48, 72, 96 and 118

hr;

\- cage wash at 118 hr.

After the last collection point animals were euthanazed by CO2

asphyxiation.

SAMPLE PROCESSING

Radioactivity present in 14CO2 and cage wash was quantified by

direct liquid scintillation counting (LSC), while faeces and

liver homogenates were combusted prior to counting. Exhaled

volatiles were extracted from charcoal traps with methanol and

an aliquot assayed by LSC.

Comment: The total dose of radioactivity was not calculated.

STATISTICAL METHODS

No specific methods or software given.

**Remark:** The study was initially designed to incorporate a repeat

dosing phase but this was cancelled.

**Result:** EXPOSURE

Achieved exposure levels throughout the 6 h period were +/-

10% of target (478, 763, 991, 1695, 2507, 5125 ppm).

ELIMINATION OF \[14C\]-ETBE

Comment: The total absorbed dose was not calculated and

elimination values are expressed as % of total radioactivity

recovered in excreta.

The majority of absorbed radioactivity was eliminated by 48 hr

with 96-98% of the total excreted in urine or as exhaled

volatile organics at all exposure levels. The majority of

exhaled volatile organics were eliminated by 12 hr with the

greatest proportions in the 1 to 3 h collection. Minor

amounts were eliminated as 14CO2 or in faeces.

Although not noted by the authors it appeared that there was

\- 59/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

no consistent gender difference in elimination

characteristics.

As exposure concentrations increased from 500 ppm to 1750 ppm

the proportion of radioactivity in urine decreased from 60% to

38% with a concomitant increase in volatile organics from 37%

to 58%. However as exposure concentrations increased from

1750 ppm to 5000 ppm these relative proportions remained quite

similar.

Blood and kidney concentrations (mg equiv. ETBE/g) increased

from 37 to 155 (blood) and 74 to 185 (kidney) as exposure

concentration increased from 500 ppm to 1750 ppm. Values then

remained similar from 1750 ppm to 5000 ppm.

**Test substance:** Non-labeled ETBE (98%) was obtained from Pittsburgh
Applied

Research Corp. (PA, USA). 14C-ETBE was obtained from Wizard

Laboratories (CA, USA), chemical purity 99.8%.

**Conclusion:** The majority (96-98%) of an inhaled dose of 14C-ETBE was

eliminated by the Fischer rat within 48 hr. The proportion of

radioactivity in urine decreased from 60% to 38% as exposure

concentrations increased from 500 ppm to 1750 ppm, with a

concomitant increase in volatile organics from 37% to 58%. The

relative proportions remained quite similar (plateau) as

exposure concentrations increased from 1750 ppm to 5000 ppm.

The authors conclude that processes responsible for the

absorption and elimination of ETBE in the rat appear to become

saturated at an exposure concentration of 1750 ppm.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

27-OCT-2003 (82)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** rat

**No. of animals, males:** 6

**Doses, males:** 5000 ppm

**Route of administration:** inhalation

**Exposure time:** 6 hour(s)

**Year:** 1996

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND TREATMENTS

Six male F-344 rats (Charles River, NC, USA; 234-249 g) were

exposed nose only to 5000 ppm \[14C\]-ETBE. Enviromental

conditions were monitored and recorded automatically. The 12

h light/dark cycle was interrupted briefly on study days for

collection of samples during a dark phase. All animals

received pelleted Standard NIH-07 rodent chow (Zeigler Bros.,

\- 60/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

PA, USA) and drinking water ad libitum prior to and following

nose-only exposures.

Each rat received approx. 3 uCi of \[14C\]-ETBE.

Immediately post-exposure, 3 animals were sacrified, carcasses

digested and the total content of radioactivity determined by

liquid scintillation counting (LSC). The remaining animals

were transferred to individual glass metabolism cages for 96

hr, then sacrificed (CO2) and carcasses digested for

quantitation of total radioactivity.

SAMPLE COLLECTION

See details for mouse study (identical reference number).

SAMPLE PROCESSING

See details for mouse study (identical reference number).

EXPOSURE APPARATUS

See details for mouse study (identical reference number).

METABOLISM

See details for mouse study (identical reference number).

DATA ANALYSIS

See details for mouse study (identical reference number).

**Result:** EXPOSURE

The mean achieved exposure level was 4927 +/- 209 ppm.

ELIMINATION OF \[14C\]-ETBE

The major pathways of elimination of radioactivity (ETBE

equivalents) were urine (43.55%) and expired volatiles

(50.54%). Approximately 90% of the absorbed dose was

eliminated within 24 hr following the end of exposure and

approximately 98% by 96 h. Only minor proportions were

eliminated as 14CO2 (0.87%) or in faeces (3.30%).

BIOTRANSFORMATION OF ETBE

In urine levels of TBA were greater than those of ETBE, the

latter decreasing quickly after exposure. In exhaled air ETBE

was present at higher concentration than TBA up to 3 hr (144.1

mmoles and 34.4 mmoles respectively at 3 hr). ETBE levels

decreased quickly and were lower thereafter. In addition the

presence of HBA and MPD were was detected in urine.

**Test substance:** Non-labeled ETBE (97.5%) was obtained from
Pittsburgh Applied

Research Corp. (PA, USA). 14C-ETBE was obtained from Wizard

Laboratories (CA, USA), chemical purity 99.2%.

**Conclusion:** Following single nose-only exposure to 5000 ppm
\[14C\]-ETBE,

rats eliminated radioactivity (mainly as TBA) in urine and

exhaled air. Amounts of unchanged ETBE were also detected

(greatest at 1 h) but decreased quickly with time whereas

levels of TBA increased to 6 hr. HBA and MPD, being further

products of metabolism of TBA, were also detected in urine.

**Reliability:** (2) valid with restrictions

\- 61/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Study available for review. GLP compliant method developmnent

study, clearly reported, acceptable for assessment.

27-OCT-2003 (14)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** rat

**No. of animals, males:** 8

**No. of animals, females:** 8

**Doses, males:** 0, 500, 1750 and 5000 ppm (single or multiple

exposures)

**Doses, females:** 0, 500, 1750 and 5000 ppm (single or multiple

exposures)

**Route of administration:** inhalation

**Exposure time:** 6 hour(s)

**Year:** 1996

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND TREATMENTS

Animals: male (200-250 g) and female (130 - 180 g) F-344 rats

Supplier: Charles River, NC, USA;

Group size: n=8/sex

Diet: Standard NIH-07 rodent chow (Zeigler Bros., PA, USA), ad

libitum;

Water: unspecified, ad libitum;

Acute treatment regime: 0, 500, 1750 and 5000 ppm \[14C\]-

ETBE, 6 hr, nose-only;

Repeat treatment regime: whole body exposure (6 hr/d) to 0,

500 and 5000 ppm unlabeled ETBE prior to a single 6 hr

nose-only to 500 or 5000 ppm 14C-ETBE;

Environmental: conditions were monitored and recorded

automatically; the 12 hr light/dark cycle was interrupted

briefly on study days for collection of samples (during dark

phase);

Exposure chamber: stainless steel Cannon nose-only unit (Lab

products, Maywood, NJ, USA). Uniformity of concentration

throughout the apparatus had been established before the

experiments and was continuously monitored during the

experiments (14C by LSC, total ETBE by GC)

Immediately post-exposure, 4 animals/sex/exposure level were

transferred to individual glass metabolism cages and 4

animals/sex/exposure level were euthanazed. The carcasses of

the latter were digested and the total carcass content of

radioactivity determined by liquid scintillation counting

(LSC). The surviving animals remained in the metabolism cages

(air flow rate 200 mL/min) for 48 hr, after which they were

sacrificed by CO2 asphyxiation and carcasses digested for

radioassay.

\- 62/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

It was estimated (from exposure levels, breathing rate and

specific activity measurements) that the rats were exposed to

45-51 mCi of \[14C\]-ETBE in the nose-only experiments.

SAMPLE COLLECTION

See details for mouse study (identical reference number).

SAMPLE PROCESSING

See details for mouse study (identical reference number).

METABOLISM

See details for mouse study (identical reference number).

DATA ANALYSIS

See details for mouse study (identical reference number).

**Result:** EXPOSURE

The mean achieved exposure levels (activity inhaled) were:

555 ppm (17.98 uCi)

1717 ppm (15.39 uCi)

4957 ppm (17.15 uCi)

ELIMINATION OF \[14C\]-ETBE

In the nose-only experiment and in the pre-exposed animals the

major pathways of elimination of radioactivity (ETBE

equivalents) were urine and expired air. The totals

eliminated increased with increasing exposure but in a less

than dose proportional fashion. At all exposure levels \>90%

of the absorbed dose was eliminated within 48 h following the

end of exposure.

Males, 6 hr nose only:

\-\--% absorbed eliminated (48 hr)\-\--

500 ppm 1750 ppm 5000 ppm

Exhaled volatiles 27.9 40.8 50.8

Exhaled CO2 1.1 1.5 1.6

Urine 58.6 53.1 44.7

Faeces 2.8 0.1 0.2

Total 90.4 95.6 97.3

(Values = mean for 8 rats/group)

Although a minor pathway the relative proportion eliminated as

14CO2 increased with increasing exposure concentration.

At the 500 ppm exposure urine accounted for majority of dose

whilst at 5000 ppm exhaled air was the major pathway for

elimination. This shift was more pronounced in females (6 hr,

nose only):

\-\--% absorbed eliminated (48 hr)\-\--

500 ppm 1750 ppm 5000 ppm

Exhaled volatiles 31.7 52.2 64.1

Exhaled CO2 1.3 1.7 2.0

Urine 59.3 41.3 30.4

Faeces 1.0 0.5 0.2

Total 93.5 95.8 96.7

\- 63/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

(Values = mean for 8 rats/group)

Pre-exposure to ETBE for 13 days prior to exposure to 14C-ETBE

decreased the percentage of absorbed material eliminated in

exhaled air and increased the proportion in urine. This effect

was most pronounced in animals exposed to 5000 ppm (results

for males given below, response in females comparable):

\--% absorbed eliminated (48 hr)\--

Treatment regime = 0/5000 5000/5000

Exhaled volatiles 48.6 24.1

Exhaled CO2 2.1 1.4

Urine 45.1 71.5

Faeces 0.5 0.3

Total 96.3 97.4

(Values = mean for 8 rats/group)

RETENTION

Carcass accounted for approx. 1.5-3% of the absorbed

radioactivity irrespective of exposure level or sex.

BIOTRANSFORMATION OF ETBE

In urine and exhaled air levels of \[14C\]-TBA were greater than

those of \[14C\]-ETBE, the latter decreasing quickly in exhaled

air after exposure.

Pre-exposure to 5000 ppm ETBE for 13 d increased the

proportion of absorbed material eliminated as \[14C\]-TBA (less

pronounced effect at 500 ppm):

\-\-\-\-\-\-\-\--uM TBA in urine \-\-\-\-\-\-\-\--

\-\-\-\-\--males\-\-\-\-\-- \-\-\-\--females\-\-\-\--

0/5000 5000/5000 0/5000 5000/5000

8 hr 2344 4666 806 1439

24 hr 1058 2982 911 2429

In addition the presence of HBA and MPD were confirmed

qualitatively in urine.

**Test substance:** Non-labeled ETBE (97.5%) was obtained from
Pittsburgh Applied

Research Corp. (PA, USA). 14C-ETBE was obtained from Wizard

Laboratories (CA, USA), chemical purity 99.2%.

**Conclusion:** Following single nose-only exposure to \[14C\]-ETBE at
500, 1750

or 5000 ppm rats eliminated radioactivity mainly in urine and

exhaled air with feces and 14CO2 being minor pathways. At 500

ppm most was eliminated in urine whilst at 5000 ppm exhaled

air accounted for the greatest proportion. Total

radioactivity eliminated increased with increasing exposure,

but in a less than exposure proportional manner. Pre-exposure

to ETBE increased the proportion eliminated as \[14C\]-TBA and

the overall weight of evidence indicated that pre-exposure to

ETBE induced its metabolism. \[14C\]-TBA was always the main

elimination product in urine and exhaled air. HBA and MPD,

being further products of metabolism were also detected in

urine.

**Reliability:** (2) valid with restrictions

Study available for review. GLP compliant near-guideline

\- 64/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

investigation, clearly reported, acceptable for assessment.

30-NOV-2003 (15)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** rat

**Year:** 2001

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND TREATMENTS

Animals: F344 rats (12 wk old; males 210-240 g; females

190-220 g)

Supplier: Harlan Winkelmann (Borchen, Germany)

Group size: 5 per sex per exposure level

Diet: no details

Water: no details

Treatment: 4 hr inhalation exposure to target concentrations

of 4 ppm or 40 ppm ETBE vapour

Exposure chamber: exposure of the rats coincided with exposure

of a group of human volunteers, within the same chamber (see

following record for further details)

Blood collection: from tail vein (100 ul)

Urine collection: animals housed in metabolism cages

(unspecified); urine (4 degrees C) collected every 6 hr for 72

hr; animals accustomed to metabolism cages for 3 d prior to

exposure; control urine collected for 12 hr prior to exposure;

report states no urine excreted during the exposure period

ANALYTICAL PROCEDURES

Chamber air was sampled every 15 min and the concentration of

ETBE vapour present determined by GC-MS. Actual concentrations

were 4.5 +/- 0.6 ppm and 40.6 +/- 3.0 ppm (mean and SD for 16

determination taken over 4 hr).

The concentration of ETBE in blood was quantified using

headspace GC-MS, with MTBE as internal standard; limit of

detection 0.1 nmol ETBE per ml blood (signal to noise ratio

5:1; variation not stated). TBA in blood was determined using

GC-MS, with deuterated TAB as standard; limit of detection 0.2

nmol per ml blood (signal to noise ratio 5:1; variation 10%).

The concentration of 2-methyl-1,2-propanediol in urine was

determined using GC-MS, with 1,2-propanediol as standard;

limit of detection 1 nmol/ml urine (signal to noise ratio 5:1;

variation \<15%). 2-Hydroxyisobutyrate in urine was quantified

after conversion to the methyl ester using GC-MS; limit of

detection 3 nmol per ml urine (signal to noise ratio 5:1;

variation \<18%).

STATISTICAL METHODS

\- 65/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Data were analysed using Student\'s t-test (P values \<0.05

considered significant). Half-lives were calculated using

exponential regression.

**Remark:** Some of this information has also been published in:

Amberg, A, Rosner, E and Dekant, W (2000) Biotransformation

and kinetics of excretion of ethyl-tert-butyl ether in rats

and humans. Toxicol Sci. 53, 194-201.

Dekant, W, Bernbauer, U, Rosner, E, Amberg, A. (2001)

Toxicokinetics of ethers used a fuel oxygenates. Toxicol Lett

124, 37-45.

**Result:** RECEIVED DOSE IN THE RAT

Received doses of 2.3 and 21 umol were calculated for rats

exposed to 4 ppm or 40 ppm ETBE for 4 hr, respectively.

TOXICOKINETICS AND BIOTRANSFORMATION OF ETBE BY RATS

-BLOOD

ETBE was not detected in blood samples taken before exposure

commenced. A maximal concentration of 1.0 uM was achieved

following 4 hr exposure to 4 ppm ETBE, with a maximum of 5.3

uM present after 4 hr exposure to 40 ppm. Elimination

half-lives of 0.4 hr and 0.8 hr were obtained in the 4 ppm and

40 ppm groups, respectively.

Following exposure to ETBE, the concentration of TBA in rat

blood increased significantly (P\<0.01; control level not

reported) reaching a maximum of 5.7 or 21.7 uM for the 4 ppm

or 40 ppm exposures, respectively (elimination half-lives not

determined). The authors note that the measured concentration

of TBA in blood was greater in the rats compared to humans

exposed under identical conditions.

\- URINE

TBA, 2MP and 2HIB were present in control urine prior to

exposure to ETBE. The concentration of urinary metabolites

increased post-exposure, with 2HIB the main metabolite. The

following recovery data were obtained over 72 hr

post-exposure:

ETBE (low exposure, high exposure)

\- background: none detected

\- corrected total excreted: none detected

\- half-life: not relevant

TBA (low exposure, high exposure)

\- background: 0.2, 0.2 umol over 6 hr

\- corrected total excreted: 0.2, 0.7 umol

\- half-life: not determined, 4.6 hr

2MP (low exposure, high exposure)

\- background: 0.02, 0.02 umol

\- corrected total excreted: 0.3, 1.6 umol

\- half-life: 4.0, 2.6 hr

\- 66/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

2HIB (low exposure, high exposure)

\- background: 1.4, 1.9 umol

\- corrected total excreted: 2.3, 10.6 umol

\- half-life: 4.7, 3.0 hr

Reviewer\'s comments:

\- urinary excretion of metabolites was less the rats than in

humans exposed under identical conditions;

\- urinary half-lives in rats were also shorter than in humans.

\- unchanged ETBE not detected in rat urine (present in humans)

**Test substance:** ETBE, \>99%, Aldrich Chemical Co., Deisenhofen,
Germany.

**Conclusion:** ETBE vapour (4 ppm or 40 ppm) is readily absorbed by the
rat

lung and distributed into the blood from which it was

eliminated with a half-life of 0.4-0.6 hr. It is metabolised

to tert-butanol (primary metabolite; half-life not determined)

which reaches a higher blood concentration in the rat than is

seen humans exposed under identical conditions. Urinary

metabolites were consistent with cytochrome P450-mediated

metabolism of tert-butanol, and comprised 2-hydroxyisobutyrate

(predominant metabolite) and 2-methyl-1,2-propanediol along

with minor amounts of TBA.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

02-JAN-2004 (25)

**In Vitro/in vivo:** In vivo

**Type:** Metabolism

**Species:** other: rats, human

**Year:** 1998

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND TREATMENTS

Male and female Fischer F344 rats (220-260g) were obtained

from Harlan Winkelmann (Borchen, Germany). The animals were

maintained on a 12h light/dark cycle, Altromin food and tap

water were available ad libitum. Animals were accustomed to

metabolism cages for 3 days before the experiments and control

urine was collected at this time.

Male (n=2) and female rats (n=2) were individually exposed to

\[2-13C\]-ETBE by inhalation for 6 h in a static exposure

chamber with an initial concentration of 2000 ppm. An

comparable study was also conducted with non-isotopic ETBE.

The concentration of ETBE in the chamber was monitored every

10 min GC-FID. After 6 h the animals were transferred to

individual glass metabolism cages with free access to food and

water where they were maintained for 72 h.

\- 67/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

In a separate experiment male animals (n=3) were dosed with

\[2-13C\]-TBA (dissolved in corn oil) at a dose level of

250g/kg. These animals were also maintained in metabolism

cages for 72 h with urine collection.

A single male human subject (aged 44 years, weight 80 kg) was

given 5 mg/kg bw \[2-13C\]-TBA (study design approved by the

institutional review board of the University of Wurzburg).

SAMPLE COLLECTION

In the animal studies urine was collected over 0-24 h and

24-48 h. In the human experiment, urine was collected up to 48

hr (12 hr intervals).

Comment: It is not clear if samples were collected at ambient

temperature or chilled, or how they were stored.

SAMPLE ANALYSIS

Urine was analysed directly by NMR in the presence of D2O.

Spectra were recorded on a Bruker AC 250 or a Bruker Avance

DMX 600 spectrometer under standard conditions. Some samples

were pre-treated with b-glucuronidase or sulfatase enzymes

(Sigma) or treated with HCl (pH 2) for 1 h at 37°C to release

putative conjugates.

Headapace GC/MS was performed on a Fisons MD 800 mass

spectrometer coupled with a Carlo Erba GC 8000 GC (split

injection ratio 1:5).

**Result:** BIOTRANSFORMATION OF ETBE

TBA glucuronide, 2-methyl-1,2-propanediol,

2-hydroxyisobutyrate and TBA sulfate were identified in 0-24

hr urine from rats exposed to \[2-13C\]-ETBE, together with

small amounts of 13C-acetone.

Comment: The authors note that these were identical to the

metabolites in urine from rats dosed with MTBE in a parallel

experiment.

After 24-48 hr, only 2-hydroxyisobutyrate was observed in

urine. There was no apparent gender difference (qualitative or

quantitative) in the metabolite pattern.

No metabolites deriving from oxidation of the b-carbon of the

ethyl group in ETBE (e.g. tert-butyl glycol or

tert-butoxyacetic acid) were observed.

BIOTRANSFORMATION OF TBA

Comment: These studies were included to confirm the structure

of metabolites \"downstream\" from TBA.

NMR and GC/MS showed the presence of \[2-13C\]-TBA and the same

range of components present in urine from rats exposed to

\[2-13C\]-ETBE. TBA-sulphate predominated.

The urine samples from the human subject given \[2-13C\]-TBA

exhibited a pattern of metabolites that was qualitatively the

\- 68/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

same as for rat. The major metabolite was however

2-hydroxyisobutyrate rather than TBA sulfate.

**Test substance:** \[2-13C\]-ETBE (\>95% pure) and \[2-13C\]-TBA (\>97%
pure) were

prepared by custom synthesis.

**Conclusion:** The metabolism of \[2-13C\]-ETBE in the rat was shown to
proceed

by oxidation of the ethyl group to TBA, with TBA-glucuronide,

TBA-sulfate and 2-hydroxyisobutyrate and trace amounts of

\[13C\]-acetone present in urine. Later time-points (24-48 h)

contained only 2-hydroxyisobutyrate suggesting that this

component had a longer half-life of elimination. Metabolism of

\[2-13C\]-TBA in rat and human gave rise to the same range of

metabolites as was noted in rats exposed to \[2-13C\]-ETBE. The

relative proportions of metabolites differed, however, with

only small amounts of TBA-sulfate and a greater preponderance

of 2-hydroxyisobutyrate in humans.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

30-NOV-2003 (8)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** human

**No. of animals, males:** 8

**Doses, males:** 0 (control), 5, 25, 50 ppm

**Route of administration:** inhalation

**Exposure time:** 2 hour(s)

**Deg. product:** yes

**Year:** 1988

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** SUBJECTS AND TREATMENTS

8 healthy male volunteers (Caucasian; age 21 to 41 years, mean

29 years; bodyweight 70 to 97 kg, mean 82 kg). All were

non-smokers, had no occupational exposure to ETBE and

refrained from alcohol and drugs 2 days before and during the

study. Exposures were conducted during 2 h of light physical

exercise in an exposure chamber. Each subject exposed on 4

occasions to nominal levels of 0, 5, 25, and 50 ppm ETBE with

at least 2 weeks between successive exposures. All sampling

of blood and expired air was performed during exercise.

