micronucleus test of etbe using bone marrow of rats.pdf

8/11/2019 Micronucleus Test of ETBE Using Bone Marrow of Rats.pdf

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Study No.7047

FINAL REPORT

Micronucleus Test of ETBE Using Bone Marrow of Rats of the

13-Week Toxicity Study of 2-Ethoxy-2-methylpropane in F344 rats

(Inhalation Study) [Preliminary Carcinogenicity Study]

Study No.: 7047

June 29, 2007

Japan Industrial Safety and Health Association

Japan Bioassay Research Center


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Study No.7047

Contents for Preface

Study Title................................................................................................................................i

Objective .................................................................................................................................i

Guidelines ...............................................................................................................................i

GLP .........................................................................................................................................i

Sponsor ...................................................................................................................................i

Test Facility and facility manager ............................................................................................i

Schedule of the Study ............................................................................................................ ii

Study Personnel ..................................................................................................................... ii

Archives of documents and samples..................................................................................... iii

Signature, seal and date of study director (person prepared the final report) ....................... iii

Statement .............................................................................................................................. iv

Certificate of Quality Assurance .............................................................................................v

Text.....................vi

Contents for text

Abstract..1

I Test material.2

I-1 Specifications of the test material...2

I-1-1 Name of the test material.......2

I-1-2 Chemical structure and molecular weight2

I-1-3 Physicochemical properties of the test material.....2

I-2 Test material used in this bioassay.......2

I-3 Specificity and identity, and stability of the test material ......3

I-3-1 Analytical method for specificity and identity...3

I-3-2 Stability of the test material....3

I-4 Animals...3

II Study methods.......4

II-1 Administration...4

II-1-1 Administration route...4

II-1-2 Methods of administration of the test material...4

II-1-3 Study period........4


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II-1-4 Administration concentration4

II-1-5 The reason for the selection of the route and method of test material

administration.... 4

II-1-6 Vaporization method and adjustment of test material levels... 5

II-1-7 Measurement of test material concentrations...............................................5II-2 Animal management... .5

II-2-1 Number of animals of each group....5

II-2-2 Allocation and individual identification methods....6

II-2-3 Animal husbandry...6

(1) Housing conditions....6

(2) Food.....7

(3) Drinking water....7

II-3 Observation and laboratory investigations, and methods..7

II-3-1 Observation of status and general conditions....7

II-3-2 Measurement of body weights..8

II-4 Micronucleus test..8

II-4-1 Preparation of specimens and microscopic observation..8

(1) Preparation of specimens.....8

(2) Microscopic observation of specimens...8

1) Microscopic observation.....8

2) Distinction of polychromatic erythrocytes from normochromatic erythrocytes....8

3) Identification (discrimination) of micronuclei....8

II-5 Numerical value processing and statistical methods......9

II-5-1 The handling of numerical values, and their display.....9

II-5-2 Statistical processing.....9

III Results and discussion....9

IV Judgement of the results...10

V Unforeseeable circumstances that might have affected the reliability of the study and

deviations from the protocol..10

References.. 10

Table 1 Results of micronucleus test males- ...12Table 1 Results of micronucleus test females- 13

Table 2 Results of micronucleus test (summary table) males-..14

Table 2 Results of micronucleus test (summary table) females-..15

Figure 1 Results of micronucleus test..16


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Study Title

Micronucleus Test of ETBE Using Bone Marrow of Rats of the 13-Week Toxicity Study of

2-Ethoxy-2-methylpropane in F344 rats (Inhalation Study) [Preliminary Carcinogenicity Study]

Objective

The mutagenic potential of ETBE to induce micronuclei in polychromatic erythrocytes was tested using

the bone marrow of rats exposed to ETBE by inhalation for 13 weeks.

Guidelines

The present study was conducted following the Guideline of Micronucleus Test with Rodents in the

Methods of Testing New Chemical Substances of the Joint Notification ofNo.1121002 of the

Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Heisei 1511

13-No.2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry and

No.031121002 of the Environmental Policy Bureau, Ministry of the Environment, Japan, November

21, 2003.

The micronucleus test was conducted using bone marrow sampled from rats which were killed on

schedule in the study entitled 13-Week toxicity study of 2-ethoxy-2-methylpropane in F344 rats

(inhalation study) [preliminary carcinogenicity study] (Study No. 0666).

GLP

The present study was performed in compliance with the Good Laboratory Practice (GLP) Standards of

the "The basis related to the examination institution which carried out an examination to modify new

chemical substance"of the Joint Notification of No.1121003 of the Pharmaceutical and Food Safety

Bureau, Ministry of Health, Labour and Welfare, Heisei 151117-No.3 of the Manufacturing

Industries Bureau, Ministry of Economy, Trade and Industry and No.031121004 of the Environmental

Policy Bureau, Ministry of the Environment, Japan, November 21, 2003, and also performed in

compliance with "OECD Principles of Good Laboratory Practice" (November 26, 1997).