The concentration of ETBE in the chamber air was analysed

every 5 min during exposure (mean measured concentrations =

4.8, 25 and 50 ppm). Other variables (temperature, humidity,

air exchanges, average rate of exercise) were closely

monitored and found to be consistent during each exposure and

between different exposures.

\- 69/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

SAMPLE COLLECTION

Complete urine collections were made prior to exposure then at

0, 2, 4, and approx. 7, 11, 20 and 22 h post-exposure with a

spot sample at 46 h. Those at 7, 11 and 20 h were collected at

home and stored at room temperature. Collection condition of

the other samples is not clear.

Blood (capillary from fingerprick) taken at multiple

time-points (\>20 samples over 24 h) commencing before

exposure, then during and up to 48 h post-exposure. Samples

(200 ul) were transferred immediately to sealed headspace

vials. Timepoints were not specified but were adequate to

provide concentration versus time profiles and comprehensive

toxicokinetic estimates.

Exhaled air was collected once before exposure, four times

during (30 min intervals) and 6 times after exposure. Samples

were carefully collected according to a set procedure and ETBE

measurement undertaken as air was being exhaled. TBA in

exhaled air was trapped in sorbent tubes for later

measurement.

SAMPLE PROCESSING

ETBE in exhaled air was assayed by direct capillary column

GC-FID. TBA in exhaled air was measured (after desorption) by

GC using FID.

ETBE, TBA and acetone in blood and urine were determined by

capillary GC-FID. All three analytes were shown to be stable

on storage in the matrices up to 24 h.

STATISTICAL METHODS

Toxicokinetic calculations for exhaled air used a previously

published method (linear four compartment model with zero

order uptake) applied previously to MTBE. The absorbed dose

was calculated as Respiratory Intake (new approach) and Net

Respiratory Intake (old approach), the former regarded by the

researchers (with good reason) as more accurate as it allowed

for material exhaled during exposure.

Elimination curves for ETBE in urine were fitted to

biexponential functions using non-linear regression analysis.

The elimination phase for TBA in blood and urine was fitted to

a monoexponential function by non-linear regression analysis.

Area under the concentration-time curve (AUC) for ETBE in

blood was calculated using the four compartment model whilst

AUCs for TBA and acetone were calculated by the trapezoidal

rule in Microsoft Excel.

Analysis of variance (SuperANOVA) and Student\'s paired t test

were used to compare results from different exposures with a

level of significance set to 0.05. Data are presented as mean

\- 70/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

values with 95% confidence intervals.

**Remark:** Half lives:

\-\--5 ppm\-\-- \-\--25 ppm\-\-- \-\--50 ppm\-\--

ETBE TBA ETBE TBA ETBE TBA

Blood

1st: 1.8 min n.d 1.2 min n.a 2.0 min n.a.

2nd: 20 min n.d 15 min n.a 1.9 min n.a.

3rd: 2.1 h n.d 1.5 h n.a 1.5 h n.a.

4th 33 h n.d. 24 h 12 h 24 h 12 h

Urine

1st 6.3 min n.d 9.1 min n.a 9.3 min n.a.

2nd 8.8 h n.d 8.7 h 8.4 h 8.2 h 7.6 h

Comment: The concentration versus time plot for blood

indicates that this long half-life probably accounts for a

relatively small proportion of absorbed ETBE and may represent

a deep compartment (body fat e.g.).

**Result:** TOXICOKINETICS IN BLOOD

The concentration of ETBE in blood increased throughout

exposure period (tending to level-off but no plateau

achieved). Average maximum concentrations of 1.1, 5.4 and 10

mM were achieved at 5, 25 and 50 ppm respectively (i.e.

essentially dose proportional). The elimination of ETBE from

blood was well described by the four compartment model used.

The apparent terminal elimination half-life was 28 h with no

statistically significant differences between exposure levels.

The AUC for ETBE was linearally related to exposure level,

indicating linear kinetics up to 50 ppm. Clearance of ETBE in

blood was in the range approximately 0.6 to 0.9 l/h/kg.

In contrast, the concentration of TBA in blood increased

steadily throughout exposure and remained high for several

hours post exposure. Average maximum blood concentrations at

30 min post exposure were 6.9 mM and 12 mM (25 ppm and 50 ppm

respectively), and began to decline slowly from 4 h after

exposure. Concentrations at 5 ppm were too low for

quantification. AUC for TBA was linear with exposure level.

Average blood elimination half-life for TBA was 12 h.

Acetone was detected at elevated levels in blood, being

greatest (concentration, AUC) after exposure to 50 ppm ETBE,

however concentrations in general were poorly correlated with

exposure.

TOXICOKINETICS IN URINE

Urinary excretion of ETBE over 24 h was low (0.120, 0.061 and

0.056 % of respiratory uptake at 5, 25 and 50 ppm

respectively). The elimination half-life of ETBE in urine was

in the range 6.3 to 9.3 h. Corresponding urinary excretion of

TBA over 24 h was 0.71, 0.66 and 0.90% at 5, 25 and 50 ppm

respectively). The elimination half-life of TBA in urine was

in the range 7.6 to 8.4 h. Acetone excretion (data not

\- 71/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

reporetd) was widely variable between subjects and treatments

but concentrations appeared exposure related.

ELIMINATION

The authors estimate that a total of approx. 50% of the

retained dose of ETBE was subsequently excreted as ETBE and

TBA in exhaled air and in urine.

**Test substance:** ETBE obtained from Ecofuel S.P.A., Italy. It was
redistilled

to a purity of 98.5% prior to use. Major impurities

(identified by GC-MS) were TBA (0.4%) and MTBE (1.1%).

**Conclusion:** ETBE was absorbed from the respiratory tract with no
evidence

of metabolic saturation (linear kinetics over the exposure

range tested), with rapid distribution and subsequent rapid

elimination (exhaled air and urine) of ETBE and its major

metabolite TBA. Approx. 50% of the absorbed dose was

eliminated as ETBE and TBA in exhaled air and urine,

suggesting that products of further further metabolism were

present but not detected in this study.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

15-JAN-2004 (65)

**In Vitro/in vivo:** In vivo

**Type:** Toxicokinetics

**Species:** human

**Year:** 2001

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** SUBJECTS AND TREATMENTS

Six healthy volunteers (3 female, age 26-29 yr, bwt 58-61 kg;

3 male, age 26-31 yr, bwt 62-83 kg) were exposed to ETBE

vapour at concentrations of 4 or 40 ppm in a dynamic exposure

chamber for 4 hr. The subjects refrained from consuming

alcohol or medication for 2 d prior to the study, did not

abuse alcohol and were non-smokers or occasional smokers.

Subjects did not refuel their cars for 2 d prior to the study.

The exposure chamber had an internal volume of 8 m3, an

air-flow rate of 28 m3/hr, a temperature of 22 degrees C and

relative humidity of 50-60%. Preliminary analyses showed that

exposure concentrations were uniform throughout the chamber.

Exposures commenced at 0800 hr, with a 4 wk interval between

the 4 ppm and 40 ppm exposures.

A group 10 rats was also placed inside the chamber to ensure

identical exposure conditions were achieved during the animal

phase of these investigations.

\- 72/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Blood collection: 10 ml samples, heparinised syringes)

Urine collection: collected (4 degrees C) every 6 hr for 72 hr

ANALYTICAL PROCEDURES

See record above

STATISTICAL METHODS

See record above

**Remark:** Some of this information has also been published in:

Amberg, A, Rosner, E and Dekant, W (2000) Biotransformation

and kinetics of excretion of ethyl-tert-butyl ether in rats

and humans. Toxicol Sci. 53, 194-201.

Dekant, W, Bernbauer, U, Rosner, E, Amberg, A. (2001)

Toxicokinetics of ethers used a fuel oxygenates. Toxicol Lett

124, 37-45.

**Result:** RECEIVED DOSE IN HUMANS

Received doses of 121 and 1092 umol were calculated for

subjects exposed to 4 ppm or 40 ppm ETBE for 4 hr,

respectively.

TOXICOKINETICS AND BIOTRANSFORMATION OF ETBE BY HUMANS

-BLOOD

ETBE was not detected in blood samples taken before exposure

commenced. A maximal concentration of 1.3 uM was achieved

following 4 hr exposure to 4 ppm ETBE, with a maximum of 12.1

uM present after 4 hr exposure to 40 ppm. An elimination

half-life of 1.1 hr was obtained for individuals exposed to 4

ppm, whereas two half-lives (1.1 hr; 6.2 hr) were present

after exposure to 40 ppm. ETBE concentrations had decreased to

the limit of detection 4 hr (4 ppm) or 24 hr (40 ppm) after

exposure ended.

TBA was present at low levels (approx. 0.5 nmol/ml) in the

pre-exposure blood samples, and increased significantly

(P\<0.01) following exposure to ETBE (max. 1.8 uM or 13.9 uM

for the 4 ppm or 40 ppm exposures, respectively). Elimination

half-lives of 8.2 hr or 9.8 hr were derived for the 4 ppm or

40 ppm exposures, respectively, with detectable amounts of TBA

remaining 24 hr after exposure ended (numerical data not

presented in report; from graphical results, the Reviewer

estimates that the concentration of TBA in blood 24 hr

post-exposure was approx. 0.5 uM or 5 uM for the low and high

exposures, respectively). Clearance followed first order

kinetics.

\- URINE

TBA, 2MP and 2HIB were present in control urine prior to

exposure to ETBE. The concentration of urinary metabolites

increased post-exposure, with 2HIB the main metabolite. The

following recovery data were obtained over 72 hr

post-exposure:

\- 73/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

ETBE (low exposure, high exposure)

\- background: none detected

\- corrected total excreted: 0.3, 0.9 umol

\- half-life: 5.6, 3.5 hr

TBA (low exposure, high exposure)

\- background: 2.0, 2.5 umol over 6 hr

\- corrected total excreted: 5.1, 22.6 umol

\- half-life: 14.6, 11.2 hr

2MP (low exposure, high exposure)

\- background: 3.5-5.8 umol

\- corrected total excreted: 13.6-96.6 umol

\- half-life: 10.2, 12.3 hr

2HIB (low exposure, high exposure)

\- background: 93.6, 167.4 umol

\- corrected total excreted: 130.4, 522.6 umol

\- half-life: 20.3, 28.3 hr

**Test substance:** ETBE, \>99%, Aldrich Chemical Co., Deisenhofen,
Germany.

**Conclusion:** ETBE vapour (4 ppm or 40 ppm) is readily absorbed by the
human

lung and distributed into the blood from which it was

eliminated in a bi-phasic manner with half-lives of approx. 1

hr and 6 hr. It is readily converted to tert-butanol (primary

metabolite) which exhibits an elimination half-life of 8-9 hr

in blood. (The authors comment that the concentration of TBA

in blood was less than that quantified in rats exposed under

identical conditions.) Urinary metabolites were consistent

with cytochrome P450-mediated metabolism of tert-butanol, and

comprised 2-hydroxyisobutyrate (predominant metabolite) and

2-methyl-1,2-propanediol along with minor amounts of TBA.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

02-JAN-2004 (25)

\- 74/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[5.1 Acute Toxicity]{.underline}**

**[5.1.1 Acute Oral Toxicity]{.underline}**

**Type:** LD50

**Species:** rat

**Strain:** other: OFA

**Sex:** male/female

**No. of Animals:** 30

**Vehicle:** CMC

**Doses:** 25, 200 and 2000 mg/kg bwt

**Value:** \> 2000 mg/kg bw

**Method:** OECD Guide-line 401 \"Acute Oral Toxicity\"

**Year:** 1981

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Male and female OFA rats (100 - 120 g) were obtained from a

commercial supplier and acclimatised for 5 d prior to

treatment.

ETBE was administered as a solution in 1% carboxymethyl

cellulose at doses of 25, 200 and 2000 mg/kg bwt. There were

5 rats/sex/dose, and a dosing volume of 10 ml/kg was

employed.

**Result:** There were no deaths during the 14 day post-treatment

observation period.

Body weight and body weight gain in males was similar for

the 3 treatment groups, whereas growth in females given 2000

mg/kg bwt appeared lower in comparison to the other treated

female groups from day 10 onward.

Macroscopic examination at necropsy revealed desquamation of

the gastric mucosae in 2 male and 1 female given 2000 mg/kg

ETBE, and also in 2 males from the 200 mg/kg bwt dose group.

**Test substance:** ETBE, purity not stated, lot no. 408-2404, Total
Raffinage

Distribution, France.

**Conclusion:** Under the conditions of the test, the acute oral LD50 of

ETBE in the OFA rat was \>2000 mg/kg bwt.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (44)

\- 75/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** other: limit test

**Species:** rat

**Strain:** Sprague-Dawley

**Sex:** male/female

**No. of Animals:** 10

**Vehicle:** other: undliuted

**Doses:** 2.67 ml/kg, equivalent to 2003 mg/kg

**Value:** \> 2003 mg/kg bw

**Method:** OECD Guide-line 401 \"Acute Oral Toxicity\"

**Year:** 1987

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Five male and five female SD rats (mean bwt 209 g and 171 g,

respectively) were given a single dose of 2.67 ml/kg bwt

undiluted ETBE by gavage. Body weights and clinical signs

were monitored for 14 days post-dosing, and the animals

taken to necropsy on day 15.

**Result:** The behaviour of the animals was subdued 1-4 hr post-dosing,

but there were no deaths and they gained weight in a normal

manner. There were no unusual macroscopic findings at

necropsy.

**Test substance:** ETBE, 95.8%, batch no. 93050601, supplied by Elf
France.

**Conclusion:** Under the conditions of the test, the acute oral LD50 of

ETBE in the SD rat was \>2000 mg/kg bwt.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

04-JAN-2004 (73)

\- 76/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** LD50

**Species:** rat

**Strain:** Wistar

**Sex:** male/female

**No. of Animals:** 16

**Vehicle:** other: administered undiluted

**Doses:** 500, 1000, 2500, 5000 mg/kg bwt

**Value:** \> 5000 mg/kg bw

**Year:** 1988

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Male and female Wistar rats (208-241 males; 212-230 females;

age 8 wk) were obtained from a commercial supplier.

ETBE was administered undiluted at doses of 500, 1000, 2500 or

5000 mg/kg bwt (variable volume: 0.15-1.6 ml/rat). There were

2 rats/sex/dose level. Animals were observed for 14 d, then

subject to gross necropsy.

**Result:** There were no deaths or clinical signs reported during the
14

day observation period. All animals appeared normal at

necropsy.

**Test substance:** Supplied by ARCO Chemical Co., clear liquid, SG 0.73
(no

further details)

**Conclusion:** Under the conditions of the test, the acute oral LD50 of

ETBE in the OFA rat was \>5000 mg/kg bwt.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant study following

US-EPA guidelines, acceptable for assessment.

30-NOV-2003 (56)

**Type:** LD50

**Species:** rat

**Value:** \> 2000 mg/kg bw

**Method:** other: no data

**GLP:** no data

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Test substance:** ETBE

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

04-JAN-2004 (23)

\- 77/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[5.1.2 Acute Inhalation Toxicity]{.underline}**

**Type:** other: limit test

**Species:** rat

**Strain:** Crj: CD(SD)

**Sex:** male/female

**No. of Animals:** 10

**Vehicle:** other: filtered air

**Doses:** 5.99 mg/l

**Exposure time:** 4 hour(s)

**Value:** \> 5.88 mg/l

**Method:** OECD Guide-line 403 \"Acute Inhalation Toxicity\"

**Year:** 1989

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Male and female SD rats (age approx. 4 wk) were obtained
from

a commercial supplier and held in quarantine for 2 wk. The

animals weighed approx. 180 g (females) to 260 g (males) at

the time of the study.

The vapour generating system comprised a U-shaped glass tube,

heated to approx. 69 degrees C. ETBE was delivered into the

tube at a constant rate (not specified) via 1/8 inch OD teflon

tubing and a Fluid Metering Inc pump. The ETBE was completely

vaporised under these conditions, and delivered to the

exposure chamber (160 l, stainless steel; nose only exposure)

at 59 l/min.

The concentration of test substance in air was monitored using

IR spectrometry every 30 min.

The animals were exposed for 4 hr, followed by a 14 day

observation period. Body weight was recorded weekly. A gross

necropsy was performed at study termination.

**Result:** IR analysis demonstrated an actual mean+/-SD concentration
of

5.88 +/- 0.077 mg/l; TWA = 5.88 mg/l.

There were no deaths during the exposure period or during the

14 d observation period.

Clinical signs comprised redness around the nose/eyes and

discoloured facial fur immediately after exposure (appearance

of most animals was normal the following day).

Weight gain was normal in all animals during the 2 wk

observation period.

There were no treatment-related findings at necropsy. (Lung

foci and/or red areas in the lung were noted in 6/10 rats. The

report states these were of a type and severity common in

control rats and therefore were considered unrelated to

treatment.)

\- 78/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Test substance:** ETBE, 99%, product no. LF-9072 from Aldrich Chemical
Co.,

Milwaukee.

**Conclusion:** Under the conditions of the test, the acute inhalation
LC50 of

ETBE in male and female SD rats was \>5.88 mg/l.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

21-NOV-2003 (41)

**Type:** other: ocular and nasal effects after acute inhalation

exposure

**Species:** human

**Year:** 1998

**GLP:** no

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** SUBJECTS AND TREATMENTS

8 healthy male volunteers (Caucasian; age 21 to 41 years, mean

29 years; bodyweight 70 to 97 kg, mean 82 kg). All were

non-smokers, had no occupational exposure to ETBE and

refrained from alcohol and drugs 2 days before and during the

study. Exposures were conducted during 2 h of light physical

exercise in an exposure chamber. Each subject exposed on 4

occasions at nominal levels of 0, 5, 25, and 50 ppm ETBE with

at least 2 weeks between successive exposures. All sampling

of blood and expired air was performed during exercise.

Reviewer\'s comment: ETBE is an odorous material, hence

subjects would have been aware of when they were exposed to

the control and test atmospheres.

The concentration of ETBE in the chamber air was analysed

every 5 min during exposure and mean measured concentrations

were 4.8, 25 and 50 ppm. Other variables such as temperature,

humidity, air exchanges and average rate of exercise were

closely monitored and found to be very consistent during each

exposure and between different exposures.

SUBJECTIVE SYMPTOMS

A questionnaire was used to assess symptoms of subjective

irritation (discomfort in the eyes, nose, throat and airways;

difficulty breathing; smell) and effects on the central

nervous system (headache, fatigue, nausea, dizziness,

intoxication). Responses were self-recorded on a visual

analogue scale graded from \"not at all\" to \"almost

unbearable\". Responses were recorded on 8 occasions

(pre-exposure, during exposure while exercising, 4 hr

post-exposure).

OCULAR MEASUREMENTS

Blinking frequency (from video tape record), eye redness

(photographic comparison; 50 ppm only), tear film break-up

time (slit-lamp with fluorescein; 50 ppm only) and

conjunctival epithelial damage (Lissamine green) were assessed

\- 79/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

using published methods (Nihlen et al (1998) Toxicol Appl

Pharmacol, 148, 281). Assessments were recorded by a single

observer to reduce variability.

NASAL MEASUREMENTS

Nasal mucosal swelling (acoustic rhinometry) and analysis of

nasal lavage (leukocytes, epithelial cells, inflammatory

markers) were assessed using published methods (Nihlen et al

\(1998\) Toxicol Appl Pharmacol, 148, 281).

PULMONARY FUNCTION

Peak expiratory flow (PEF), forced vital capacity (FVC),

one-second forced expiratory volume (FEV1) and vital capacity

(VC) were assessed following guidelines of the American

Thoracic Society Medical Section of the American Lung

Association (1994, Am j Respir Crit Care med, 152, 1107). Lung

transfer factor (TLco; a measure of the gas exchange

characteristics of lung parenchyma) was determined using

carbon monoxide (0.3%) as marker) (Cotes et al. (1993) Eur

Respir 6, 41-52).

STATISTICAL METHODS

The data were analysed using repeated ANOVA.

**Result:** SUBJECTIVE SYMPTOMS

Graphical data indicated that subjective discomfort of the

throat and airways was increased significantly after 40

exposure to 50 ppm ETBE, and remained elevated for the

remainder of the 2 hr exposure period (P=0.02; control score =

approx. 2.5 units; exposed score = approx. 6-8 units).

Symptoms had resolved 80 min after exposure ceased. The taste

and odour of the test atmosphere was unpleasant and

unacceptable to the subjects.

Reviewer\'s comment: The authors state that subjective symptoms

of ocular and nasal discomfort, fatigue, nausea dizziness and

intoxication were greater in the 50 ppm group, but no

dose-response relationship or statistically significant

difference was present. The intensity of the response at 50

ppm appeared to be very mild (stated to be between \"not at

all\" and \"hardly at all\").

OCULAR MEASUREMENTS

There was no statistically significant effect on eye redness,

tear-film break-up, conjunctival damage or blinking frequency.

NASAL MEASUREMENTS

Nasal volume was decreased by 6-15% (P = 0.001) in all groups,

including controls, compared to pre-exposure values. Exposure

to ETBE vapour was without effect on the composition of nasal

lavage fluid (i.e. leukocytes, epithelial cells, inflammatory

markers etc).

PULMONARY FUNCTION

A small (3-4%) but significant (P\<0.05) decrease in FVC and VC

was reported in subjects exposed to 25 or 50 ppm ETBE vapour.

\- 80/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Reviewer\'s comment: The author\'s state that FEV1 and TLco were

also decreased significantly (approx. 2% and 2-4%,

respectively) after exposure, however data included in the

publication are assigned P values of 0.06-0.5 ie

non-significant. The reason for this discrepancy is not known.

**Test substance:** ETBE, technical grade, supplied by Ecofuel S.P.A,
Italy.

**Conclusion:** The authors conclude that exposure to 50 ppm ETBE vapour
for 2

hr was associated with subjective irritation of the throat and

airways and slightly impaired lung function. This reviewer

notes that these changes were mild in nature and minor in

extent.

**Reliability:** (4) not assignable

Study available for review. Highly odourous nature of the test

substance sample suggests that subjective findings may be

unreliable. Inconsistencies present in reporting of

statistical significance of changes in key lung parameters.

15-JAN-2004 (66)

**[5.1.3 Acute Dermal Toxicity]{.underline}**

**Type:** other: limit test

**Species:** rabbit

**Strain:** New Zealand white

**Sex:** male/female

**No. of Animals:** 10

**Vehicle:** other: applied undiluted

**Doses:** 2000 mg/kg bwt, under occlusion

**Value:** \> 2000 mg/kg bw

**Method:** other: comparable to OECD 402

**Year:** 1988

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Male and female New Zealand rabbits (5 per sex; age approx.
8

wk) were obtained from a commerical supplier. The animals

weighed 2.3-2.9 kg (males) and 2.5-2.8 kg (females) at the

start of the study.

ETBE (6.9-7.9 ml, equivalent to 2 g/kg bwt) was applied

undiluted to at least 10% of the body surface (clipped free of

fur), covered by gauze and then secured with tape (occlusion)

for 24 hr. The treatment site was cleaned (water) after

removal of the patch and observed for signs of dermal

irritation on 1, 7 and 14 d after post-dosing.

Animals were also observed 1, 2, 4 hours post-dosing and twice

daily thereafter for 14 days for mortality, toxicity and

pharmacological effects. Dermal responses were recorded using

the Draize scoring system.

Body weights recorded pre-test, weekly and at termination.

\- 81/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

All animals were subject to a gross examination at necropsy on

day 14. Abnormal tissues preserved for future microscopic

examination.

**Result:** One male rabbit died on day 3.

Clinical signs included one male with diarrhoea from day 8;

one female with yellow nasal discharge from day 2; one female

with lachrymation on days 6-9.

Dermal reactions included:

\- very slight to slight erythema (grade 1-2) on day 1, very

slight to severe erythema (grade 1-4) on day 7, and absent to

severe erythema (grade 0-4) on day 14;

\- slight to moderate-to-severe oedema (grade 2-3) present day

1, very slight to moderate-to-severe oedema (grade 1-3) on 7,

and absent or slight oedema (grade 0-2) on day 14.

\- flaking skin was present at days 7 and 14.

All animals exhibited skin irritation and poor hair regrowth

(considered indicative of injuries in-depth by the report) at

necropsy. Males which died had congested lungs with brown

areas, excess fluid in pleural and peritoneal cavities, pale

margins in liver.

**Test substance:** ETBE, purity not stated, SG 0.73, clear liquid, from
ARCO

Chemical Co., PA (no further details).

**Conclusion:** Under the conditions of this test, the acute dermal LD50
of

ETBE in male and female New Zealand white rabbits was \>2000

mg/kg bwt.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant near-guideline

investigation, clearly reported, acceptable for assessment.

02-JAN-2004 (59)

**Type:** other: limit test

**Species:** rabbit

**Strain:** New Zealand white

**Sex:** male/female

**No. of Animals:** 10

**Vehicle:** other: applied undiluted

**Doses:** 2000 mg/kg bwt, under occlusion

**Value:** \> 2000 mg/kg bw

**Method:** other: comparable to OECD 402

**Year:** 1989

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Male and female New Zealand rabbits (5 per sex; age approx.
8

wk) were obtained from a comerical supplier. The animals

weighed approx. 3.7 kg (males) to 4.0 kg (females) at the

start of the study.

ETBE (undiluted, equivalent to 2 g/kg bwt) was applied

uniformly to the shaved dorsum of each rabbit, covered with a

\- 82/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

surgical dressing under plastic film (occlusion) and held in

place by elastic adhesive bandage for 24 hr. The application

site was cleaned with light mineral oil and towel dried on

removal of the patch. The treatment site was observed for

signs of dermal irritation on 1, 7 and 14 d after post-dosing.

Animals were observed regularly for up to 5 hr post-dosing,

then daily for 14 days.

Body weights recorded pre-test, weekly and at termination.

All animals were subject to a gross examination at necropsy on

day 14.

**Result:** There were no deaths.

Clinical signs included a transient increase in respiration in

3 male and 4 female rabbits approx. 30 minutes after

treatment.

Dermal irritation (erythema and oedema) was present at the

application site of all rabbits on removal of the patch.

Eschar formation was present at the test site of most animals

within 5-7 days, and eventually in all the animals at day 14.

(Comment: no quantitative information on the severity of the

response is given in the report)

No other overt signs of toxicity were present.

Mean bwt increased slightly during the study (no further

details).

Three females had multiple red foci on the lungs at necropsy,

but this was not considered to be treatment related.

**Test substance:** ETBE, 99%, product no. LF-9072 from Aldrich Chemical
Co.,

Milwaukee.

**Conclusion:** Under the conditions of this test, the acute dermal LD50
of

ETBE in male and female New Zealand white rabbits was \>2000

mg/kg bwt.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant near-guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (40)

\- 83/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** LD50

**Species:** rat

**Value:** \> 20 mg/kg bw

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

**[5.1.4 Acute Toxicity, other Routes]{.underline}**

**-**

**[5.2 Corrosiveness and Irritation]{.underline}**

**[5.2.1 Skin Irritation]{.underline}**

**Species:** rabbit

**Concentration:** 100 %

**Exposure:** Semiocclusive

**Exposure Time:** 4 hour(s)

**No. of Animals:** 6

**Result:** not irritating

**EC classificat.:** not irritating

**Method:** OECD Guide-line 404 \"Acute Dermal Irritation/Corrosion\"

**Year:** 1981

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** The skin irritation potential of ETBE was investigated in 6

male New Zealand White rabbits (2.7 +/- 0.2 kg bwt).

0.5 ml ETBE was applied to clipped skin (6 cm2) on the right

flank for 4 hr under a semi-occlusive dressing.

**Result:** Mean scores at: 1 hr 24 hr 48 hr 72 hr

Erythema 1.0 1.5 0.5 0.0

Oedema 1.3 0.0 0.0 0.0

**Test substance:** ETBE, purity not stated, supplied by Total Raffinage

Distribution.

**Conclusion:** Under the conditions of the test, ETBE was not
irritating to

rabbit skin.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (20)

\- 84/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Species:** rabbit

**Concentration:** undiluted

**Exposure:** Occlusive

**Exposure Time:** 4 hour(s)

**No. of Animals:** 6

**Vehicle:** other: applied undiluted

**PDII:** 3.08

**Result:** moderately irritating

**Method:** other: comparable to Guideline 404

**Year:** 1988

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** New Zealand white rabbits (2 male, 4 females; age approx. 8

wk; bwt 2-3 kg) were obtained from a commercial supplier.

The dorsum and flanks were clipped free of fur, and the left

side lightly abraded with a needle.

0.5 ml of test substance was applied to two regions areas of

intact skin and two of abraded skin (are not specified),

covered with gauze under plastic (occlusion), and held in

place with adhesive tape.

The patch was removed after 4 hr, and dermal responses

recorded at 4, 24, 48 and 72 hr using the Draize scoring

system. The primary irritation index was calculated, based

upon the mean score for erythema/eschar and oedema formation

at both intact and abraded sites at the 4 time points.

The post-treatment observation period was extended to 7 d and

14 d to examination reversibility of any skin effects present.

**Result:** Erythema, generally absent at 4 hr, was well-defined at 24
hr

and 48 hr, and slight-to-well defined at 72 hr. By day 7,

erythema was absent to well-defined. At day 14, no erythema

was present.

Oedema was slight to well defined at 4, 24 or 48 hr, and

absent to well defined at 72 hr.

Flaking skin was present at majority of sites on day 7 (absent

at preceding and later timepoints).

The PII was 3.08.

The following results were recorded at the intact skin sites:

Mean scores at: 4 hr 24 hr 48 hr 72 hr

Erythema 0.00 2.00 2.00 1.83

Oedema 1.75 1.75 1.42 1.25

**Test substance:** ETBE, purity not stated, clear liquid, from ARCO
Chemical Co.,

PA (no further details).

\- 85/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Conclusion:** Under the conditions of the study, ETBE was moderately

irritating to intact and abraded rabbit skin following 4 hr

occluded contact.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant near-guideline

investigation, clearly reported, acceptable for assessment.

30-NOV-2003 (57)

**Species:** rabbit

**Concentration:** 100 %

**Exposure:** Semiocclusive

**Exposure Time:** 4 hour(s)

**No. of Animals:** 3

**Result:** not irritating

**EC classificat.:** not irritating

**Method:** OECD Guide-line 404 \"Acute Dermal Irritation/Corrosion\"

**Year:** 1992

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** The skin irritation potential of ETBE was investigated in 3

male New Zealand White rabbits (2 - 3 kg bwt).

0.5 ml ETBE was applied to a gauze pad (2.5 x 2.5 cm) which

was held in contact with clipped healthy skin under a

semi-occlusive dressing for 4 hr.

**Result:** Mean scores at: 1 hr 24 hr 48 hr 72 hr

Erythema 1.0 1.0 0.67 0.33

Oedema 0.67 0.33 0.0 0.0

One animal showed slight desquamation at the application

site on day 7

**Test substance:** ETBE, 95.8%, batch no. 93050601, supplied by Elf
France.

**Conclusion:** Under the conditions of the test, ETBE was not
irritating to

rabbit skin.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (72)

\- 86/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Species:** rabbit

**Result:** slightly irritating

**EC classificat.:** not irritating

**Method:** other: EEC Directive 92/69

**GLP:** no data

**Test substance:** other TS

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Test substance:** ETBE

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

04-JAN-2004 (23)

**[5.2.2 Eye Irritation]{.underline}**

**Species:** rabbit

**Concentration:** 100 %

**Dose:** .1 ml

**Exposure Time:** 24 hour(s)

**No. of Animals:** 6

**Result:** not irritating

**EC classificat.:** not irritating

**Method:** OECD Guide-line 405 \"Acute Eye Irritation/Corrosion\"

**Year:** 1987

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** The eye irritation potential of ETBE was investigated in 6

male New Zealand White rabbits (2.9 +/- 0.25 kg bwt).

0.1 ml ETBE was instilled into the conjunctival sac of the

left eye, the right eye serving as a control. Ocular

examinations were carried out 1 hr after administration,

then at 24, 48 and 72 hr post treatment.

**Result:** Chemosis (mean score = 0.8) present at 1 hr post treatment

had resolved by the 24 hr observation period. Scores for

discharge, iridial effects, corneal opacity and surface

opacity were zero at all time points included in the

investigation.

**Test substance:** ETBE, purity not stated, supplied by Total Raffinage

Distribution.

**Conclusion:** Under the conditions of the test, ETBE was not
irritating to

rabbit eye.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (21)

\- 87/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Species:** rabbit

**Concentration:** 100 %

**Dose:** .1 ml

**Exposure Time:** 24 hour(s)

**No. of Animals:** 3

**Result:** not irritating

**EC classificat.:** not irritating

**Method:** OECD Guide-line 405 \"Acute Eye Irritation/Corrosion\"

**Year:** 1987

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** The eye irritation potential of ETBE was investigated in 3

male New Zealand White rabbits (2 - 3 kg bwt).

0.1 ml ETBE was instilled into the conjunctival sac of the

right eye, the left eye serving as a control. Ocular

examinations were carried out 1 hr after administration,

then at 24, 48 and 72 hr post treatment. Readings were

continued on days 7 and 14 to determine the reversibility of

any lesions present at 72hr.

**Result:** Mean individual scores at (24+48+72hr):

1 2 3

Chemosis 0.00 0.67 0.33

Redness 0.33 1.67 0.33

Congestion 0.33 1.00 0.67

Degree of opacity 0.00 0.67 0.00

Residual redness in one animal (score = 1) and residual

congestion in another (score = 1) had resolved completely by

day 7.

**Test substance:** ETBE, 95.8%, batch no. 93050601, supplied by Elf
France.

**Conclusion:** Under the conditions of the test, ETBE was not
irritating to

rabbit eye.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (71)

**Species:** rabbit

**Concentration:** undiluted

**Dose:** .1 ml

**Exposure Time:** 24 hour(s)

**Comment:** rinsed after (see exposure time)

**No. of Animals:** 9

**Vehicle:** other: used undiluted

**Result:** moderately irritating

**Method:** OECD Guide-line 405 \"Acute Eye Irritation/Corrosion\"

**Year:** 1988

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

\- 88/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Method:** Male and female New Zealand white rabbits (age approx. 8 wk;

2-3 kg at study initiation) were purchased from a commercial

supplier.

0.1 ml ETBE was instilled into the conjunctival sac of the

left eye of 6 rabbits, the right eye serving as a control.

Ocular examinations were carried out 1 hr after

administration, then at 24, 48 and 72 hr post treatment.

Treated eyes of a further 3 rabbits were flushed with water

one minute after dosing.

The eyes were subject to an ophthalmic examination for

corneal, iridial and conjunctival effects on days 1, 2, 3, 7

and 14 using a Draize scoring system.

**Result:** UNWASHED

In the unwashed eyes corneal opacity (grade 2) was noted in 1

out of 6 eyes on days 1-3, fully resolved by day 7. Minor

iridial involvement (score 1) was also present (same animal)

at day 1 only.

Conjunctival redness was present in all animals, resolved by

day 7:

Day 1 1, 1, 2, 2, 2, 1

Day 2 0, 1, 2, 2, 2, 1

Day 3 0, 1, 1, 1, 1, 1

Day 7 0, 0, 0, 0, 0, 0

Day 14 0, 0, 0, 0, 0, 0

Conjunctivae swelling (chemosis) was present in all animals,

resolved by day 7:

Day 1 2, 2, 2, 2, 2, 1

Day 2 0, 0, 2, 2, 2, 1

Day 3 0, 0, 2, 0, 0, 0

Day 7 0, 0, 0, 0, 0, 0

Day 14 0, 0, 0, 0, 0, 0

Scores for 3 washed eyes were generally slightly lower.

Overall mean irritation Draize score (maximum possible 110):

1 day 12.2

2 days 8.3

3 days 4.0

7 days 0

14 days 0

**Test substance:** ETBE, purity not stated, clear liquid, from ARCO
Chemical Co.,

PA (no further details).

**Conclusion:** Under the conditions of this investigation, ETBE was

moderately irritating to rabbit eye.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

30-NOV-2003 (58)

\- 89/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Species:** rabbit

**Result:** slightly irritating

**EC classificat.:** not irritating

**Method:** other: EEC Directive 92/69

**GLP:** no data

**Test substance:** other TS

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Test substance:** ETBE

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

04-JAN-2004 (23)

**[5.3 Sensitization]{.underline}**

**Type:** Guinea pig maximization test

**Species:** guinea pig

**Concentration 1st:** Induction 10 % intracutaneous

**2nd:** Challenge 100 % occlusive epicutaneous

**No. of Animals:** 30

**Result:** not sensitizing

**Classification:** not sensitizing

**Method:** OECD Guide-line 406 \"Skin Sensitization\"

**Year:** 1992

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS

Hartley guinea pigs (approx. 6 wk old and weighing 250 - 550

g\) were randomly assigned to control (5 male + 5 female) and

test (10 male + 10 female) groups.

INDUCTION

\- intradermal injection:

\- 50% Freund\'s complete adjuvant (2 x 0.1 ml)

\- 10% v/v ETBE in sterile liquid paraffin (2 x 0.1 ml)

\- 10% v/v ETBE in 50% Freund\'s complete adjuvant (2 x 0.1

ml\)

The control animals received sterile liquid paraffin (2 x

0.1 ml), also by intradermal injection.

\- topical occlusive application:

0.5 ml undiluted ETBE was applied to the injection site

(under occlusion) after treating the skin with 10% SLS. The

control animals received sterile liquid paraffin in place of

ETBE.

CHALLENGE

\- 90/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

0.5 ml undiluted ETBE was applied to a 2 x 2 cm area of

clipped and depilated skin on the left flank of each

control and test animal. Skin responses were recorded at 24

and 48 hr post-challenge.

**Result:** Visual examination of the challenge site revealed no

evidence of any redness or oedema in any of the control or

test animals (all scores = 0).

**Test substance:** ETBE, 95.8%, batch no. 93050601, supplied by Elf
France.

**Conclusion:** Under the conditions of the test, ETBE did not provoke
any

sign of cutaneous sensitisation in the guinea pig.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (70)

**Type:** no data

**Species:** other: presumably rabbit

**Result:** not sensitizing

**Classification:** not sensitizing

**Method:** other: EEC Directive 92/69

**GLP:** no data

**Test substance:** other TS

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Test substance:** ETBE

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

04-JAN-2004 (23)

\- 91/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[5.4 Repeated Dose Toxicity]{.underline}**

**Type:** Sub-chronic

**Species:** rat **Sex:** male/female

**Strain:** Fischer 344

**Route of administration:** inhalation

**Exposure period:** 90 d

**Frequency of treatment:** 6 h/d, 5 d/wk for 13 wk

**Doses:** 0 (filtered air), 500, 1750 or 5000 ppm

**Control Group:** yes, concurrent vehicle

**NOAEL:** = 500 ppm

**LOAEL:** = 1750 ppm

**Method:** EPA OTS 798.2450

**Year:** 1996

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE

Animals: Fischer F344 rats (Charles River Laboratories,

Raleigh, NC, USA), males and females, age approx. 5 wk on

receipt

Acclimation period: 10 d

Housing: individually housed, stainless steel cages (wire mesh

bottom) within exposure chamber (one per sex per exposure

level) for duration of study

Diet: NIH-07 (Zeigler Brothers Inc), ad libitum

Water: deionised water, ad libitum

Light cycle: 12 hr light/dark period (lights on 0700-1900)

Assignment to treatment groups: computer-generated weight

randomisation

GENERAL EXPERIMENTAL DESIGN

Animals were exposed to target concentrations of 0 (filtered

air), 500, 1750 or 5000 ppm. The highest exposure level was

approx. 50% of the lower explosive limit for ETBE. All animals

were observed for mortality and overt clinical signs just

before and shortly after exposure, and once daily on

non-exposure days. An ophthtalmic examination was performed on

all rats prior to the start of the study and again at study

termination. Body weights from all rats were recorded weekly.

The study comprised the following elements:

\- main study group (11 rats/sex/exposure level)

\- neurotoxicity subgroup (12 rats/sex/exposure level; see

separate record)

\- interim clinical chemistry and haematology sub-group (10

rats/sex/exposure level)

\- cell replication subgroup (15 rats/sex/exposure level)

MAIN STUDY

Main study animals were exposed to target concentrations of 0,

500, 1750 or 5000 ppm ETBE vapour 6 hr/d, 5 d/wk for at least

13 wk (equivalent to at least 65 exposures). Animals were

anaesthetised (sodium pentobarbital) and sacrificed by

\- 92/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

exsanguination. All major organs were subject to macroscopic

examination, and samples retained in 10% neutral buffered

formalin (NBF). Organ weights were recorded for selected

tissues (adrenals, brain, heart, kidneys, liver, lungs,

ovaries, spleen, testes). All retained tissues were processed

(H&E staining) and those from the control and high dose groups

subject to microscopic examination. Lungs, liver, kidneys,

testes(\* see below) and femoral bone marrow (females only)

from low and intermediate level animals were also examined.

\*Note: one hundred round cross sections of seminiferous

tubules were examined per testis and assigned to a specific

group of developmental stages according to Hess (1990)

(Biology of Reproduction, 43, 525-542). The number of tubules

that contained debris and degenerating or necrotic cells was

also recorded.

CLINICAL CHEMISTRY AND HAEMATOLOGY DETERMINATIONS

Animals (10/sex/group) scheduled for interim clinical

chemistry and haematology investigations were exposed to 0,

500, 1750 or 5000 ppm for 6 wk (32-33 exposures for males or

females, respectively). Analyses were also performed on main

study animals at termination (wk 13). Blood was collected by

cardiac puncture (sodium pentabarbital anaesthesia).

Haematological evaluations were performed on blood containing

EDTA as anticoagulant using a Serono Hematology System 910

analyser, and included haematocrit, total leukocyte count,

haemoglobin concentration, erythrocyte count, platelet count,

mean corpuscular volume, mean corpuscular haemoglobin and mean

corpuscular haemoglobin concentration. Differential leukocyte

counts were performed on a blood smears.

Clinical chemistry determinations (serum) were performed using

a Roche Cobass Fara II analyser and included glucose

(non-fasting), urea, nitrogen, bilirubin, creatinine, calcium,

total protein, albumin, sodium, chloride, potassium,

phosphorus, aspartate aminotransferase, alkaline phosphatase,

alanine aminotransferase and gamma-glutamyl transferase.

CELL REPLICATION SUBGROUP

Five animals/sex/group were exposed to 0, 500, 1750 or 5000

ppm ETBE for 1, 4 or 13 wk (equivalent to 4, 19 or 63

exposures). Alzet 2ML1 pumps were implanted (s.c.) after the

first exposure in each investigation week and delivered BrdU

(16 ug/hr) for 3.5 d prior to sacrifice. Animals were removed

from the exposure chamber on the morning of day 5 of each

investigation week (prior to exposure), anaesthetised (sodium

pentobarbital) and perfused in situ with 10% NBF. The kidneys

(potential target tissue) and a section of duodenum (control

tissue) were removed, fixed (10% NBF) and embedded in paraffin

wax. BrdU-labelled cells (5 um sections) were identified by

immunohistochemical staining. Labelling index (LI; the

percentage of total nuclei counted that were positive for

\- 93/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

BrdU) was determined by counting a minimum of 1000 renal

cortical epithelial cells from 2 kidneys per animal using the

Cytology/Histology Recognition System (Sverdrup Technology

Inc, Walton Beach, FL).

OTHER RENAL HISTOPATHOLOGY

Additional studies were performed on individual kidneys to

determined the presence and extent of protein droplet

accumulation (one section haematoxylin and eosin; another

Mallory Heidenhain stain) and the presence of a2u-globulin

(immunohistochemical staining) in renal cortical cells.

EXPOSURE AND GENERATION OF TEST ATMOSPHERE

Animals were exposed in 1000 litre multi-tier, stainless

steel, whole body exposure chambers (one chamber per sex per

exposure level). Vapour concentration was determined (GC-FID)

at 5 positions within each chamber prior to introduction of

animals to check uniformity of exposure (variation \<8%), with

cages rotated weekly to compensate for any minor variations

that might be present. Airflow was 200-250 l/min (12-15

changes per hr). Environmental conditions were maintained in

the range 71.0-72.1 degrees C, relative humidity 48.0-53.1%.

The test atmosphere was generated by passing liquid ETBE

through a metering pump and into a heated stainless steel

J-tube; nitrogen gas carried the vapour into the exposure

chamber. The concentration of ETBE within the chamber

(animals\' breathing zone) was analysed by GC-FID at least 6

times per day.

STATISTICAL METHODS

The Shapiro-Wilks\' test (normality) and the O\'Brien and

Brown-Forsythe (homogeneity) test were followed by ANOVA and

Dunnetts\'s test (parametric data) or the Kruskal-Wallis test

(non-parametric data) on group means. A P value \<0.05 was

considered significant. Histopathology incidence data were

evaluated using Fisher\'s exact test with Bonferroni

correction. Labelling index and testicular data were

transformed (arcsine) prior to analysis using William\'s test.