Sponsor

Japan Petroleum Energy Center

Sumitomo Shin-Toranomon Building, 4-3-9, Toranomon, Minato-ku, Tokyo, Japan

Test Facility and Facility Manager

Japan Bioassay Research Center


2445 Hirasawa, Hadano, Kanagawa, Japan

Director: Shoji Fukushima, M.D., Ph. D.


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Study No.7047

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Schedule of the Study

Commencement: August 24, 2006

Receipt of animals: September 4, 2006

Allocation of animals: September 18, 2006

Initiation of test material treatment: September 19, 2006

Termination of test material treatment: December 18, 2006

Scheduled necropsy and Slide preparation: December 19 and 20, 2006

Completion: June 29, 2007

Study Personnel

Study director:

Tadashi Noguchi (Animal Care Section, Department of Experimental Toxicology)

Analysis of test substance, administration and management of test substance:

Tomoji Nishizawa (Inhalation Test Section, Department of Experimental Toxicology)

Tatsuya Kasai (Inhalation Test Section, Department of Experimental Toxicology)

Arata Saito (Inhalation Test Section, Department of Experimental Toxicology)

Makoto Onishi (Analytical Chemistry Section, Department of Experimental Toxicology)

Makoto Take (Analytical Chemistry Section, Department of Experimental Toxicology)

Animal care:

Taku Katagiri (Animal Care Section, Department of Experimental Toxicology)

Masaaki Suzuki (Animal Care Section, Department of Experimental Toxicology)

Toshiaki Sasaki (Animal Care Section, Department of Experimental Toxicology)

Tomoyuki Kamigaito (Animal Care Section, Department of Experimental Toxicology)

Kenji Takanobu (Animal Care Section, Department of Experimental Toxicology)


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Archives of documents and samples

Protocol, specimens, raw data, test material, record documents, final report, certificate of quality

assurance, the other documents and samples related to this study are to be stored in archives. Furthermore,

50 g of test compound are to be maintained under storage.

The retention period is for ten years (in principle) after submission of .the final report. However,

storage of specimens and test material will be restricted to the period for which their quality is maintained.

Signature, seal and date of study director (person who prepared the final report)

Animal Care Section, Department of Experimental Toxicology

Study Director:

Tadashi Noguchi


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Statement

Study title: Micronucleus Test of ETBE Using Bone Marrow of Rats of the 13-Week Toxicity Study of2-Ethoxy-2-methylpropane in F344 rats (Inhalation Study) [Preliminary Carcinogenicity

Study]

The present study was performed in compliance with the good laboratory practice (GLP) Standards of

the "The basis which related to the examination institution which carried out an examination to modify new

chemical substance" of the Joint Notification of No.1121003 he Pharmaceutical and Food Safety Bureau,

Ministry of Health, Labour and Welfare, Heisei 151117-No.3 of the Manufacturing Industries

Bureau, Ministry of Economy, Trade and Industry and No.031121004 of the Environmental Policy

Bureau, Ministry of the Environment, Japan, November 21, 2003, and also performed in compliance

with "OECD Principles of Good Laboratory Practice" (November 26, 1997).

We consider the data generated by Japan Bioassay Research Center during the course of this study to be

valid and that the final report fully and accurately reflect these raw data.

Japan Bioassay Research Center,

Japan Industrial Safety and Health Association.

Study director


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Certificate of Quality Assurance

Study title: Micronucleus Test of ETBE Using Bone Marrow of Rats of the 13-Week Toxicity Study of

2-Ethoxy-2-methylpropane in F344 rats (Inhalation Study) [Preliminary Carcinogenicity

Study]

Study No.: 7047

Test Substance: 2-ethoxy-2-methylpropane (ETBE)

The study was conducted essentially in compliance with The Standards Concerning Test Facilities

Conducting the Tests Relating to New Chemical Substances of the Joint Notification of No.1121003 he

Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Heisei 1511

17-No.3 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry and

No.031121004 of the Environmental Policy Bureau, Ministry of the Environment, Japan, November

21, 2003and "OECD Principles of Good Laboratory Practice" (November 26, 1997).

This report presents a full and accurate account of the results of the study.