**Result:** GENERAL

Gas chromatographic analysis showed that the mean

concentration of ETBE within the test chambers over the course

of the study was 505(13), 1748(59) and 4971(155) ppm (SD given

in brackets). No ETBE was detected in the control chamber.

All treated animals survived to scheduled study termination.

Transient ataxia the only clinical sign noted (high dose males

only, post-exposure only). Absolute body weight was decreased

significantly in males (3-6%) and females (2-5%) during study

weeks 1-3, while body weight gain was decreased by approx. 25%

in intermediate and high dose animals of both sexes during the

first week of treatment.

HAEMATOLOGY

\- 94/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Platelet counts were increased 14-16% (P\<0.05) and MCHC

decreased 2-4% (P\<0.05) in high dose males at wk 6 and wk 13.

MCHC was also slightly decreased (2.5%; P\<0.05) in

intermediate dose males at study termination only. MCV was

increased 1% (P\<0.05) in high dose females at the same time

points (but RBC count unaffected, hence not indicative of

regenerative anaemia). There were no other treatment related

alterations in haematological parameters in either sex.

CLINICAL CHEMISTRY

Serum chloride was decreased 3-6% (P\<0.05) and total protein

increased 9-11% (P\<0.05) in male rats exposed to 1750 ppm or

5000 ppm ETBE for 6 wk or 13 wk. Serum phosphorus (decreased

17%) and bilirubin (decreased 39%) were altered in high dose

females at wk 6 (both P\<0.05), however these parameters were

unremarkable at study termination. There were no other

treatment related alterations in clinical chemistry endpoints

in either sex.

NECROPSY OBSERVATIONS AND ORGAN WEIGHTS

No treatment related gross abnormalities were present in

either sex at scheduled necropsy. Absolute mean kidney

(10-19%) and liver (22-32%) weights were increased

significantly (P\<0.05) in intermediate and high dose males

following 13 wk exposure to ETBE, while adrenal weights were

increased 34% (P\<0.05) in high dose males only. For females,

absolute heart (12%) and liver (26%) weights were increased

significantly in high dose animals, while kidney weights were

elevated 12-21% (P\<0.05) in the 1750 ppm and 5000 ppm groups,

respectively.

HISTOPATHOLOGY

Kidneys and testes from males and femoral bone marrow from

females were the only tissues to exhibit treatment related

microscopic lesions.

Reviewer\'s comment: The source references contain only

incidence data, and generally no information on severity.

\- males, kidney (LOAEL 500 ppm)

The incidence of nephropathy (occurrence of regenerative foci)

was increased significantly (P\<0.05) in all treatment male

groups relative to the controls. Results summarised by

exposure level:

4/11 (36%), 10/11 (91%) 11/11 (100%), 11/11 (100%)

\- testes (NOAEL 500 ppm)

Exposure to 1750 ppm or 5000 ppm ETBE resulted in a

significant (P\<0.05) increase in the incidence of degeneration

of the seminiferous tubules of the testes of male rats.

Results (mean incidence per 100 examined with SD in brackets)

summarised by exposure level:

2(1), 2(2), 8(4), 13(11)

Reviewer\'s comment: The large standard deviation indicates

\- 95/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

that this finding was inconsistently expressed in high dose

males. Overall range by exposure level:

1-4, 1-7, 3-16, 0-39.

Review of the individual animal data (pathology report)

reveals that some animals were unaffected by treatment

(incidence 0 or 2) while others exhibited a pronounced 10-20

fold increase the incidence of degeneration.

ETBE treatment was without effect on the percentage of tubules

with debris was present in the lumen. Results (mean percent

with SD in brackets) summarised by exposure level:

2(2), 1(2), 3(8), 1(1)

There was no change in the percentage of seminiferous tubules

within the various functional stages of development when

treated animals were compared with controls, consistent with

no treatment related effect on spermatocyte maturation.

Results (mean percent with SD in bracket) summarised by

exposure level:

Stage I-V: 36(5), 32(5), 34(6), 36(6)

Stage VI-VIII: 30(6), 27(4), 31(3), 29(6)

Stage IX-XIII: 29(7), 37(3), 30(5), 31(6)

Stage XIV: 5(2), 5(2), 5(2), 5(2))

The report notes in those testes with tubular degeneration,

aberrant spermatids appeared most common in seminiferous

tubules in stages IX-XIII (no further details).

\- females, femoral bone marrow (NOAEL 500 ppm)

An increased incidence of congestion was observed in femoral

bone marrow from female rats exposed to 1750 ppm or 5000 ppm

ETBE for 13 wk. Incidence by exposure level:

0/10, 0/11, 5/11 (45%), 11/11 (100%)

TIMECOURSE AND CELL PROLIFERATION STUDIES IN KIDNEY

\- MALES

H&E staining revealed a concentration dependent increase in

nephropathy, as determined from the increase in mean number of

regenerative foci. Results (mean number of regenerative foci

per 2 kidney sections/rat) by week and exposure level:

\- 1 wk: 0, 0, 0, 0

\- 6 wk: 0, 0, 2, 4

\- 13 wk: 2, 11, 17, 34

Proximal tubule labelling indices was increased in high dose

male rats after 1 wk or 4 wk exposure to ETBE, and in all

treated males at wk 13. Results by week and exposure level:

\- 1 wk: 3.5, 4.9, 4.3, 7.1 (P\<0.01)

\- 4 wk: 3.3, 4.0, 2.8, 9.0 (P\<0.001)

\- 13 wk: 0.9, 2.2 (P\<0.01), 3.4 (P\<0.001), 2.5 (P\<0.001)

(The report notes that tissue growth and maturation accounts

for the variation in the control data from the different

timepoints.)

\- 96/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Mallory-Heidenhain stain demonstrated increased severity of

protein droplet accumulation in the renal tubule from male

rats exposed to ETBE. Results by week and exposure level:

\- 1 wk: 1.2, 3.4, 4.0, 4.6

\- 4 wk: 1.8, 2.6, 3.4, 3.8

\- 13 wk: 1.8, 3.0, 3.2, 3.8

Grading: 1 = minimal; 2 = \<10% of cortex involved; 3 = 10-25%

of cortex; 4 = 25-50% of cortex; 5 = \>50% of cortex.

Immunoreactivity toward a2u-globulin was present in the

protein droplets present in kidneys from all exposed males.

\- FEMALES

There was no microscopic evidence of any treatment related

increase in nephropathy in female rats.

Proximal renal tubule labelling indices were elevated in all

females rats for all treatment groups at wk 1 and wk 4 but

were indistinguishable from controls at wk 13. Results by week

and exposure level:

\- 1 wk: 2.6, 4.2 (P\<0.05), 5.0 (P\<0.05), 4.0 (P\<0.05)

\- 4 wk: 1.4, 1.4, 1.6, 1.8 (P\<0.05)

\- 13 wk: 0.6, 1.0, 1.0, 0.9

The Mallory-Heidenhain stain was negative for protein droplet

accumulation in proximal tubules.

There was no a2u-globulin immunoreactivity in kidneys from any

of the females exposed to ETBE.

**Test substance:** ETBE, 97.5% pure (by GC-FID) was supplied by the
Sponsor (ARCO

Chemical Company, PA, USA) and manufactured by Phillips 66 (no

further details). The chemical composition of the test

substance was verified using NMR.

**Conclusion:** Survival, body weight and the majority of clinical
chemistry

and haematological parameters were unremarkable in male and

female F344 rats exposed to 5000 ppm ETBE by inhalation for up

to 13 weeks. Degenerative changes in testicular seminiferous

tubules were present in males exposed to 1750 ppm or 5000 ppm,

while concurrently exposed females responded with increased

congestion of femoral bone marrow. An increased incidence of

renal nephropathy, cell proliferation and accumulation of

protein droplets in proximal tubules from all exposed males

was associated with positive immunoreactivity toward

a2u-globulin, suggesting the findings were specific to the

male rat. Overall these results are consistent with a

sub-chronic NOAEL for ETBE in the rat of 500 ppm, based upon

histopathological changes in testes and bone marrow at 1750

ppm.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

30-NOV-2003 (13) (61)

\- 97/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** Sub-chronic

**Species:** mouse **Sex:** male/female

**Strain:** CD-1

**Route of administration:** inhalation

**Exposure period:** 90 d

**Frequency of treatment:** 6 h/d, 5 d/wk for 13 wk

**Doses:** 0 (filtered air), 500, 1750 or 5000 ppm

**Control Group:** yes, concurrent vehicle

**NOAEL:** = 1750 ppm

**Method:** EPA OTS 798.2450

**Year:** 1996

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE

Animals: CD-1 mice (Charles River Laboratories, Raleigh, NC,

USA), males and females, age approx. 5 wk on receipt

All other details identical to the preceding record (rat 90 d

inhalation study)

GENERAL EXPERIMENTAL DESIGN

Animals were exposed to target exposure concentrations of 0

(filtered air), 500, 1750 or 5000 ppm. The study comprised the

following elements:

\- main study group (15 mice/sex/exposure level)

\- clinical chemistry and haematology sub-group (10

mice/sex/exposure level)

\- cell replication subgroup (15 mice/sex/exposure level)

See preceding record (rat 90 d inhalation study) for other

details.

MAIN STUDY

Main study animals were exposed to target concentrations of 0,

500, 1750 or 5000 ppm ETBE vapour 6 hr/d, 5 d/wk for at least

13 wk (equivalent to at least 65 exposures). Animals were

anaesthetised (sodium pentobarbital) and sacrificed by

exsanguination. All major organs were subject to macroscopic

examination, and samples retained in 10% neutral buffered

formalin (NBF). Organ weights were recorded for selected

tissues (adrenals, brain, heart, kidneys, liver, lungs,

ovaries, spleen, testes). All retained tissues were processed

(H&E staining) and those from the control and high dose groups

subject to microscopic examination. Lungs, liver (with gall

bladder), kidneys and all gross lesions from low and

intermediate level animals were also examined.

CLINICAL CHEMISTRY AND HAEMATOLOGY DETERMINATIONS

Ten mice per sex per exposure level were subject to clinical

chemistry and haematological evaluation at study termination

(see preceding record, rat 90 d inhalation study). Animals

were subsequently subject to a full necropsy.

CELL REPLICATION SUBGROUP

\- 98/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Fifteen animals/sex/group were exposed to 0, 500, 1750 or 5000

ppm ETBE for 1, 4 or 13 wk (equivalent to 4, 19 or 63

exposures). Alzet 2001 pumps were implanted (s.c.) after the

first exposure in each investigation week and delivered BrdU

(16 ug/hr) for 3.5 d prior to sacrifice. Animals were removed

from the exposure chamber on the morning of day 5 of each

investigation week (prior to exposure), anaesthetised (sodium

pentobarbital). The livers (potential target tissue) and a

section of duodenum (control tissue) were removed, fixed (10%

NBF) and embedded in paraffin wax. BrdU-labelled cells (5 um

sections) were identified by immunohistochemical staining.

Labelling index (LI; the percentage of total nuclei counted

that were positive for BrdU) was determined by counting a

minimum of 1000 hepatocytes per animal.

EXPOSURE AND GENERATION OF TEST ATMOSPHERE

See preceding record (rat 90 d inhalation study)

STATISTICAL METHODS

See preceding record (rat 90 d inhalation study)

**Result:** GENERAL

Gas chromatographic analysis showed that the mean

concentration of ETBE within the test chambers over the course

of the study was 505(13), 1748(59) and 4971(155) ppm (SD given

in brackets). No ETBE was detected in the control

chamber. Six male and six female mice died during the study;

five were found dead (controls + exposed animals) and 7 died

from accidental trauma. Transient ataxia was occasionally

observed post-exposure in high dose animals of both sexes.

Absolute body weight and body weight gain were unaffected by

treatment.

HAEMATOLOGY

Haemoglobin and haematocrit values were increased

significantly in male mice exposed to 1750 ppm ETBE relative

to the controls, however values for the high dose group and

intermediate or high dose females were unaffected suggesting

these findings were unrelated to treatment. There were no

other treatment related alterations in haematological

parameters in either sex.

CLINICAL CHEMISTRY

All changes in ETBE exposed mice were minimal in extent and

considered not indicative of any adverse toxicological process

by the authors of the report.

NECROPSY OBSERVATIONS AND ORGAN WEIGHTS

No treatment related gross abnormalities were present in

either sex at scheduled necropsy. Absolute liver weights were

increased significantly in male and female mice exposed to

1750 ppm or 5000 ppm ETBE vapour. Mean values (g) by exposure

level were as follows:

\- males: 2.1, 2.3, 2.4 (P\<0.05), 2.5 (P\<0.05)

\- 99/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\- females: 1.6, 1.6, 1.9 (P\<0.05), 2.1 (P\<0.05)

HISTOPATHOLOGY

\- liver (NOAEL 1750 ppm)

An increased incidence of centrilobular hypertrophy was

observed in livers of mice exposed to ETBE. This was

characterised by enlargement of hepatocytes in the

centrilobular to midzonal areas, with homogeneously

eosinophilic cytoplasm. Results summarised by exposure level:

\- males: 0/15, 0/15, 2/15, 8/15 (P\<0.05)

\- females: 0/13, 2/15, 1/15, 9/14 (P\<0.05)

\- kidney (NOAEL, females, 1750 ppm)

The incidence of minimal renal nephropathy (characterised by

thickened tubular basement membranes, lymphocytic interstitial

infiltrates, fibrosis and/or regenerative, basophilic tubules)

was slightly increased in exposed males and in 5000 ppm

females. Results summarised by exposure level:

\- males: 4/15 (27%), 7/15 (47%), 8/15 (53%), 4/10 (40%)

\- females: 4/13 (31%), 6/15 (40%), 6/15 (40%), 9/14 (64%)

Reviewer\'s comment: The study report notes these findings were

not considered indicative of a definitive effect of ETBE on

mouse kidney. No statistical analysis was performed on these

findings. In the opinion of this Reviewer, no obvious

dose-response relationship is present in males, while in

females the response appears limited to the high exposure

group only.

TIMECOURSE AND CELL PROLIFERATION STUDIES IN LIVER

The liver of male and female mice exhibited a treatment and

concentration dependent increase in cell proliferation. LI

remained elevated in males following 4 wk treatment but

returned to control levels by wk 13. In females, LI was

comparable to control values at wk 4, but elevated at wk 13

(males unaffected). Results (LI) are summarised below by sex

and by exposure level:

\- males:

\- 1 wk: 0.41, 1.64, 6.16 (P\<0.001), 18.08 (P\<0.001)

\- 4 wk: 0.52, 0.99, 1.37 (P\<0.05), 2.49 (P\<0.001)

\- 13 wk: 0.98, 0.89, 1.13, 0.67

\- females:

\- 1 wk: 2.75, 4.10, 20.24 (P\<0.001), 38.31 (P\<0.001)

\- 4 wk: 2.17, 3.80, 1.65, 2.56

\- 13 wk: 1.49, 1.63, 3.40 (P\<0.05), 4.91 (P\<0.05)

Reviewer\'s comment: The study report notes that a transient

increase in hepatocyte LI is a common response observed in

mice following exposure to mitogenic substances, with a more

exaggerated response typically seen in females.

**Test substance:** ETBE, 97.5% pure (by GC-FID) was supplied by the
Sponsor (ARCO

Chemical Company, PA, USA) and manufactured by Phillips 66 (no

further details). The chemical composition of the test

\- 100/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

substance was verified using NMR.

**Conclusion:** Survival, body weight, clinical chemistry determinations
and

haematological parameters were unremarkable in male and female

CD-1 mice exposed to 500, 1750 or 5000 ppm ETBE vapour for up

to 13 weeks. Absolute liver weights were increased in animals

exposed to 1750 ppm and above, while an increased incidence of

centrilobular hypertrophy was present in high dose animals of

both sexes. Hepatic cell proliferation (a common non-adverse

change seen in mouse liver after exposure to mitogenic

substances) was increased following 1-13 weeks exposure to

1750 ppm and above. These findings are consistent with a

sub-chronic NOAEL for ETBE in the mouse of 1750 ppm, based on

the occurrence of increased liver weight and mild

histopathological changes present in animals exposed to 5000

ppm.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

30-NOV-2003 (12) (61)

**Type:** Sub-chronic

**Species:** rat **Sex:**

**Strain:** Fischer 344

**Method:** EPA OTS 798.2450

**Year:** 1966

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** GENERAL

See record by Bond et al (1996) for further experimental

details.

NEUROLOGICAL EVALUATION

A functional observation battery (FOB) evaluation was

performed 4 days prior to the first exposure, and after 1, 6,

10, 20, 42, 65 days. Data were collected at least 18 hr after

ETBE exposure.

Animals evaluated for posture, tremors, spasms, convulsions,

palpebral closure, handling reactivity and muscle tone

together with an assessment of general condition

(piloerection, fur appearance, skin temperature and colour,

breathing pattern, salivation and lachrymation).

During then open field test, rats were observed for 2 min and

spontaneous activity and behaviour recorded; arousal, ataxia,

gait, body position, excessive vocalization, stereotypy and

any unusual behaviour were also noted at this time.

An assessment of visual approach response, auditory startle

response, tail pinch response, surface righting reflex, visual

placing response, forelimb and hindlimb grip strength, hind

\- 101/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

leg splay and papillary reflex were also performed.

Motor activity was measured during ten 6 min intervals using a

photobeam activity system.

ORGANS EXAMINED AT NECROPSY

Brains were perfused in situ by intra-aortic perfusion (0.7%

sodium nitrite) and fixed in situ with glutaraldehyde and

formaldehyde. Brains were then weighed and size measured.

Gasserian ganglia, dorsal root ganglia, spinal root fibres,

brain, spinal cord, eye, optic nerve, sciatic nerve were

subject to histopathological examination.

STATISTICAL METHODS

Levene\'s test for homogeneity followed by ANOVA and Dunnett\'s

t-test were applied on homogeneous data. Non-homogeneous data

were log-transformed. If transformed data were

non-homogeneous, Kruskall-Wallis H-test and Wilcoxon

two-sample rank-sum test were used. Categorical FOB data were

analysed using a log-linear model.

**Result:** There were no gross macroscopic changes in brain or nervous

tissue.

The FOB evaluation found no evidence of sensorimotor

dysfunction, neuromuscular dysfunction, ataxia, piloerection,

excessive vocalization, muscle tremors or spasms, clonic or

tonic seizures, increased salivation, abnormal respiration or

abnormal pupil reflex.

Decreased mean hindlimb grip strength and increase in mean

hindlimb splay were observed in low exposure group males at

first exposure, with a statistically significant increase in

mean forelimb grip strength in high exposure males on day 10.

A statistically significant decrease in mean forelimb grip

strength was observed in low dose females following 65

exposures. The absence of any consistent dose-related trend

suggest these observations are of doubtful toxicological

significance.

There was no treatment-related effect on overall motor

activity during any observation period.

**Test substance:** ETBE, 97.5% pure (by GC-FID) was supplied by the
Sponsor (ARCO

Chemical Company, PA, USA) and manufactured by Phillips 66 (no

further details). The chemical composition of the test

substance was verified using NMR.

**Conclusion:** No gross, functional or microscopic abnormalities were

observed in males and female rats exposed to 5000 ppm ETBE

vapour for up to 13 wk.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

02-NOV-2003 (13) (27)

\- 102/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** Sub-acute

**Species:** rat **Sex:** male/female

**Strain:** Sprague-Dawley

**Route of administration:** inhalation

**Exposure period:** 28 d

**Frequency of treatment:** 6 hr/d, 5 d/wk

**Doses:** 0 (air), 500, 2000 or 4000 ppm

**Control Group:** yes, concurrent vehicle

**NOAEL:** = 500 ppm

**Method:** OECD Guide-line 412 \"Repeated Dose Inhalation Toxicity:

28-day or 14-day Study\"

**Year:** 1990

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE:

Animals: SD rats (Charles River Breeding Laboratories Inc.,

Portage, MI), males and females, age approx. 9 wk on receipt

Acclimation: 3 wk

Weight at study initiation: mean 309g (males), 206g (females)

Housing: individually housed, stainless steel cages (wire mesh

bottom)

Diet: Purina Rodent Chow 5001 (Ralston Purina Co.) ad libitum

(except during inhalation exposure)

Water: Reverse osmosis tap water, automatic watering system,

ad libitum (except during inhalation exposure)

Light cycle: 12 hr light, 12 hr dark

Assignment to treatment groups: random allocation of rats with

bwt deviation \<3 SD from population mean

GENERAL EXPERIMENTAL DESIGN

Animals (n=10/sex/treatment level) were exposed whole body in

1 m3 glass and stainless steel chamber to target

concentrations of 0, 500, 2000 or 4000 ppm ETBE 6 hr/d, 5 d/wk

for 28 d. The concentration of ETBE vapour in each exposure

chamber was determined by IR analysis (Wilkes Miran 1A-1R

analyser). Animals were observed for morbidity/mortality (7

d/wk) and clinical signs (5 d/wk) once daily. Body weight was

recorded at study initiation, weekly thereafter and at

termination. Comment: no food/water intake data collected.

CLINCIAL CHEMISTRY

Blood (abdominal aorta) was collected following an 18 hr fast

under sodium pentobarbital anaesthesia, prior to necropsy.

Analyses were performed for glucose, creatine kinase, alanine

aminotransferase, aspartate aminotransferase, alkaline

phosphatase, urea nitrogen, sodium, potassium, calcium,

chloride, total protein, triglyceride, cholesterol, albumin,

creatinine (methods not given).

HAEMATOLOGY DETERMINATIONS

Blood (as above) examined for total erythrocyte count,

haemoglobin, mean corpuscular volume, total leucocytes,

\- 103/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

differential leucocytes and platelets (methods not given).

POST-MORTEM OBSERVATIONS

Animals were sacrificed (pentobarbital) 18 hours after the

final exposure, following an overnight fast, and subject to a

macroscopic examination. Organ weights were recorded for

adrenal glands, brain, gonads, heart, kidneys, liver, lungs

and spleen. Lung volume was assessed from liquid displacement.

Samples of adrenals, brain, epididymides, eyes, oesophagus,

femur and bone marrow, gonads, heart, duodenum, jejunum,

ileum, caecum, colon, kidneys, larynx, liver, lungs, lymph

nodes, mammary gland, nasal turbinates, pancreas,

parathyroids, pituitary, prostate and seminal vesicles,

salivary glands, sciatic nerve, skeletal muscle, skin, spinal

cord, spleen, sternum, stomach, thymus, thyroids, tongue,

trachea, urinary bladder, uterus, ear and any gross lesions

were preserved in 10% neutral buffered formalin. Tissues from

the control and high exposure groups were subject to

microscopic evaluation (no details of tissue processing or

staining).

NEUROLOGICAL EVALUATION

A functional observation battery evaluation (FOB) was

performed one week before first exposure and following 1, 5,

20 exposures. It included home-cage observation

(posture/appearance), hand-held observation, open field

(mobility/gait), eye blink, pupil response, extensor thrust,

visual placing, tactile placing, negative geotaxis, grip

strength, foot splay, hind limb extension, startle response,

catalepsy, righting reflex, body temperature, rotorod

performance and tail pinch.

STATISTICAL METHODS:

Data were log-transformed and analysed by ANOVA. Body

weights, body temperatures, hind limb splay, grip strength and

rotorod performance evaluated using a multivariate repeated

measures analysis of variance to determine dose-response

relationship over time. For analysis of interactions Dunnett\'s

test used.

**Remark:** No NOAEL is identified in the report from IIT Research

Institute (1991).

White et al (1995) nominate a NOAEL of 500ppm but do not

identify the critical effect(s). It is assumed that this was

derived from an increase in relative liver weight in females

exposed to 2000 ppm or 4000 ppm, however an absence of

accompanying changes in clinical chemistry or histopathology

suggests these alterations are of questionable toxicological

significance.

A more appropriate NOAEL appears to be 2000ppm, based on the

CNS effects (sedation and ataxia) seen in animals exposed to

4000 ppm ETBE vapour.

**Result:** GENERAL

\- 104/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Exposure concentrations in a range 350-555ppm were recorded in

low exposure chamber (mean 501+/-17.8), with 1720-2370 in

intermediate chamber (2090+/-111) and 3200-4540 ppm in the

high exposure chamber (3920+/-206).

All animals survived to scheduled study termination. Signs of

general sedation and reduced motor activity were noted in

animals exposed to 4000 ppm ETBE vapour, with some animals

exhibiting mild to moderate ataxia. After 3 hours exposure no

startle response was evident in the majority of high exposure

animals. All treated animals appeared to be in \'sleeping

position\' (muscle relaxation not evident) during exposure but

were normal 15 minutes post-exposure. Other treatment-related

clinical signs included salivation, redness around

nose/mouth/face and discoloured paws/forelimbs.

There were no statistically significant effects of treatment

on bw or bw gain.

CLINICAL CHEMISTRY

No treatment related effects or trends were apparent in the

data.

HAEMATOLOGY

White blood cell counts were significantly increased in

intermediate (elevated 61%; P\<0.05) and high exposure

(elevated 81%; P\<0.05) females. All other parameters were

comparable to control for both sexes. Histopathological

examination found no changes in bone marrow or evidence of

inflammation which could account for these observation.

NECROPSY OBSERVATIONS

Lung foci, present in all groups (hence not considered

treatment related) were the only macroscopic observation of

note. Absolute and relative liver weights were significantly

increased in high exposure male (16% inc; P\<0.05) and female

rats (abs. 10% inc; rel. 12% inc; P\<0.05 for both). Relative

liver weight was also increased in females from the

intermediate exposure group (10% inc; P\<0.05). Absolute kidney

(inc 12%; P\<0.05) and adrenal weights (inc 14%; P\<0.05)were

also increased in high exposure males.

HISTOPATHOLOGY

Lymphoid and plasma cell hyperplasia in mandibular lymph

nodes, and lymphoid hyperplasia and haemorrhage in the

respiratory lymph nodes was commonly seen but not treatment

related. There were no histopathological changes in liver,

kidney or adrenal gland (ie those tissues showing weight

changes). Comment: pyelonephritis, unilateral hydropelvis and

tubular basophilia, present sporadically in both sexes

(including controls), were not considered related to

treatment. Unilateral tubular atrophy of the testes was

present in one control male, while males from the 4000 ppm

exposure group were unremarkable. Bone marrow had a normal

\- 105/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

microscopic appearance. Overall there were no treatment

related histopathological changes.

FUNCTIONAL OBSERVATION BATTERY

There were no differences in clinical signs, mobility/gait,

sensory perception and reflex responses. Body temperature was

decreased by approx. 0.5 degrees C (P\<0.05) in 4000 ppm males

on day 5 only. Hind limb splay at day 20 was significantly

increased (+27%; P\<0.05) when results for high exposure males

and females were combined and compared with the controls

(individual sex-specific values were comparable to control).

Comment: Combined values at day 20 (7.51+/-1.98) are

essentially identical to combined day 0 control values

(8.36+/-1.91) and combined day 0 high exposure animal values

(7.45+/-1.94). These observations therefore appear of marginal

biological significance.

**Test substance:** ETBE, supplied by Amoco Corporation, purity not
stated.

**Conclusion:** Survival, body weight and the majority of clinical
chemistry

and haematological parameters were unremarkable in male and

female F344 rats exposed to 4000 ppm ETBE by inhalation for up

to 28 weeks. An increase in relative liver weight in females

exposed to 2000 ppm with no accompanying clinical chemistry or

histopathological changes appears of doubtful toxicological

relevance. Overall the results are consistent with a sub-acute

NOAEL for ETBE in the rat of 2000 ppm, based upon CNS

depression (sedation and ataxia) seen in animals exposed to

higher exposure levels.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

05-OCT-2003 (42) (87)

**Type:** Sub-acute

**Species:** rat **Sex:** male/female

**Strain:** Sprague-Dawley

**Route of administration:** inhalation

**Exposure period:** 4 wk

**Frequency of treatment:** 6 h/d, 5 d/wk for 4 wk

**Doses:** 0, 500, 2000 or 4000 ppm

**Control Group:** yes, concurrent vehicle

**NOAEL:** = 2000 ppm

**Year:** 1991

**GLP:** no data

**Test substance:** as prescribed by 1.1 - 1.4

**Remark:** Published abstract:

Crl:CD rats (n=10/sex/treatment level) exposed to 0, 500, 2000

or 4000 ppm ETBE vapour 6 hr/d, 5 d/wk for 4 wk.

Parameters monitored included bwt, organ wt, lung volume,

haematology, clinical chemistry, gross and microscopic

histopathology as well as function observation battery for

\- 106/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

neuromuscular function, reflex response and sensory perception

(before and after 1, 5 and 20 exposures).

CNS depression (ataxia, sedation) observed immediately

post-exposure in 4000 ppm group, with rapid and full recovery

post-exposure.

Changes in body temperature and hindlimb splay pattern present

in high dose group. (The authors note that the latter finding

is \'possibly\' indicative of an effect on hindsplay.)

Liver, kidney, and adrenal wt (absolute and/or relative)

increased in high dose males, and liver wt increased in

intermediate and high dose females. No histopathological

changes were associated with these findings.

White cell counts increased in females exposed to 2000 or 4000

ppm ETBE vapour for 4 wk.

**Test substance:** No details available.

**Conclusion:** The author\'s conclude that repeated exposure to 4000 pm
ETBE

produced transient signs of CNS depression and associated

changes in body temperature and possibly hindlimb splay.

**Reliability:** (4) not assignable

Study available for review. Abstract, highly limited

information on methods and findings.

30-NOV-2003 (76)

**[5.5 Genetic Toxicity \'in Vitro\']{.underline}**

**Type:** Ames test

**System of testing:** Salmonella typhimurium TA 1535, TA 1537, TA 1538,
TA98,

TA 100 (with independent repeat)

**Concentration:** First test: 0, 8, 40, 200, 1000 and 5000 ug/plate;

Second test: 0, 313, 625, 1250, 2500 and 5000 ug/plate

**Cytotoxic Concentration:** \> 5000 ug/plate

**Metabolic activation:** with and without

**Result:** negative

**Method:** OECD Guide-line 471

**Year:** 1983

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Commercially-available freeze-dried S9 fraction (MOLTOX Inc)

from Arochlor 1254-induced rats was used in these studies.

Absolute ethanol was used as carrier solvent, and added at a

final volume of 0.05 ml/plate.

Each dose was tested in triplicate, with independent repeat.

2-Nitrofluorene (TA 98 and TA 1538), 9-aminoacridine (TA 1537)

and sodium azide (TA 1535 and TA 100) were used as positive

control substances in the absence of S9 mix, and 2-amino

\- 107/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

anthracene (all strains) as positive control in the presence

of S9.

Results were considered positive if a suitable response had

been obtained with the negative and positive control

substances, and the number of revertants in the presence of

ETBE was at least twice that of the negative control, with a

dose-response correlation.

**Result:** Results from a preliminary test using TA 100 (without S9)

revealed no toxicity at doses up to 5000 ug/plate.

All data met the acceptability criteria established for the

test. No increase in the number of revertants was seen with

any of the tester stains, both in the absence and presence

of S9 fraction, in either the first or second study. A

satisfactory response was obtained with the negative and

positive controls.

**Test substance:** ETBE, 95.8%, batch no. 93050601, supplied by
Elf-Antar,

France.

**Conclusion:** Under the conditions of these tests, ETBE was not
mutagenic

to Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98

or TA 100 at doses up to 5000 ug/plate, both in the absence

and presence of Arochlor 1254-induced rat S9 fraction.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (69)

**Type:** Ames test

**System of testing:** TA 1535, TA 1537, TA 1538, TA98, TA 100 (with

independent repeat)

**Concentration:** 0, 5, 15, 50, 150 and 500 ug/plate

**Cytotoxic Concentration:** \>1500 ug/plate for TA 1537, TA 1538 and TA
98; \> 500

ug/plate for TA 1535 and TA 100

**Metabolic activation:** with and without

**Result:** negative

**Method:** other: Maron and Ames, Mut Res 113, 173 - 215,

**Year:** 1983

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** S9 fraction was prepared from the livers of Arochlor

1254-induced male Sprague Dawley rats. 95% ethanol was used

as carrier solvent, and added at a final volume of 0.1

ml/plate.

Each dose was tested in triplicate, with independent repeat.

B-propiolactone (TA1535), hycanthone (TA 1537),

2-nitrofluorene (TA 1535 and TA 98) and sodium azide (TA 100)

were used as positive control substances in the absence of S9

mix, and 2-amino anthramine (all strains) was used as

positive control in the presence of S9.

\- 108/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Statistically significant increases in revertant numbers were

identified using Dunnett\'s test.

**Result:** Results from a preliminary test demonstrated that doses \>

1500 ug/plate were cytotoxic to TA 1537, TA 1538 and TA 98,

while doses \> 500 ug/plate were cytotooxic to TA 1535 and TA

100\.

No increase in the number of revertants was seen with any of

the tester stains, both in the absence and presence of S9

fraction, in either the first or second study. A

satisfactory response was obtained with the negative and

positive controls.

**Test substance:** ETBE, purity not stated, lot no. 408-2404, Total
Raffinage

Distribution, France.

**Conclusion:** Under the conditions of these tests, ETBE was not
mutagenic

to Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98

or TA 100 at doses up to 500 ug/plate, both in the absence

and presence of Arochlor 1254-induced rat S9 fraction.

**Reliability:** (2) valid with restrictions

Study available for review. Non-GLP near-guideline

investigation, clearly reported, acceptable for assessment.

02-NOV-2003 (45)

**Type:** Ames test

**System of testing:** Salmonella typhimurium TA 97, TA 98, TA 100, TA
1535

(no independent repeat)

**Concentration:** 0, 100, 333, 1000, 3333, 10000 ug/plate

**Cytotoxic Concentration:** \>10000 ug/plate

**Metabolic activation:** with and without

**Result:** negative

**Year:** 1992

**GLP:** no

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** S9 fraction was prepared from the livers of Arochlor

1254-induced rat or hamster. Assays performed using 10% or 30%

S9 from each source.

Initial toxicity was determined using TA100 only (test

performed on single occasion.

Each dose was tested in triplicate (no independent repeat).

Sodium azide (TA100 and TA1535), 9-aminoacridine (TA97 and

TA1537) and 4-nitro-o-phenyldimaine (TA98) were used as

positive control substances in the absence of S9, while

2-aminoanthracene was used in the presence of S9.

A result was judged to be indicative of mutagenicity if there

was a reproducible, dose-related response over the solvent

control, under a single metabolic condition, in replicate

trials.

**Result:** Only limited results are reported in this publication.

\- 109/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Results are presented for TA 100 and 1535 only in absence of

S9 in a single test using 2 concentrations of S9 from rat and

hamster. No increases in numbers of revertants were seen in

either strain.

Note: the report states that ETBE was evaluated using all

strains, but results are only presented for TA 100 and TA

1535\.

**Test substance:** ETBE, 99%, Aldrich Chemical Co., Milwaukee, WI.

**Conclusion:** Under the conditions of this test, ETBE was not
mutagenic in a

bacterial reverse mutation assay at concentrations up to 10000

ug/plate, in the absence or presence of rat or hamster S9.

**Reliability:** (2) valid with restrictions

Study available for review. Briefly reported experimental

investigation from US-NTP, acceptable for assessment.

02-NOV-2003 (91)

**Type:** Ames test

**System of testing:** no data

**Concentration:** no data

**Metabolic activation:** no data

**Result:** negative

**Method:** other: no data

**GLP:** no data

**Test substance:** other TS

**Remark:** Chatin, L. (Elf Antar France).

As reported in the Acts of 6th EFOA Conference \"ETBE in

Europe\", Brussels, October 27-28 1994, p. 9

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Test substance:** ETBE

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003

**Type:** Chromosomal aberration test

**System of testing:** Metaphase analysis in CHO cells

**Concentration:** 0, 300, 1000, 3000, 5000 ug/ml

**Cytotoxic Concentration:** \>5000 ug/ml

**Metabolic activation:** with and without

**Result:** negative

**Method:** OECD Guide-line 473

**Year:** 1995

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Logarithmically growing cultures were treated (in duplicate)

for 4 hours with ETBE and harvested 10 hr after onset of

\- 110/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

treatment. Optimal harvest time determined in a preliminary

study by cell cycle kinetics using bromodeoxyuridine.

Incubations were performed in the absence or presence of

Aroclor induced rat liver S9 fraction. CAs were scored in 50

cells per culture.

Toxicity determined by colony-forming potential and mitotic

inhibition in the preliminary toxicity test and by mitotic

index analysis in the main study.

All treatments and culturing performed in glass bottles to

avoid solvent effects of ETBE.

Mitomycin C (minus S9) and cyclophosphamide (plus S9) were

used a positive control substances.

Results were analysed using Fisher\'s exact test, and

considered positive if there was:

\- a statistically significant, dose-related increase in

chromosome aberrations (excluding gaps);

\- a statistically significant increase at one dose level which

was reproducible between experiments and of large magnitude.

**Result:** No cytotoxicity was present as judged by the lack of mitotic

inhibition and cell cycle delay.

No statistically significant increases in chromosome

aberrations were seen at any treatment level.

Positive and negative control results were acceptable.

**Test substance:** ETBE, 98% pure (containing 13 ppm antioxidant)
supplied by the

Sponsor (ARCO Chemical Company, PA, USA) and manufactured by

Phillips 66 (no further details).

**Conclusion:** Under the conditions if this investigation, ETBE did not

induce chromosomal aberrations in CHO cell in vitro at

concentrations up to 5000 ug/ml, in the presence and absence

of Arochlor-induced S9.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, some minor deviations, well documented methods

and results, raw data available, acceptable for assessment.

02-NOV-2003 (17)

\- 111/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** HGPRT assay

**System of testing:** mammalian gene mutation assay in CHO cells

**Concentration:** 100, 300, 1000, 3000, 5000 ug/ml

**Cytotoxic Concentration:** \>5000 ug/ml

**Metabolic activation:** with and without

**Result:** negative

**Method:** OECD Guide-line 476

**Year:** 1995

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** Duplicate cultures (8 x 10e5 cells) were grown for 20-24
hours

before treatment with ETBE (up to 5000 ug/plate; 4 hr) in

presence and absence of S9 (rat, Arochlor 1254-induced).

Subcultures were made at 2-3 day intervals (5-10 x 10e5 cells

per culture). At 8 days after treatment 2 x 10e5 cells plated

into each of 5 plates containing selective medium for each

culture. Counted after 6-8 days.

There were three independent repeat tests in the absence of S9

and two independent repetitions in the presence of S9.

Cytotoxicity was determined by comparing the numbers of cells

in treated cultures with those in controls 24 hours after

treatment (cloning efficiency).

Ethylmethanesulphonate and dimethylnitrosamine were used as

positive control substances, DMSO as negative control.

Data were log transformed and analysed by the method of Irr

and Snee (1979, Banbury Report II, 263).

A result was considered valid if the spontaneous mutation

frequency less than 20 x 10e-6, the positive control frequency

was at least 3 times control and control cloning efficiency of

at least 80%.

**Result:** No cytotoxicity was present at concentrations up to the

maximum recommended for the assay (5000ug/ml). A satisfactory

response was obtained with the positive control cultures.

No statistically significant or dose-related increases in

mutation frequency were seen in any test.

Comment: the second test without S9 was repeated due to high

values in a vehicle control culture and in the cultures

treated with 100, 3000 and 5000ug/ml. A negative result was

obtained. Results from all three tests performed in the

absence of S9 are summarised below:

Mutation frequency (x 10e-6):

Test 1, -S9:

Control - 0.45

100ug/ml - 0.5

\- 112/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

300ug/ml - 1.3

1000ug/ml - 2.9

3000ug/ml - 1.2

5000ug/ml - 2.8.

Test 2, -S9:

Control - 17.7

100ug/ml - 26.7

300ug/ml - 12.2

1000ug/ml - 14.9

3000ug/ml - 25.9

5000ug/ml - 31.7

Test 3, -S9:

Control - 6.2

100ug/ml - 0.6

300ug/ml - 0.5

1000ug/ml - 2.3

3000ug/ml - 1.1

5000ug - 3.0

**Test substance:** ETBE, 98% pure (containing 13 ppm antioxidant)
supplied by the

Sponsor (ARCO Chemical Company, PA, USA) and manufactured by

Phillips 66 (no further details).

**Conclusion:** Under the conditions of the assay, ETBE (up to 5000
ug/ml) did

not induce mutations at the HGPRT locus in CHO cells in the

absence or presence of Arochlor-induced rat S9.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

02-NOV-2003 (18)

**Remark:** The structures of methyl- and ethyl-ter-buthyl-ethers were

analyzed by CASE, an expert system, and compared to the

structural determinants previously recognized as being

associated with carcinogenicity in rodents, mutagenicity in

Salmonella or the induction of sister chromatid exchanges

and chromosomal aberrations in cultured mammalian cells.

On the basis of this analysis the two chemicals are

predicted to be neither genotoxicants nor carcinogens.

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

23-DEC-2003 (75)

\- 113/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[5.6 Genetic Toxicity \'in Vivo\']{.underline}**

**Type:** Micronucleus assay

**Species:** mouse **Sex:** male/female

**Strain:** other: OF1

**Route of admin.:** gavage

**Exposure period:** 24 and 48 hr

**Doses:** 5000 mg/kg bwt

**Result:** negative

**Method:** Directive 84/449/EEC, B.12 \"Other effects - Mutagenicity

(micronucleus test)\"

**Year:** 1984

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS

Groups of 5 male and 5 female OF1 mice (22 - 29g, age 4 - 6

wk) were given 0 or 5000 mg/kg ETBE by gavage in 1%

carboxymethyl cellulose (dose volume 25 ml/kg bwt) and

sacrificed at 24 hr and 48 hr post-treatment.

PROCEDURE

Femoral bone marrow smears were prepared from each mouse,

and 2000 PCEs scored for the presence of micronuclei. The

ratio of PCE:NCE was quantified in 1000 erythrocytes.

POSITIVE CONTROL

Cyclophosphamide (50 mg/kg bwt in distilled water by ip

injection, dose volume 25 ml/kg bwt) was used as a positive

control substance, with animals sacrificed at 24 hr only.

STATISTICS

Results were analysed using the Mann Whitney U test and

Student\'s T test.

**Result:** CLINICAL SIGNS AND TOXICITY

All animals given ETBE survived 48 hr post-dosing with

no apparent signs of toxicity.

MICRONUCLEUS TEST

There was no increase in the number of micronuclei per 1000

PCEs in mice treated with 5000 mg ETBE/kg bwt in either sex

at either time point, whereas the incidence of micronuclei

was increased significantly (P \< 0.01) following

cyclophosphamide treatment.

**Test substance:** ETBE, purity not stated, lot no. 408-2404, Total
Raffinage

Distribution, France.

**Conclusion:** Under the conditions of the test, no increase in
micronuclei

was found in male and female mice 24 hr and 48 hr after oral

administration of 5000 mg ETBE/kg bwt (oral limit dose).

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, clearly reported, acceptable for assessment.

05-OCT-2003 (43)

\- 114/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Type:** Micronucleus assay

**Species:** mouse **Sex:** male/female

**Strain:** CD-1

**Route of admin.:** inhalation

**Exposure period:** 6 hr/d for 5 d

**Doses:** 0, 400, 2000 or 5000 ppm

**Result:** negative

**Method:** OECD Guide-line 474 \"Genetic Toxicology: Micronucleus
Test\"

**Year:** 1995

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND TREATMENTS

Groups of 5 male (approx. 28g) and 5 female (approx. 25 g)

CD-1 mice were exposed whole body to 0, 400, 2000 or 5000 ppm

ETBE vapour (6 hr/d for 5d). The highest exposure represented

one half of the lower explosive limit for ETBE. Body weights

were recorded before first exposure and at scheduled

termination. Individual clinical observations were recorded

before and after exposure, and on a group basis during

exposures.

GENERATION OF TEST ATMOSPHERE

Liquid ETBE was delivered by a metered piston pump into a

heated glass evaporator and the resultant vapour delivered

into a 900l stainless steel and glass exposure chamber. The

nominal vapour concentration in the chambers was calculated

from the volume of ETBE vapourised and the total airflow. The

actual vapour concentration was determined using GC-FID (23-24

measurements during each 6 hr exposure period).

MICRONUCLEUS EVALUATIONS

Femoral bone marrow smears were prepared from each mouse 24 hr

after the final exposure, and 2000 PCEs scored for the

presence of micronuclei. The ratio of PCE:NCE was quantified

in 1000 erythrocytes. Toxicity was determined from the number

of PCEs per 1000 erythrocytes.

POSITIVE CONTROL

Cyclophosphamide (15 mg/kg bwt in distilled water; ip

injection) was administered 24 hr prior to sacrifice.

STATISTICS

Results were analysed using the Mann Whitney U test.

**Result:** ACHIEVED CONCENTRATION

Daily exposure concentrations (mean+/-SD) were 400+/-8.5,

1996+/-37 and 5053+/-80 ppm (limit of detection 10 ppm).

CLINICAL SIGNS AND TOXICITY

All animals survived until study termination. Clinical signs

included abnormal gait, hypoactivity, incoordination,

abdominal breathing and lack of startle reflex in the 5000ppm

group and to a lesser extent in the 2000ppm group.

\- 115/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Hypoactivity was also present following 4.5 hr exposure to

400ppm ETBE vapour. There were no differences in body weight

between the groups.

MICRONUCLEUS TEST

No statistically significant increase in micronucleus

frequency were seen in animals exposed to ETBE. A

statistically significant increase (P\<0.01) was observed in

the positive control group.

There was no significant change in proportion of PCEs to NCEs.

**Test substance:** ETBE, 98% pure (containing 13 ppm antioxidant)
supplied by the

Sponsor (ARCO Chemical Company, PA, USA) and manufactured by

Phillips 66 (no further details).

**Conclusion:** Under the conditions of the test, no increase in
micronuclei

was found in male and female mice following 5 consecutive

daily exposures to 400-5000 ppm ETBE vapour.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

05-OCT-2003 (16)

**[5.7 Carcinogenicity]{.underline}**

**Remark:** The source reference (Maltoni, C (1995) The contribution of

experimental (animal) studies to the control of industrial

carcinogenesis. Appl. Occup. Environ. Hyg. 10, 749-760.)

includes reference to a carcinogenicity study on ETBE.

At the time of preparation of this IUCLID dataset (November

2003), the study was not located on ToxLine following a

literature search using the first named author.

**Source:** Maltoni, C (1995) The contribution of experimental (animal)

studies to the control of industrial carcinogenesis. Appl.

Occup. Environ. Hyg. 10, 749-760.

**Reliability:** (3) invalid

Study available for review. Contains no relevant information.

30-NOV-2003

\- 116/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Remark:** The source reference (Belpoggi, G, Soffriti, M, Minardi,F,

Bua, L, Cattin, E and Maltoni, C (2002). Ann. NY Acad. Sci.,

982, 7-86.) includes reference to a carcinogenicity study on

ETBE. This is cited as:

Maltoni, C et al. (1999) Comprehensive long-term experimental

project of carcinogenicity bioassay on gasoline oxygenated

additives: plan and first report of results from the study on

ethyl-tertiary butyl ether (ETBE). Eur J Oncol, 4, 493-508.

At the time of preparation of this IUCLID dataset (November

2003), the article does not appear to have been published in

this journal, nor was the study located on ToxLine following a

literature search using the first named author.

**Source:** Belpoggi, G, Soffriti, M, Minardi,F, Bua, L, Cattin, E and

Maltoni, C (2002). Ann. NY Acad. Sci., 982, 7-86.

**Reliability:** (3) invalid

Study unavailable for review.

02-JAN-2004

**Species:** rat **Sex:** male/female

**Strain:** Sprague-Dawley

**Route of administration:** gavage

**Exposure period:** Treated for 104 wk

**Frequency of treatment:** Once daily, 4 days per week (Monday,
Tuesday,

Thursday, Firday)

**Post exposure period:** Maintained until natural death

**Doses:** 0, 250 or 1000 mg/kg bwt/d

**Control Group:** yes, concurrent vehicle

**Year:** 1999

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE:

Animals: SD rats (CRC/RF laboratory colony, Bologna, Italy;

bred in-house), males and females, age 8 wk at start of study.

Housing: group housed (5 per sex per cage)

Diet, water, environmental conditions: no details.

GENERAL EXPERIMENTAL DESIGN:

Animals (n=60/sex/treatment) were administered ETBE at doses

of 0, 250 or 1000 mg/kg bwt in virgin olive oil (Oliaria

Toscana), by gavage, 4 days per week for 104 weeks.

Comment: the report notes that more frequent treatment would

not have been tolerated by the animals.

All animals received a constant daily volume of 1 ml of test

solution or olive oil each day.

Dosing solutions were prepared weekly and stored at 4 degrees

C until use.

Comment: no information is provided on analysis for stability

\- 117/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

or concentration achieved in the dosing solutions.

After the final treatment, animals were observed until natural

death, with the experimental phase of the study ending after

137 weeks (September 28, 1995), with the death of the last

surviving animal at age 145 weeks.

OBSERVATIONS

Food and water consumption and body weights were recorded

regularly throughout the study (weekly for the first 13 weeks,

then every 8 weeks until study week 104, then every 8 weeks

until death).

Animals were examined for massess and gross lesions at weekly

intervals during study weeks 1-13, and at two weekly intervals

thereafter.

NECROPSY

Animals were subject to a full necropsy on natural death. All

gross lesions, as well as an extensive selection of internal

and external tissues, were sampled for subsequent microscopic

examination.

STATISTICAL METHODS:

Differencs between control and treated groups was analysed

using Chi-square.

**Result:** Food and water intake and mean body weights were comparable

between the groups.

Survival in males decreased in a treatment dependent manner

from around wk 40, with the death of the last surviving

animals occurring on study week 152, 136 or 120 for the

control, low or high dose groups (equivalent to age 144, 128

0r 112 wk, respectively).

Survival in high dose females was lower than in other groups

from week 40, however from around week 96 there was little

difference between the groups. Death of the last surviving

female occurred at wk 136 for the control and low dose groups,

and at wk 152 for the high dose group (at age 128 or 144 wk,

respectively).

TUMOUR FINDINGS

There was no statistically significant difference in the

number of animals with benign tumours or the total number of

benign tumours or their incidence per 100 animals.

There was no statistically significant difference in the

number of animals with malignant tumours or in the total

number of malignant tumours, although the incidence was

significantly increased (P\<0.05) in the females from the 250

mg/kg bwt/d group (55 tumours per 100 rats) versus the

controls (25 per 100 rats) (high dose group unaffected).

\- 118/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

The combined number of acanthomas, squamous cell dysplasias,

borderline carcinomas and squamous cell carcinomas of the oral

cavity (including tongue and lips) was increased (P\<0.05) in

males only from the 1000 mg/kg bwt/d group (total = 25 per 100

rats) relative to the controls (total = 10 per 100 rats),

however there was no difference when these lesions were

analysed individually.

Three squamous cell carcinomas were identified in each of the

male- and female groups given 250 mg ETBE/kg bwt/day, with

none present in the controls or in the 1000 mg/kg bwt/d

groups.

There was a significant increase in the number of Schwannomas

of the uterus-vagina in low (n = 8 tumours in 60 rats) and

high dose females (n = 2 tumours per 60 rats) versus the

controls (zero), however no dose-response relationship was

present.

A high incidence of haemolymphoreticular neoplasia (primarily

lymphoimmunoblastic lymphoma) was recorded both in the control

and the treated animals but no statistically significant

differences or dose-trend was present. Lympohblastic lymphoma

and myeloid leukemia occurred in animals (both sexes) from the

250 mg/kg bwt/d group only.

Comment: no information is presented on time-to-tumour, nor

are the results corrected for intercurrent mortality. These

analyses would help interpretation of the findings given the

presence of dose-related differences in survival and the

inclusion of an extended (non-guideline) post-treatment phase

in this study.

**Test substance:** ETBE (AGIP Petroli (Euron) SpA), 94% pure, density
0.74.

**Conclusion:** No obvious carcinogenic response was present in male and

female SD rats following oral (gavage) administration of ETBE

in olive oil at doses of 250 or 1000 mg/kg bwt/d for 104 wk,

with follow-up until natural death (up to study week 152).

Inadequate statistical analysis (i.e. no information on

time-to-tumour or on the impact of intercurrent mortality)

confounds interpretation of these data, which are therefore

considered unreliable.

**Reliability:** (3) invalid

24-FEB-2006 (55)

\- 119/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Remark:** The structures of methyl- and ethyl-ter-buthyl-ethers were

analyzed by CASE, an expert system, and compared to the

structural determinants previously recognized as being

associated with carcinogenicity in rodents, mutagenicity in

Salmonella or the induction of sister chromatid exchanges

and chromosomal aberrations in cultured mammalian cells.

On the basis of this analysis the two chemicals are

predicted to be neither genotoxicants nor carcinogens.

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

23-DEC-2003 (75)

**[5.8.1 Toxicity to Fertility]{.underline}**

**Type:** Two generation study

**Species:** rat

**Sex:** male/female

**Strain:** Sprague-Dawley

**Route of administration:** gavage

**Premating Exposure Period**

**male:** 10 wk

**female:** 10 wk

**No. of generation studies:** 2

**Doses:** 0 (corn oil), 250, 500 or 1000 mg/kg bw/d

**Control Group:** yes, concurrent vehicle

**NOAEL Parental:** = 250 mg/kg bw

**NOAEL F1 Offspring:** \>= 1000 mg/kg bw

**NOAEL F2 Offspring:** \>= 1000 mg/kg bw

**other: NOAEL Reproduction** **:**

\>= 1000 mg/kg bw

**Method:** OECD Guide-line 416 \"Two-generation Reproduction Toxicity

Study\"

**Year:** 2004

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE

Animals: time-mated female Sprague Dawley rats (Charles River

Laboratories, L\'Arbresle, France);

Age at start of treatment: approx. 7 wk (mean bw 203 g males,

157 g females)

Acclimation period: 6 d

Housing: individually housed, stainless steel cages (wire mesh

bottom) for duration of study

Diet: A04 C pelleted maintenance diet (UAR, Villemoisson,

\- 120/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

France), ad libitum

Water: deionised water, ad libitum

Light cycle: 12 hr light/dark period (lights on 0700-1900)

Assignment to treatment groups: computer-generated weight

randomisation

DESIGN

ETBE was administered to groups of 25 male and 25 female rats

by gavage at doses of 0 (corn oil; 4 ml/kg bw) 250, 500 or

1000 mg/kg/day as follows:

F0 generation:

\- males: from 10 wk before mating, during 2 wk mating period

and until sacrifice (after weaning of F1 pups)

\- females: from 10 wk before mating, during 2 wk mating

period, pregnancy and lactation until day 21 post-partum

F1 and F2 generations:

\- as above, commencing day 22 post-partum

DOSING SOLUTIONS

Solutions were prepared weekly (based upon stability data

demonstrating no losses of ETBE over 9 d) and samples taken

for analysis (GC) to confirm received dose.

MATING PROCEDURE

Mating followed a 10 wk treatment period. Dosing of F0 animals

began at age 7 wk, with mating from age approx. 17 wk. Dosing

of F1 animals began at weaning when the animals were 22 d old,

with mating from age approx. 13 wk.

Females were monitored for estrous stage (microscopic

examination of fresh vaginal lavage, stained with methylene

blue) each morning during the last 3 weeks of the pre-mating

period and during the mating period (until mating confirmed by

presence of a vaginal plug or sperm in a vaginal lavage).

Females were paired overnight with males from the same dose

group until mating had occurred or 14 days had elapsed,

whichever occurred first. Parental F1 animals were selected

from different litters to avoid brother-sister matings.

PREGNANCY AND PARTURITION

Pregnant females were housed individually, allowed to litter

normally and raise progeny until weaning.

PARENTAL OBSERVATIONS

Parental animals were observed at least twice daily for

clinical signs, morbidity or mortality. Body weight and food

consumption was recorded weekly for males, and at least weekly

for females, commencing during the pre-mating period.

PUP OBSERVATIONS

Total litter size, number of live/dead pups and sex of each

pup was recorded as soon as possible after birth, and the pups

observed daily for clinical signs. Anogential distance was

\- 121/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

measured (both generations) on day 1. Any gross malformations

were noted. The size of each litter was adjusted to 4 males

and 4 females on post-partum day 4. Pup body weight was

recorded on days 1, 4, 7, 14 and 21 post-partum.

Reflex development was recorded in F1 and F2 pups as follows:

\- surface righting reflex on day 5 post-partum

\- cliff avoidance on day 11 post-partum

\- air-righting reflex on day 17 post-partum

Acoustic startle response and pupil constriction reflex were

assessed in F1 pups at 4 wk of age. Spontaneous motor activity

was evaluated in F1 pups twice, at weekly intervals, when the

animals were 7-8 wk old.

Males monitored between 32-47 d old for preputial separation,

females monitored between 28-40 d old for vaginal opening

(pups from both generations).

SPERM COUNTS

Sperm collected (under isoflurane anaesthesia, prior to

terminal sacrifice) from F0 and F1 parental males for

quantitative evaluation of:

\- epididymal sperm motility (x40 magnification), sperm count

(cauda sperm count, Malassez cell) and sperm morphology (eosin

staining, x40 magnification)

\- homogenisation resistant testicular sperm (Neubauer cell)

NECROPSY OBSERVATIONS

Parental animals sacrificed (carbon dioxide) after weaning of

the litters (that is, between day 22-25 post-partum), weighed,

and subject to a gross internal examination (including

examination of the uterus for implantation scars).

Following organs weighed:

Males:

\- testes (separately), epididymides (separately), prostate,

seminal vesicles together with coagulating glands, brain,

liver, kidneys, spleen, pituitary gland, thyroids with

parathyroids, adrenals

Females:

\- uterus, ovaries, brain, liver, kidneys, spleen, pituitary

gland, thyroids with parathyroids, adrenals,

Three pups/sex/litter born to the F0 and F1 mothers subject to

gross necropsy at weaning. Brain, spleen and thymus removed

and weighed.

PRESERVATION OF TISSUES

The following organs preserved from all F0 and F1 parental

animals:

ovaries and oviduct,

\- 122/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

uterus (with cervix and horns)

vagina

testis (right)

epididymis (right)

seminal vesicles

prostate

coagulating glands

pituitary gland

adrenal gland

A vaginal smear taken from all females and stained (Harris

Schorr staining).

Any abnormal tissue found at necropsy in the weanling pups was

preserved.

EXAMINATION OF TISSUES

The following tissues from control and high dose F0 and F1

animals stained with haematoxylin-eosin prior to microscopic

examination:

any macroscopic abnormalities

ovaries and oviducts (including qualitative assessment of

primordial follicle population)

uterus (with cervix and horns)

vagina

epididymis

seminal vesicles

prostate

coagulating glands

pituitary gland

adrenal gland

The vaginal smear taken from females at terminal sacrifice

also examined microscopically.

The testis from all treated males plus the controls was

stained with haematoxylin/PAS and subject to a detailed

histopathological examination (including an assessment of

retained spermatids, missing germ cell layers or types,

multinucleated giant cells or sloughing of spermatogenic

cells).

Any macroscopic abnormalities present in the pups born to F0

and F1 mothers were prepared for histopathological evaluation.

STATISTICAL ANALYSIS

Mean values were compared by one-way ANOVA and Dunett\'s test

(mean values being considered as normally distributed and

variances being considered homogenous). Percentage values were

compared by the Fisher exact probability test.

Mean percentages of motility and morphology sperm parameters

were Arcsine transformed and then compared by ANOVA. Mean

values for anogenital distance, ratio of anogenital distance

to pup body weight and anogenital distance normalized to the

\- 123/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

cube root of the individual pup\'s body weight were compared by

analysis of variance.

**Result:** ANALYSIS OF DOSING SOLUTIONS

Stability and homogeneity studies returned satisfactory

results (all analysed concentration within 10% of nominal).

PARENTAL/ADULT OBSERVATIONS

F0 PARENTAL GENERATION

No deaths in either sex during the pre-mating, mating or

gestation periods. One female given 250 mg/kg bw/d and three

females given 500 mg/kg bw/d sacrificed during the first week

of lactation because their entire litters died or were in poor

condition. Considered unrelated to treatment by study director

since no comparable findings in the high dose group.

Ptyalism (excessive salivation) observed in all treated groups

(dose-related trend), considered related to unpalatable nature

of the dosing solutions by study director.

Body weight gain decreased significantly in mid (-29%; P\<0.01)

and high (-22%; P\<0.001) males on study days 85-113 (ie final

28 days of treatment, following mating). Considered

treatment-related by study director. Female body weights

unremarkable. Food intake not adversely affected by treatment

in either sex.

F1 PARENTAL GENERATION

One control male sacrificed due to poor condition on study day

36, one female given 1000 mg/kg bw/d found dead on day 47, and

one low dose female and one high dose female sacrificed early

because their entire litters were found dead a few days after

birth. These findings considered unrelated to treatment by the

study director.

Ptyalism (excessive salivation) observed in all treated groups

(dose-related trend), considered related to the unpalatable

nature of the dosing solutions by study director.

Body weight and food consumption unaffected by treatment in

both sexes.

F2 GENERATION

One control female found dead on day 25, necropsy signs

consistent with mis-dosing.

Ptyalism (excessive salivation) observed in all treated groups

(dose-related trend), considered related to the unpalatable

nature of the dosing solutions by study director.

Body weight and food consumption unaffected by treatment in

both sexes.

REPRODUCTIVE DATA FOR PARENTAL GENERATIONS

\- 124/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

F0 GENERATION

Mating indices for males and females comparable among the

groups and unaffected by treatment (no trend or statistically

significant differences present).

Fertility indices for males and females comparable among the

groups and unaffected by treatment (no trend or statistically

significant differences present).

The majority of pregnancy and parturition parameters

unremarkable, with implantation, fecundity, neo-natal loss,

gestation and delivery unaffected by treatment. The exception

was a slightly greater post-implantation loss in the low and

mid dose groups, reflecting litters found dead or sacrificed

prematurely due to poor clinical condition. Considered

unrelated to treatment by study director since no similar

findings in high dose group.

F1 GENERATION

Mating indices for males and females comparable among the

groups and unaffected by treatment (no trend or statistically

significant differences present).

Fertility indices for males and females comparable among the

groups and unaffected by treatment (no trend or statistically

significant differences present).

Pregnancy and parturition parameters unremarkable, with

implantation, fecundity, post-implantation loss, neo-natal

loss, gestation and delivery unaffected by treatment. A single

pregnant female from the 250 mg/kg bw/d group did not deliver,

and was found to have only a single implantation site at

necropsy. Considered unrelated to treatment by study director

since only one animal from the low dose group affected.

LITTER SIZE AND PUP PARAMETERS DURING LACTATION

F0 LITTERS/F1 PUPS

The number of litters delivered by the F0 dams and number of

F1 pups per litter unaffected by treatment. At 250 mg/kg/d and

500 mg/kg/d there was a slight increase in the number of pups

that died during the first 4 days of lactation, however

findings for the high dose group were unremarkable and the

findings were considered unrelated to treatment by the study

director. Viability and lactation indices were unaffected by

treatment at all dose levels.

0 250 500 1000 mg/kg/d

Litters obtained 23 21 22 25

Pups/dam 14.3 14.1 14.9 14.2

Viability index on LD4 (%) 97.6 92.9 82.3 97.7

Total decedent pups (LD1-21) 18 35 64 10

Lactation index (%) 94.6 91.7 96.1 99.0

F1 LITTERS/F2 PUPS

\- 125/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

The number of litters delivered by the F1 dams and number of

F2 pups delivered per litter unaffected by treatment. At 250

mg/kg/d and 1000 mg/kg/d, the number of pups that died during

the lactation period was slightly higher than controls during

the first 4 days of lactation. Considered unrelated to

treatment by study director since only one (particularly

large) litter per dose group affected. Viability and lactation

indices unaffected by treatment at all dose levels.

0 250 500 1000 mg/kg/d

Litters obtained 21 21 22 20

Pups/dam 13.7 13.7 13.7 14.0

Viability index on LD4 (%) 97.6 94.8 97.0 92.9

Total decedent pups (LD1-21) 11 17 9 21

Lactation index (%) 97.6 98.8 100.0 99.3

PUP CLINICAL SIGNS, BODY WEIGHTS AND EXTERNAL FINDINGS

F1 PUPS

Pups from one low dose litter and three mid dose litters

emaciated in appearance, cold to the touch or dehydrated, and

were found dead or prematurely sacrificed for ethical reasons.

Similar findings also observed in four surviving pups from

three litters at 250 mg/kg/day, three surviving pups from one

litter at 500 mg/kg/day and six pups from four different

litters at 1000 mg/kg/day. Most of these pups displayed lower

body weight. Since incidence was not dose related and did not

correlate with lower mean group body weight gain, findings

considered unrelated to treatment by the study director.

Body weight and body weight gain unaffected by treatment.

No gross external abnormalities present in F1 pups born to F0

mothers.

F2 PUPS

Tremors and ataxia present from day 15 post-partum in one pup

from the 250 mg/kg/day group (sacrificed for humane reasons).

Other clinical signs (necrosis of the tail or of the limbs,

cold to the touch, emaciated appearance) were low in

incidence, not dose-related and randomly distributed between

the litters and treatment groups.

No effect on body weight or body weight gain noted at 250 and

500 mg/kg/day, while high dose pups exhibited a transient

non-significant decrease (-11%) in body weight gain during the

first 4 days after birth.

At 1000 mg/kg/day, absence of tail (acaudia) observed at birth

in two female pups from two different litters, with anal

atresia also present in one of these pups. The incidence of

acaudia (2/280 pups; 0.7%) was outside the historic control

range for the laboratory (minimum = 0.0%; maximum = 0.05%;

data from 16 studies, October 2000-December 2003) but close to

the background incidence recorded in the MARTA \[\*\] historical

\- 126/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

control database (0.31% for 22147 fetuses from 1575 control

litters during the period 1990-2004). The study director

concluded that no clear relationship to treatment was

established. No historical control data were available from

the laboratory performing the study for anal atresia, however

the observed incidence (1/280 pups; 0.35%) was close to that

recorded in the MARTA historical control database (0.32% for

22147 fetuses from 1575 control litters during the period

1990-2004). Occurrence of anal atresia in this investigation

considered spontaneous in origin by the study director.

\[\* A historical control database of preclinical developmental

teratology and reproductive toxicity parameters, a joint

project of MARTA and MTA; www.hcd.org\]

Comment: No instance of acaudia or anal atresia in off-spring

from dams treated with ETBE at a dose of 1000 mg/kg/day during

a preliminary dose-range finding study (zero incidence in 164

fetuses from 12 litters and 144 pups from 11 litters; CIT

study 24168 RSR, October 2003; summarised in this IUCLID

dataset) or a developmental toxicity study (zero incidence in

258 fetuses from 22 litters; CIT study 24860 RSR, January

2004; summarised in this IUCLID dataset).

PUP ANOGENITAL DISTANCE, REFLEX DEVELOPMENT AND SEXUAL

MILESTONES

F1 PUPS

Anogenital distance, the ratio of anogenital distance to pup

body weight and anogenital distance normalized to the cube

root of pup body weight not significantly altered by

treatment.

Mean AGD values (in mm, SD in brackets) were:

0 250 500 1000

Males 4.71 (0.43) 4.62 (0.33) 4.47 (0.39) 4.64 (0.30)

Females 2.84 (0.36) 2.77 (0.24) 2.63 (0.26) 2.68 (0.37)

No evidence of any adverse treatment-related changes in

surface righting, cliff avoidance and air righting (95 to 100%

positive responses). (Observations performed on F1 pups only.)

Mean age at preputial separation unaffected by treatment (i.e.

occurred on day 35, 34, 35 or 35 for pups from the control,

250, 500 and 1000 mg/kg/day groups, respectively).

Mean age for vaginal opening unaffected by treatment (i.e.

occurred on day 34, 34, 35 or 33 for pups from the control,

250, 500 and 1000 mg/kg/day groups, respectively).

Auditory function (acoustic startle reflex) and visual

function (pupil constriction reflex) at 4 weeks of age, and

spontaneous locomotor activity (measured twice at 7 and 8

weeks old) unaffected by treatment. (Observations performed on

F1 pups only)

\- 127/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

F2 PUPS

Anogenital distance, the ratio of anogenital distance to pup

body weight and anogenital distance normalized to the cube

root of pup body weight not significantly altered by

treatment. Mean AGD values (in mm, SD in brackets) were:

0 250 500 1000

Males 4.57 (0.39) 4.56 (0.42) 4.54 (0.42) 4.63 (0.38)

Females 2.80 (0.35) 2.72 (0.29) 2.82 (0.29) 2.90 (0.33)

No evidence of any treatment-related effect on surface

righting, cliff avoidance and air righting (between 96.0 and

99.4% positive responses).

Mean age at preputial separation similar in the control and

the treated groups (i.e. occurred on day 36, 35, 35 or 36 for

pups from the control, 250, 500 and 1000 mg/kg/day groups,

respectively).

Mean age for vaginal opening similar in the control and the

treated groups (i.e. occurred on day 35, 34, 34 or 33 for pups

from the control, 250, 500 and 1000 mg/kg/day groups,

respectively). Although pups from two high dose dams displayed

acaudia and anal atrasia, anogenital distance was normal.

SPERM ANALYSES FOR F0 AND F1 PARENTAL MALES

No statistically significant or dose-related changes in

seminology data for F0 and F1 males. A slightly lower

testicular sperm head count noted in all treated F0 males

appeared to be the consequence of a high control value.

Overall, sperm parameters unaffected by treatment in both

generations at all dose-levels.

\-\-\-\-\-- mg/kg bw/d \-\-\-\-\--

0 250 500 1000

F0 MALES

Epididymal sperm

Spermatozoa (10\^3/mm\^3) 923 938 938 918

Motility (%) 99.7 100 98.6 97.6

Normal morphology (%) 93 93 97 96

Testicular sperm

Sperm heads (10\^6/g testis) 148 109 108 110

Sperm production (10\^6/g/d) 18.8 17.9 17.1 18.0

F1 MALES

Epididymal sperm

Spermatozoa (10\^3/mm\^3) 725 673 701 688

Motility (%) 84.6 87.1 93.3 88.3

Normal morphology (%) 84 86 86 88

Testicular sperm

Sperm heads (10\^6/g testis) 100.6 97.8 105.3 99.8

Sperm production (10\^6/g/d) 16.5 16.0 17.3 16.4

\- 128/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

NECROPSY AND HISTOPATHOLOGY

F0 GENERATION PARENTS

Absolute and relative liver weights significantly increased in

high dose males, and correlated with liver enlargement noted

during necropsy and hepatocellular hypertrophy (considered

adaptive response to treatment by the study director) in

livers from three animals selected for histopathological

examination. Liver weights from females given 1000 mg/kg/day

slightly (increased 4-6%) but non-significantly increased.

Kidney weights significantly increased in high dose males and

correlated with the presence of microscopic acidophilic

globules in cortical tubular epithelium from 5 of 6 animals

selected for microscopic examination (considered related to

alpha 2 µ globulin accumulation by study director). Kidney

weights also significantly increased in male rats given 500

mg/kg/day (no microscopic examination performed).

Summary table: percentage change in F0 liver and kidney

weights

\-\-- Males \-\-- \-- Females \--

250 500 1000 250 500 1000

Liver

\- absolute +2 +2 +17\*\* -1 +4 +6

\- relative +3 +6 +24\*\* +10 +8 +4

Kidney

\- absolute +11 +15\*\* +21\*\* -1 +2 +5

\- relative +11 +18\*\* +28\*\* +9 +5 +3

All other organ weight and macroscopic findings consistent

with those observed in untreated rats of this strain and age,

and considered of no toxicological importance by the study

director.

F1 PUPS

No treatment-related or statistically significant changes in

pups found dead or sacrificed early, or in pups subject to

scheduled necropsy at weaning.

F1 GENERATION PARENTS

Absolute and relative liver weights significantly greater in

males given 500 or 1000 mg/kg/day and correlated with liver

enlargement recorded at necropsy. Microscopic examination of

liver tissue from two selected high dose males revealed slight

or moderate hepatocellular hypertrophy, considered an adaptive

response to treatment by the study director. Absolute and

relative kidney weights significantly increased in males given

1000 or 500 mg/kg/day, with slight to marked accumulation of

acidophilic globules (considered related to alpha 2 µ globulin

accumulation by study director) detected microscopically in

tissue from one selected mid dose male and four selected high

dose males.

\- 129/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Summary table: percentage change in F1 liver and kidney

weights

\-\-- Males \-\-- \-- Females \--

250 500 1000 250 500 1000

Liver

\- absolute +0 +14\*\* +27\*\* +1 +3 +10\*\*

\- relative +0 +11\*\* +25\*\* +3 +6 +9\*

Kidney

\- absolute +10 +22\*\* +58\*\* +4 +3 +11\*\*

\- relative +10\*\* +19\*\* +58\*\* +6 +6 +10\*\*

All other organ weight and macroscopic findings consistent

with those observed in untreated rats of this strain and age,

and considered of no toxiciological importance by the study

director.

F2 PUPS

No treatment-related or statistically significant changes

present in pups found dead or sacrificed early, or in pups

subject to scheduled necropsy at weaning.

MICROSCOPIC EXAMINATION OF REPRODUCTIVE ORGANS

F0 GENERATION

Minimal degeneration of seminiferous tubules present in two

control and two high dose animals, with desquamated

spermatocytes present in control (8/25 animals) and treated

high dose animals (1/25 animals). These findings commonly

recorded in rats of this strain and age and considered of no

toxicological importance by the study director. Interstitial

mononuclear cell aggregation and subacute prostatitis of the

prostate and spermatic granuloma in the epididymides occurred

with equal incidence and severity in both control and treated

animals in this study, and considered of no toxicological

importance by the study director.

Microscopic examination of the ovaries, uterus and vagina from

control and high dose females revealed morphological changes

consistent with a regular estrous cycle in both control and

treated animals. Quantitative analysis of the primordial and

growing follicles in the ovaries showed no perceptible

differences between control and treated animals.

F1 GENERATION

A reduced number of tailed and round spermatids,

spermatocytes, and spermatogonia present in 3/25 low dose

males, 1/24 mid dose males and 3/25 high males versus 0/25 in

the controls. In the majority of instances, changes correlated

with seminiferous tubules lined with Sertoli cells only

(minimal severity), with oligospermia or aspermia in the

epididymides of the males receiving 1000 mg/kg/day

(epididymides not examined in the low and intermediate

dose-level groups). Minimal focal degeneration of germinal

\- 130/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

epithelium observed in 1/24, 1/25 or 1/25 males from the

control, low- or high-dose groups, respectively. Minimally or

slightly degenerated/necrotic cells observed in the lumen of

the testis from 9/24 control males, 4/25 low dose males, 8/24

mid dose males and in 6/25 high dose males. Although some of

these observations not present in the controls, the study

director considered there was no evidence of a dose-response

relationship in their incidence and/or severity, and that

similar findings can occur spontaneously in the untreated rats

of this strain and age. Consequently, findings considered

unrelated to treatment.

Microscopic examination of the ovaries, uterus and vagina from

control and high dose females revealed morphological changes

consistent with a regular estrous cycle in both control and

treated animals. Quantitative analysis of the primordial and

growing follicles in the ovaries showed no perceptible

differences between control and treated animals.

**Test substance:** ETBE, CAS No. 637-92-3, \>98% pure, Total France.

**Conclusion:** The following No Observed Adverse Effect Levels were

established from the results of this study.

Systemic toxicity in the parental (adult) F0, F1 and F2

generations: 250 mg/kg body weight/day (based on body weight,

organ weight and histopathological changes present after

treatment at 500 and 1000 mg/kg bw/d).

Fertility, gonadal function, reproductive performance,

parturition, lactation and development of the off-spring to

weaning or sexual maturity: 1000 mg/kg body weight/day, the

highest dose tested in this study.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

13-JUL-2004 (33)

**Type:** other: fertilisation in vitro

**Route of administration:** drinking water

**Exposure Period:** 2 wk

**Year:** 2003

**GLP:** no

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE

Animals: Simonson albino rats (Sprague-Dawley derived;

supplier not specified); males at least 100 days old, females

28-45 days old.

No further details available.

TREATMENTS

Females received either 0.3% ETBE in drinking water or

drinking water alone for 2 wk preceeding oocyte recovery (n=6

\- 131/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

per group). Female bwt, number of females ovulating and the

number of ooctyes collected was recorded.

RECOVERY OF OOCYTES

Ovulation was induced using 15 IU pregnant mare\'s serum

gonadotropin i.p. followed 2 d later by 15 IU human chorionic

gonadotrophin i.p. Animals were sacrificed by cervical

dislocation 16-18 hr later, oviducts and attached ovaries

removed and the oviducal cumulus dispersed with hyaluronidase

in saline-BSA. Isolated oocytes were rinsed with 3 drops of

saline-BSA, the zona pellucida removed by incubation with acid

Tyrone\'s medium (pH 2.5) for 10 seconds followed by further

rinsing.

RECOVERY OF SPERM

Males were sacrificed (carbon dioxide), and cauda epididymides

and attached vas deferens dissected, rinsed in modified

Tyrode\'s medium and placed in warmed medium in a culture dish.

Sperm were removed by flushing with medium from a blunt needle

inserted into the vas deferens. Sperm motility was evaluated

subjectively using phase contrast microscopy (no further

details) and sperm concentration using a haemocytometer. The

suspension was diluted to 7 or 0.5 x 10\^6 sperm/ml and

incubated for 3 hr (37 degrees C, 5% CO2) before use.

OOCYTE PENETRATION ASSAY

Following removal of the zona pellucida, oocytes were mixed

with diluted sperm in modified Tyrone\'s medium (final volume

of 120 ul). Oocytes in all treatments within a replicate were

inseminated with aliquots from the same suspension. After 20

hr incubation (37 degrees C, 5% CO2) oocytes were rinsed to

remove loosely adherent spermatozoa, incubated (10 min) with

0.08 mg/ml Hoechst 33342 then placed on slides and stored (in

the dark) until microscopic examination (UV illumination, x400

magnification) for decondensed sperm heads and attached sperm.

STATISTICAL METHODS

Data were analysed by ANOVA.

**Remark:** The author\'s note that in vitro fertilising potential of a

fixed concentration of sperm was found to vary from male to

male, and was affected by other assay conditions such as

composition of the medium. Higher sperm concentrations (7 x

10\^6/ml) typically had more than enough high quality sperm to

produce 100% oocyte penetration, and fertilisation was not

affected by minor reductions in oocyte quality. A reduced

sperm concentration (0.5 x 10\^6/ml) was necessary in order to

detect variations in oocyte quality.

**Result:** ETBE treatment was without significant effect on any of the

parameters evaluated in this study:

Control ETBE

Final bwt (g) 155 148

Percentage females ovulating 72 84

No. oocytes recovered 30 29

per ovulating female

\- 132/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Percentage oocytes remaining 57 66

after removal of zona pellucida

Percentage oocytes fertilised 84 82

Penetrated sperm/oocyte 1.84 1.72

**Test substance:** ETBE, purity not specified, Aldrich Chemical Co.,
Milwaukee,

WI.

**Conclusion:** Under the conditions of this study, administration of
ETBE

(0.3% in drinking water) to female rats was without effect on

oocyte fertilisation in vitro.

**Reliability:** (2) valid with restrictions

Study available for review. Experimental research study,

acceptable for assessment however reliability/reproducibility

of methodology not known.

02-JAN-2004 (7)

**[5.8.2 Developmental Toxicity/Teratogenicity]{.underline}**

**Species:** rat **Sex:** female

**Strain:** Sprague-Dawley

**Route of administration:** gavage

**Exposure period:** GD5-19

**Frequency of treatment:** daily

**Doses:** 0 (corn oil), 250, 500 or 1000 mg/kg bw/d

**Control Group:** yes, concurrent vehicle

**NOAEL Maternal Toxity:** = 500 mg/kg bw

**NOAEL Teratogenicity:** = 1000 mg/kg bw

**NOAEL Embryotoxicity** **:** = 1000 mg/kg bw

**NOAEL Fetotoxicity** **:** = 1000 mg/kg bw

**Result:** No effect on fetal development

**Method:** OECD Guide-line 414 \"Teratogenicity\"

**Year:** 2003

**GLP:** yes

**Test substance:** as prescribed by 1.1 - 1.4

**Method:** ANIMALS AND MAINTENANCE

Animals: time-mated female Sprague Dawley rats (Charles River

Laboratories, L\'Arbresle, France);

Age at start of treatment: approx. 11 wk (bw 215-297 g)

Acclimation period: 5 d

Housing: individually housed, stainless steel cages (wire mesh

bottom) for duration of study

Diet: A04 C pelleted maintenance diet (UAR, Villemoisson,

France), ad libitum

Water: deionised water, ad libitum

Light cycle: 12 hr light/dark period (lights on 0700-1900)

Assignment to treatment groups: computer-generated weight

randomisation

DESIGN

ETBE was administered by gavage (dosing needle) at doses of 0

(corn oil; 4 ml/kg bw) 250, 500 or 1000 mg/kg/day on GD 5-19,

inclusive.

\- 133/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

DOSING SOLUTIONS

The dosing solutions were prepared weekly (based upon

stability data demonstrating no losses of ETBE over 9 d) and

samples taken for analysis (GC) to confirm received dose.

GENERAL OBSERVATIONS

The dams were examined twice daily mortality, and for the

occurrence of clinical signs 3 times per day on GD5-14, and

twice daily thereafter. Body weight and food consumption were

recorded on 10 occasions between GD2-20.

TERMINAL OBSERVATIONS

Dams were sacrificed by carbon dioxide asphyxiation on GD20

and subject to a macroscopic evaluation of internal organs and

placenta. The gravid uterine weight recorded, and the ovaries

and uterue examined for:

\- no. corpora lutea

\- no./distribution of dead/live fetuses

\- no./distribution of early/later resorptions

\- no./distribution of implantations sites/uterine scars

(ammonium sulphide)

FETAL EXAMINATION

All litters with at least 1 live fetus were weighed and

subject to a detailed external examination (including oral

cavity). Any findings were recorded according to the glossary

of the International Federation of Teratology Societies, and

classified as malformations or variations. Dead fetuses were

discarded, live fetuses were sacrificed with thiopental sodium

(s.c.) and the sex recorded. Approx. half the fetues were

fixed in Harrison\'s fluid and examined using a free-hand

serial sectioning technique (Wilson, 1965, Teratology

Principles and Techniques, Univ. Chicago Press, pp 262-277).

The remainder were fixed (ethanol), eviscerated and bone and

cartilage examined after staining with alizarin red S and

alcian blue (Peters, 1977, Methods in Prenatal Toxicology,

Georg Thieme Publ., pp 153-154).

STATISTICAL METHODS

Data were expressed as group means (+/- SD) or as percentages.

The unit of comparison was the litter, with fetal body weight

analysed by sex as well as for both sexes combined. Means

(normally distributed) were compared by ANOVA and Dunnett\'s

test, percentage values by Fisher\'s exact probability test.

**Result:** ANALYSIS OF DOSING SOLUTIONS

Solutions displayed good homogeneity, and deviations from

nominal concentration were +/- 10%.

PREGNANCY STATUS

There were no significant or biologically relevant differences

in pregnancy status between the groups:

Dose-level (mg/kg/day) 0 250 500 1000

Mated females 24 24 24 24

\- 134/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Non-pregnant females 3 5 4 2

Pregnant, alive at GD20 21 19 20 22

Pregnancy rate 87% 79% 83% 92%

Litters 21 19 20 22

MATERNAL OBSERVATIONS

There were no deaths in any group. Excess salivation was

present immediately post-dosing (resolved by one hour) in 5,

13 or 17 females from the 250, 500 and 1000 mg/kg/day dose

groups at various times during the study.

Comment: the report considers this a response to the

unpalatable \'taste\' of ETBE rather than an adverse clinical

effect.

There was a transient reduction in maternal body weight gain

of high dose dams on GD 5-9 (-20%), with mean body weight gain

decreased significantly over the whole dosing period (-11%,

p\<0.05). Net body weight gain in the 1000 mg/kg/day group

(corresponding to the maternal carcass weight at day 20 minus

maternal bw on GD5) was also significantly lower (-17%,

p\<0.01) when compared with the controls.

Dose-level (mg/kg/day) 0 250 500 1000

Bw change GD 5-9 24 23 23 20

\% diff. from control -4 -4 -20

Bw change GD 5-20 135 132 134 120\*

\% diff. from control -2 -1 -11

Net bw change GD 5-20 61.8 59.4 60.0 51.5\*\*

\% diff. from control -4 -3 -17

\* = p\<0.05; \*\* = p\<0.01

The food consumption was unaffected by the treatment.

No treatment-related macroscopic changes were present in the

dams at necropsy.

LITTER DATA

There were no differences in the number of corpora lutea and

implantation sites between the groups (hence no effects on

pre-implantation losses).

There was no total litter resorption in any group.

The number (21) and rate (1 per female) of early resorptions

was slightly greater in the 1000 mg/kg/day when compared to

the control group (number = 14, rate = 0.7 per female), hence

the extent of post-implantation loss was minimally increased

in the high-dose females (7.5%) versus controls (5.2%;

non-significant). The report notes that this incidence was in

the range of historical control data for the laboratory (mean

post-implantation loss=3.4%, minimum=1.0%, maximum= 8.5%) and

was therefore considered unrelated to treatment.

The number of live fetuses were in a range 11.7 to 12.3 per

\- 135/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

female, with no treatment-related or statistically

significant differences present between the groups. No dead

fetuses were found in any group.

The sex ratio in the low dose group (53.5% male, 46.5% female)

was significantly different from the control value. This

difference appeared principally related to an unusually low

proportion of males (40.8%) and a correspondingly higher

number of females (59.2%) in the control group. The sex ratio

in the other treated groups (47.3-48.8% male, 51.2-52.7%

female) was within the normal range (no dose-response

relationship present). The report considered this observation

in the low dose litters a chance event, unrelated to treatment

with ETBE.

Fetal weight was unaffected by the treatment.

FETAL EXAMINATION

A single occurrence of umbilical hernia (malformation) and a

bent tail (variation) were present in the 250 mg/kg/day group

(no other findings in any group). These were considered

unrelated to treatment due by the study report due to isolated

nature of the findings and absence of any dose-response.

SOFT TISSUE EXAMINATION

\- MALFORMATIONS

There were no malformations in the control or the low or

intermediate treatment groups. At 1000 mg/kg/day an absence of

kidneys, ureters and adrenals was observed in single fetus.

These were considered unrelated to treatment by the study

report due to isolated nature of the findings and absence of

any dose-response.

\- VARIATIONS

The following findings were noted among the groups:

\- dilated ureters in one fetus from the control, 250 m/kg/day

and 1000 mg/kg/day groups;

\- short uterine horns in one fetus from the 500 mg/kg/day;

\- dilated renal pelvis in one fetus from the 250 and 1000

mg/kg/day.

These few findings, which were not dose-related and randomly

distributed across the groups, were considered by the report

to be unrelated to the treatment.

SKELETAL EXAMINATION

\- MALFORMATIONS

The following malformations were recorded:

\- misaligned sternebra in one control fetus;

\- at 250 mg/kg/day, fused rib and misshapen sacral vertebra in

one fetus; split sternebra and fused ribs in one other fetus;

\- at 500 mg/kg/day, bilateral misshapen ilium noted in one

fetus;

\- at 1000 mg/kg/day, absence of one pair of ribs observed in

\- 136/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

two fetuses, one of which also had an absence of thoracic

vertebra.

Because of their low incidence and the absence of any

dose-relationship, these malformations were not considered by

the report to be related to the treatment with ETBE.

\- VARIATIONS

There was a significant increase in the incidence of

unossified 4th metacarpal at the highest dose: 43/136 fetuses

(31.6%, p\<0.05) were affected versus 27/135 (20%) in the

control group. This was not statistically significant when

expressed on a fetus/litter basis. Cartilage was generally

present suggesting that this was due to slightly delayed

ossification rather than to a persistent alteration. As the

findings were isolated, not statistically significant or

dose-related at the level of the litter and not associated

with any significant delay in ossification of other bones, and

since the total incidence of skeletal variations in treated

animals did not increase, the report concludes these are of no

toxicological significance. There were no other findings.

**Test substance:** ETBE, CAS No. 637-92-3, \>98% pure, Total France.

**Conclusion:** No evidence of maternal or embryo-fetal toxicity was
recored

when ETBE was administered to pregnant SD rats at 250 or 500

mg/kg bw/d (gavage) on GD 5-19. Maternal toxicity

(statistically significant decrease in bw gain and net bw

gain) was observed at 1000 mg/kg/day. There were no adverse

effects on embryofetal development at this dose-level.

Consequently, under the conditions of the study, the NOAEL for

maternal toxicity was 500 mg/kg/day and the NOAEL for

embryofetal development was 1000 mg/kg/day.

**Reliability:** (1) valid without restriction

Study available for review. GLP compliant guideline

investigation, well documented methods and results, raw data

available, acceptable for assessment.

30-NOV-2003 (32)

\- 137/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[5.8.3 Toxicity to Reproduction, Other Studies]{.underline}**

**Type:** other: dose range-finding study

**In Vitro/in vivo:** In vivo

**Method:** ANIMALS AND MAINTENANCE

Animals: Male Fischer F344 rats; male and female Sprague

Dawley rats (Charles River Laboratories, L\'Arbresle, France);

Age at start of treatment: Males approx. 7 wk, females approx.

11 wk

Acclimation period: 1 wk

Housing: individually housed, stainless steel cages (wire mesh

bottom) for duration of study

Diet: A04 C pelleted maintenance diet (UAR, Villemoisson,

France), ad libitum

Water: deionised water, ad libitum

Light cycle: 12 hr light/dark period (lights on 0700-1900)

Assignment to treatment groups: computer-generated weight

randomisation

DESIGN

ETBE was administered by gavage (dosing needle) at doses of 0

(corn oil; 3 ml/kg bw) 50, 250, 500 or 1000 mg/kg/day.

Male F344 rats: n=12/dose level; daily administration for 12

weeks

Male SD rats: n=12/dose level; daily administration for 12

weeks

Female SD rats: n=24/dose level; daily administration through:

\- pre-mating (2 weeks)

\- mating (2 weeks)

\- pregnancy (until day 19 post-coitum for 12 females/dose

level)

\- until the end of lactation (day 21 post-coitum, remainder of

females per dose level).

Dosing solutions were analysed (GC) to confirm received dose.

GENERAL OBSERVATIONS

The parental animals were examined for mortality and clinical

signs daily, and body weight and food consumption were

recorded weekly.

MATING

The mating index, pre-coital time and fertility index were

calculated. After mating, the males were sacrificed and the

females divided into two sub-groups.

PREGNANCY AND REPRODUCTION PARAMETERS

One half of the females were subjected to hysterectomy on day

20 of pregnancy. The gravid uterus was weighed to allow

calculation of the net body weight gain of the pregnant

females. The fetuses were removed by hysterectomy and the

\- 138/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

litter parameters recorded: namely number of corpora lutea,

implantation sites, early and late resorptions, dead and live

fetuses. The fetuses were weighed, sexed and examined

externally.

The remainder of the females were allowed to deliver normally.

Duration of gestation and fetal parameters were recorded, and

the litters observed daily during the lactation period (to

post-natal day 21) for survival and clinical signs.

TERMINAL OBSERVATIONS IN PARENENTAL ANIMALS

At study termination, parental animals were subjected to a

macroscopic post-mortem examination, with particular attention

paid to the reproductive organs. In addition, in males, the

testes and epididymides were weighed and sperm sampled and

examined for total count and viability.

STATISTICAL METHODS

Data were analysed by ANOVA and Dunnett\'s test. Percentage

values were compared using the Fisher exact probability test.

**Remark:** The objective of this study was to provide preliminary

information on the potential effects of ETBE on reproductive

and developmental indices in the rat. Particular attention was

paid to the male reproductive system since published results

(see record for Medinsky et al., 1999; Section 5.4) suggested

this was a possible target in Fischer rats.

The report notes, however, that the investigation of Medinsky

et al. (1999) used F344 rats; this is an inbred strain of low

fecundity and therefore unacceptable for use in reproduction

studies designed to meet international regulatory requirements

(e.g. OECD Guideline 416; EU Annex V method B35; US-EPA

40CFR-798.4700).

For this reason, testis and epididymal evaluations included in

the current investigation were conducted in two strains of

male rat: Fisher 344 (included for consistency with Medinsky

et al.) and Sprague Dawley (for compliance with regulatory

testing requirements).

**Result:** GENERAL

There were no deaths in any group. Excessive secretion of

saliva post-dosing with ETBE was the only clinical sign

present, affecting primarily high-dose animals of both sexes.

There were no toxicologically relevant changes in body weight

or food consumption for any of the treated males, however

female SD rats treated with 1000 mg ETBE/kg bw/d exhibited a

slight (-11%) but statistically significant (P\<0.05) decrease

in body weight gain although food intake was unaffected.

MATING DATA - SD rats

The pre-mating period was of similar duration in the control

and treated groups. The male and female mating and fertility

indexes were similar in the control and the treated groups:

Dose level (mg/kg/d) 0 50 250 500 1000

\- 139/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Mating index (%) 95.8 95.8 100 83.3 100

Fertility index (%) 95.7 95.7 95.8 95.0 95.8

SPERM PARAMETERS - SD and F344 rats

The count, viability and morphology of spermatozoa were not

affected by treatment in either strain at any dose-level:

Dose level (mg/kg/d) 0 50 250 500 1000

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--F344
rats\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

Epididymal sperm

Spermatozoa (10\^3/mm\^3) 337 338 367 330 364

Motility (% motile) 75 91 86 82 89

Normal morphology (%) 97 96 96 97 96

Testicular sperm

Sperm heads (10\^6/g) 125 126 127 129 123

Production (10\^6/g/d) 20.5 20.7 20.8 21.1 20.0

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--SD
rats\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

Epididymal sperm

Spermatozoa (10\^3/mm\^3) 378 336 315 345 324

Motility (% motile) 90 87 91 96 86

Normal morphology (%) 97 97 97 97 96

Testicular sperm

Sperm heads (10\^6/g) 129 134 123 104 123

Production (10\^6/g/d) 21.1 21.9 20.2 17.0 20.1

HYSTEROCTOMY, DELIVERY AND LITTER DATA - SD rats

Treatment with the test item had no influence on ovulation,

implantation, fecundity or embryo-fetal development.

Treatment with the test item had no adverse effect on

post-implantation or neonatal losses, delivery litter size,

fetal weight or sexual differentiation.

OBSERVATION OF PROGENY DURING LACTATION

The survival, growth and development of the pups were similar

in the control and the treated groups. There were no relevant

clinical signs or any weight change in any pup.

PATHOLOGY

No relevant macroscopic post-mortem findings were noted in

males of either strain or females at any dose-level.

**Test substance:** ETBE, CAS No. 637-92-3, \>98% pure, Totalfinaelf
France.

**Conclusion:** The test item, ETHYL TERTIARY BUTYL ETHER (ETBE), was

administered daily by oral gavage at 50, 250, 500 or 1000

mg/kg/day to male Fischer rats for 12 weeks or to male and

female Sprague-Dawley rats through the pre-mating and mating

periods and until sacrifice (males, 12 weeks treatment) or

until day 19 post-coitum for half of the females or day 21

post-partum for the other half.

Minor signs of maternotoxicity (lower body weight gain) were

noted at 1000 mg/kg/day during pregnancy whereas no effect was

observed at lower dose-levels. There were no adverse changes

\- 140/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

in male gonadal function, mating behavior, fertility,

embryo-fetal development or parturition at any dose-level. The

survival and development of the pups was also unaffected.

Consequently, under the conditions, the author concluded the

results supported a parental NOAEL of 1000 mg/kg bw/d (for

parental and reproductive effects), a fetal NOAEL of 1000

mg/kf bw/d and maternal NOAEL of 500 mg/kg bw/day (based on

significantly decreased bw gain at 1000 mg/kg bw/d).

**Reliability:** (2) valid with restrictions

Study available for review. GLP compliant dose-range finding

study, clearly reported methods and results, suitable for

assessment.

07-NOV-2003 (31)

**[5.9 Specific Investigations]{.underline}**

**Endpoint:** other: Odour and taste threshold studies with ETBE

**Method:** SUBJECTS

Seven individuals (1 male, six female; no further details)

were selected from a panel of experienced subjects maintained

by the test laboratory. Based on preliminary screening, the

individuals were said to be representative of the \"normal

distribution of olfactory sensitivity\" found in the general

population. Prior to the odour threshold study, the olfactory

response of each subject was calibrated using 1-butanol.

ODOUR THRESHOLD IN AIR

A known volume of ETBE (0.6 ul) was mixed with a known volume

of carbon-filtered, hydrocarbon-free air (0.380 cubic feet) in

a Tedlar bag (final concentration = 9.25 ppm; confirmed by

GC). This vapour sample was mixed with additional

hydrocarbon-free air using a dynamic dilution triangle

olfactometer (IITRI System, 1979 Model) to give a further 6

diluted samples, spaced at log3 intervals (range: 0.0068-1.156

ppm). Each ETBE/air sample was accompanied by two samples of

filtered air. The subjects were asked to \'sniff\' all three

samples and identify the one that contained ETBE. The results

were used to derive:

\- detection threshold: the dilution at subjects could

differentiate between the ETBE/air sample and filtered air;

\- recognition threshold: the dilution at which subjects could

rate the intensity of the odour relative to that of 1-butanol.

ODOUR THRESHOLD IN WATER

A series of dilutions of ETBE in distilled water (5 samples,

range = 0.066-0.866 ppm, confirmed by GC) was presented to 7

panellists in ascending concentration order. Each sample was

accompanied by two flasks that contained plain distilled

water. The panellist sniffed the headspace of each flask, and

indicated which one differed from the other two. The odour

\- 141/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

threshold in water was calculated from a linear regression

plot.

TASTE THRESHOLD IN WATER

An aliquot of the samples used for the aqueous odour

threshold tes was presented to the subjects in order of

increasing concentration. Each sample was paired with a

water reference. The panellists were required to taste the

sample and decide if it tasted different to the water blank.

The taste threshold in water was calculated from a linear

regression plot.

**Result:** The mean odour detection and recognition threshold values
for

ETBE in air were 0.013 ppm and 0.024 ppm, respectively.

(MTBE was also included in the study, and returned values of

0.053 ppm and 0.08 ppm for detection and recognition

thresholds, respectively).

The mean odour detection threshold for ETBE in water was

0.049 ppm, with a recognition threshold of 0.106 ppm. (The

equivalent values for MTBE in water were 0.095 ppm and 0.193

ppm, respectively.)

The mean taste detection threshold for ETBE was 0.047 ppm,

and was described as highly objectionable. (The taste

detection threshold for MTBE was 0.134 ppm).

**Test substance:** ETBE, 99.0-99.5% pure supplied by ARCO Chemical
Company, PA,

USA.

**Reliability:** (2) valid with restrictions

Study available for review. Well documented research

investigation, meets generally accepted scientific principles,

acceptable for assessment.

05-OCT-2003 (84)

**[5.10 Exposure Experience]{.underline}**

**-**

\- 142/156 -

date: 22-MAR-2006

5\. Toxicity Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[5.11 Additional Remarks]{.underline}**

**Type:** other: SAR modelling

**Remark:** This paper predicts toxicological properties of ETBE using a

SAR model (CASE/MULTICASE). It considers the potential

biological reactivity of ETBE, along with that of putative

predicted metabolites (derived using META).

The authors conclude that several metabolites are potential

sensory irritants, contact sensitisers, mutagens,

developmental toxicants or carcinogens.

Reviewer\'s comment: The reliability of some of the metabolic

predictions included in publication (i.e. putative formation

of ethylene oxide from ETBE) is unknown, but appears

inconsistent with results from metabolism and toxicokinetic

studies summarised elsewhere in this IUCLID dataset. As a

result, the toxicological profile proposed by these authors

cannot be considered reliable or suitable for assessment.

**Reliability:** (3) invalid

Study available for review. Modelled data of unknown

reliability, not suitable for assessment.

02-NOV-2003 (92)

**Type:** other: Narcotic effects

**Remark:** General anesthetic effects (Inhalation, Mouse):

LC50 = 123 g/m3/15\'

**Source:** Agip Petroli SpA ROMA

EUROPEAN COMMISSION - European Chemicals Bureau Ispra (VA)

**Test substance:** ETBE

**Reliability:** (4) not assignable

This information originates from the Eurpean Chemicals Bureau

IUCLID dataset for CAS No. 637-92-3. Its reliability has not

been assessed.

02-NOV-2003 (3)

\- 143/156 -

date: 22-MAR-2006

6\. Analyt. Meth. for Detection and Identification Substance ID:
637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[6.1 Analytical Methods]{.underline}**

**-**

**[6.2 Detection and Identification]{.underline}**

**-**

\- 144/156 -

date: 22-MAR-2006

7\. Eff. Against Target Org. and Intended Uses Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[7.1 Function]{.underline}**

**-**

**[7.2 Effects on Organisms to be Controlled]{.underline}**

**-**

**[7.3 Organisms to be Protected]{.underline}**

**-**

**[7.4 User]{.underline}**

**-**

**[7.5 Resistance]{.underline}**

**-**

\- 145/156 -

date: 22-MAR-2006

8\. Meas. Nec. to Prot. Man, Animals, Environment Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**[8.1 Methods Handling and Storing]{.underline}**

**-**

**[8.2 Fire Guidance]{.underline}**

**-**

**[8.3 Emergency Measures]{.underline}**

**-**

**[8.4 Possib. of Rendering Subst. Harmless]{.underline}**

**-**

**[8.5 Waste Management]{.underline}**

**-**

**[8.6 Side-effects Detection]{.underline}**

**-**

**[8.7 Substance Registered as Dangerous for Ground Water]{.underline}**

**-**

**[8.8 Reactivity Towards Container Material]{.underline}**

**-**

\- 146/156 -

date: 22-MAR-2006

9\. References Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

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\(3\) Anesthesiology 11, 455, 50, as reported in RTECS.

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\(5\) Becker, KH and Thuner, L (1997) Produktanalysen und

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its products.) Wuppertal University (Gesamthochschule),

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\(6\) Bennett, PJ and Kerr, A (1990) Kinetics of the reactions of

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\(7\) Berger, T and Horner, CM (2003) In vivo exposure of female

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\(8\) Bernauer U, Amberg A, Schentow D and Dekant W (1998)

Biotransformation of 12C- and 2-13C-labeled methyl

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\(9\) Boeri, RL, Kowalski, PL and Ward, TJ (1994) Acute toxicity

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\(10\) Boeri, RL, Kowalski, PL and Ward, TJ (1994) Acute toxicity

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\(11\) Boeshertz, D (2002) The promotion of biofuels in the

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date: 22-MAR-2006

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\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\(12\) Bond, JA, Medinsky, MA, Wolf, DC, Cattley, R, Farris, G,

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\(13\) Bond, JA, Medinsky, MA, Wolf, DC, Dorman, DC, Cattley, R,

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\(14\) Borghoff, SJ (1966) Ethyl tertiary-butyl ether (ETBE): A

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\(15\) Borghoff, SJ and Ashgarian, B (1966) Ethyl tertiary-butyl

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\(16\) Bushy Run Research Center (1995) Ethyl tertiary butyl ether:

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\(17\) Bushy Run Research Center (1995) Ethyl tertiary butyl ether:

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\(18\) Bushy Run Research Center (1995) Ethyl tertiary butyl ether:

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\(19\) CEMC (2002) Level III model, Canadian Environmental

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\(20\) Centre International de Toxicologie (1992) Ether effet

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\(21\) Centre International de Toxicologie (1992) Ether irritation

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\(22\) Chatin L, Fombarlet C, Bernasconi C, Gauthier A and

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\(23\) Chatin, L. (Elf Antar France). As reported in the Acts of

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\(24\) Cowan, RM and Park, K (1996) Biodegradation of the gasoline

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\(26\) Domanska, U, Lachwa, J, Morawski, P and Malamowski, SK

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\(28\) Evans, TW and Edlund, KR (1936) Tertiary alkyl ether:

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\(29\) Fayolle, F, Hernandez, G, Le Roux, F and Vandecasteele, J-P

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\(30\) Furey, RL and Perry, KL (1990) Volatility characteristics of

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Washington DC, May 1990.

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\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\(31\) Gaoua, W (2003) Ethyl tertiary butyl ether (ETBE), CAS No.

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\(32\) Gaoua, W (2003) Ethyl tertiary butyl ether (ETBE): Prenatal

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\(33\) Gaoua, W (2004) Ethyl tertiary butyl ether (ETBE):

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the oral route (gavage) in rats. CIT Study No. 24859 RSR,

unpublished study for Totalfinaelf on behalf of the ETBE

Producers\' Consortium.

\(34\) Glave, WR and Hansch, C (1972) Relationship between

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61, 589-591.

\(35\) Hong J-Y, Wang Y-Y, Bondoc FY, Lee M, Yang CS, Hu W-Y and

Pan J (1999) Metabolism of methyl tert-butyl ether and other

gasoline ethers by human liver microsomes and

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\(36\) Hong, J-Y, Wang, Y-Y, Bondoc, FY, Yang, CS, Gonzalez, FJ,

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\(38\) Hong, J-Y, Wang, Y-Y, Mohr, SN, Bondoc, FY and Deng, C

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\(39\) Hong, J-Y, Yang, CS, Lee, M, Wang, Y-Y, Huang, W-Q, Tan, Y,

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\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\(40\) IIT Research Institute (1989) Acute dermal toxicity study of

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\(41\) IIT Research Institute (1989) Acute inhalation toxicity

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Report 1496 for Amoco Corporation, November 1989.

\(42\) IIT Research Institute (1991) Four-week inhalation toxicity

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Study no 1544 for Amoco Corporation, 30 April 1991.

\(43\) Institut Pasteur de Lille (1992) Etude de l\'activite

genotoxique par la technique du micronucleus chez la souris

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\(44\) Institut Pasteur de Lille (1992) Etude de toxicite aigue aux

doses limites (seion CEE annexe VI J 08-7-92) par voie orale

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\(45\) Institut Pasteur de Lille (1992) Recherche de mutagenicite

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\(46\) Japar, SM, Wallington, TJ, Rudy, SJ and Chang, TY (1991)

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\(47\) Johanson, G, Nihlen, A, and Lof, A (1995) Toxicokinetics and

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\(48\) Kaneko, T, Wang, P-Y and Sato, A (2000) Partition

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\(50\) Kharoune, M, Kharoune, T, Ribero, T, Pauss, A and Lebeault,

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\(51\) Kharoune, M, Pauss, A and Lebeault, JM (1998) Ethyl

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\(56\) MB Research Laboratories (1988) Test article: ethyl tertiary

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\(57\) MB Research Laboratories (1988) Test substance ethyl

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\(58\) MB Research Laboratories (1988) Test substance: ethyl

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\(59\) MB research Laboratories, Inc (1988) Test substance: ethyl

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date: 22-MAR-2006

9\. References Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

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\(62\) Montgomery, CR (1994) n-Octanol/water partition coefficient

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\(68\) Noureddini, H (1999) Ethyl tertiary butyl ether (ETBE) and

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\(69\) Pharmakon Europe (1994) Test article: ETBE. Salmonella

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\(70\) Pharmakon Europe (1994) Test article: ETBE. Test to evaluate

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\(71\) Pharmakon Europe (1994) Test article: ETBE. Test to evaluate

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date: 22-MAR-2006

9\. References Substance ID: 637-92-3

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\(72\) Pharmakon Europe (1994) Test article: ETBE. Test to evaluate

the acute primary cutaneous irritation and corrosivity in

the rabbit (3 animals). Report No 78193 for Elf, 1 April

1994\.

\(73\) Pharmakon Europe (1994) Test article: ETBE. Test to evaluate

the acute toxicity following a single oral administration

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1994\.

\(74\) Rhodes, IAL and Verstuyft, AW (2001) Selecting analytical

methods for the determination of oxygenates. Environmental

Testing and Analysis, 10(2), pages 24-26, 28-31 and 43.

\(75\) Rosenkrantz, HS, Klopman, G (1991) Predictions of the lack

of genotoxicity and carcinogenicity in rodents of two

gasoline additives: methyl- and ethyl-tert-buthyl-ethers. In

Vitro Toxicology 4, 49-54.

\(76\) Ryan, BM, Ferguson, JS, Hatoum, NS, Klan, MJ and Farmer, RG

\(1991\) Toxicologist, 11, Abstract 385.

\(77\) Skahan, DJ (1994) Letter to Dr CR Montegomery, Associate

Director of Analytical Chemistry, US-EPA; dated 11 January

1994\.

\(78\) SRC EPIWIN experimental database (2003)

\(79\) Steffan RJ, McClay, K, Vainberg, S, Condee, CW and Zhang, D

\(1997\) Biodegradation of the gasoline oxygenates methyl

tert-butyl ether, ethyl-tert butyl ether and tert-amyl

methyl ether by propane oxidizing bacteria. App Env

Microbiol 63, 4216-4222.

\(80\) Sulfita, JM and Mormille, MR (1993) Anaerobic biodegradation

of known and potential gasoline oxygenates in the

terrestrial subsurface. Environ Sci technol 27, 976-978.

\(81\) Sun JD and Beskitt JL (1995) Ethyl tertiary-butyl ether

(ETBE) pharmacokinetics after single and repeated inhalation

exposure of mice. Bushy Run Research Center Project 94N1455.

Unpublished study for ARCO Chemical Co., 19 June 1995.

\(82\) Sun JD and Beskitt JL (1995) Ethyl tertiary-butyl ether

(ETBE) pharmacokinetics after single and repeated inhalation

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\(83\) Turini A, Amato G, Longo V, and Gervasi, PG (1998) Oxidation

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date: 22-MAR-2006

9\. References Substance ID: 637-92-3

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\(84\) Vetrano, KM (1993) Final report to ARCO Chemical Company on

the odor and taste threshold studies performed with methyl

tertiary butyl ether (MTBE) and ethyl tertiary butyl ether

(ETBE). TRC Environmental Corporation Project No. 13442-M31,

April 29, 1993.

\(85\) Wallington, TJ and Japar, SM (1991) Atmospheric chemistry of

diethyl ether and ethyl tert-butyl ether Environ Sci Technol

25, 410-415.

\(86\) Wetton, PM and McKenzie, J (2003) Ethyl tert-butyl ether

(ETBE): acute toxicity to Daphnia magna. Safepharm project

no. 1811/001, unpublished report for Total France (on behalf

of the ETBE Producer\'s Consortium), 8 December 2003.

\(87\) White, RD, Daughtrey, WC and Wells, MS (1995) Health effects

of inhaled tertiary amyl methyl ether and ethyl tertiary

butyl ether. Toxicology Letters 82/83, 719-724.

\(88\) Wildey, RJ (2003) Fugacity modelling for ETBE, unpublished

report, 14 October 2003.

\(89\) Yeh, CK and Novak JT (1994) Anaerobic biodegradation of

gasoline oxygenates in soils. Water Environ Res 66, 744-753.

\(90\) Yeh, CK and Novak, JT (1995) The effect of hydrogen peroxide

on the degradation of methyl and ethyl tert-butyl ether in

soils. Water Environ Res 67, 828-834.

\(91\) Zeiger, E, Anderson, B, Haworth, S, Lawlor, T and

Mortelmans, K (1992) Salmonella Mutagenicity Tests: V.

Results from the testing of 311 chemicals. Environ.

Molecular Mutagenesis, 19 (S21), 2-141.

\(92\) Zhang, YP, Macina, OT, Rosenkranz, HS, Karol MH, Mattison,

DR and Klopman, G (1997) Prediction of the metabolism and

toxicological properties of gasoline oxygenates. Inhal.

Toxicol. 9, 237-254.

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date: 22-MAR-2006

10\. Summary and Evaluation Substance ID: 637-92-3

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**[10.1 End Point Summary]{.underline}**

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**[10.2 Hazard Summary]{.underline}**

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**[10.3 Risk Assessment]{.underline}**

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