Date of audit and

inspection

Date of submitting the reports to the

test facility management and the

study director

Object

Protocol August 24, 2006 August 24, 2006

September 27, 2006 September 27, 2006

March 14 and 30, 2007 March 14 and 30, 2007Protocol (Amendment)

April 12, 2007 April 12, 2007

December 19, 2006 December 19, 2006Slide preparation and counting ofmicronuclei January 11, 2007 January 11, 2007

Management of the test substance June 20, 2007

Handling of data June 18 20, 2007

June 18 20, 2007

June 20, 2007

Final reportJune 29, 2007 June 29, 2007

June 29, 2007

Quality Assurance Director

Attached to: Japan Bioassay Research Center


Title: Quality Assurance Manager

Signature:

Mitsuru Haresaku


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Text

Micronucleus Testing of ETBE Using Bone Marrow of Rats for the13-Week Toxicity Study of 2-Ethoxy-2-methylpropane in F344 rats

(Inhalation Study) [Preliminary Carcinogenicity Study]

Study No. 7047


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Abstract

The mutagenic potential of 2-ethoxy-2-methylpropane (ETBE) was assessed with reference to the

frequency of micronucleated polychromatic erythrocytes (MNPCE) in polychromatic erythrocytes (PCE) in

the bone marrow of rats exposed to ETBE by inhalation for 13 weeks.The micronucleus test was conducted using rats killed on schedule in the study entitled 13-Week

Toxicity Study of 2-Ethoxy-2-methylpropane in F344 rats (Inhalation Study) [Preliminary Carcinogenicity

Study] (Study No. 0666). Both sexes of rats were exposed (whole body) to air containing the test

chemical ETBE for 13 weeks (6 hours/day, 5days/week). Animal husbandry and administration of the test

chemical were appropriately managed.

Although no mortality was found during the treatment period of 13 weeks, retardation of body weight

gain was noted in the males treated with 5000 ppm ETBE.

The micronucleus test was conducted for both sexes of the control and three treated (500, 1500 and 5000

ppm ETBE) groups. Each group consisted of 10 rats of either sex. At sacrifice, the bone marrow cells

were collected from the femur, smeared on slides and then the frequencies of micronucleated PCE in PCE

(MNPCE/PCE) and the ratios of PCE to the total number of erythrocytes (PCE/E) were calculated.

The frequencies of MNPCE/PCE in males were 0.140.06% in the control group, 0.160.07% in the 500

ppm ETBE group, 0.170.07% in the 1500 ppm ETBE group, and 0.180.10% in the 5000 ppm ETBE

group. Those in females were 0.160.13% in the control group, 0.180.08% in the 500 ppm ETBE group,

0.160.09% in the 1500 ppm ETBE group, and 0.160.08% in the 5000 ppm ETBE group. As the values

of the controls were within the background data of F344 rats in our facility, it was evident that this study

was well conducted. No statistical significant differences in the ratios (MNPCE/PCE) of MNPCE to PCE

were observed in either sex of the treated groups, as compared to the control values. In addition, no

dose-dependent increased tendencies in MNPCE/PCE were found in either sex of any of the treated groups.

In addition, no alterations of the ratio of PCE/E were noted.

It was considered that the highest level of ETBE was appropriately selected for this study, since

retardation of body weight gain was observed in males exposed to 5000 ppm ETBE.

Thus, from these results, ETBE was assessed to be negative on micronucleus induction for bone marrow

in rats exposed to ETBE by inhalation for 13 weeks.


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I Test material

I-1 Specifications of the test material

I-1-1 Name of the test material

Name: 2-Ethoxy-2-methylpropane

Synonym: Ethyl tertiary-butylether (ETBE)

CAS No.: 637-92-3

I-1-2 Chemical structure and molecular weightChemical formula:

Molecular weight: 102.17

H3C C

CH3

CH3

O CH2 CH3

I-1-3 Physicochemical properties of the test material

Appearance: Colorless transparent liquid.

Boiling point: 70C

Vapor pressure: 17kPa (25C)

Specific gravity: 0.74 (25C/4C)

Solubility: Slightly soluble in water 1.2g/100 g, 20C)

Storage condition: At room temperature and dark place.

I-2 Test material used in this bioassay

Supplier: Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan)

Grade: Grade 1 (Highly purified product) of Tokyo Chemical Industry Co., Ltd.

Purity: 992-99.3% (Report from the supplier)

Lot no.: R74EE (September 29, 2006 - October 19, 2006)

BVQ3B (October 20, 2006 - December 18, 2006)


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I-3 Specificity and identity, and stability of the test material

I-3-1 Analytical method for specificity and identity

The identity of test material was assessed by electron impact mass spectrometry (Agilent Technologies

5973N) and confirmed by comparison with reported values.

As a result, the mass spectrum of the test material showed a fragment peak the same as the reported

value (Reference 1), confirming its nature as 2-ethoxy-2-methylpropane (Reference 2).

I-3-2 Stability of the test material

Stability of the test material was assessed using gas chromatographs (Hewlett Packard 5890A) before the

beginning and after the end of use, and confirmed by comparing the data.

There were no difference between the results for the beginning and the end of use, and the stability of the

test material during the treatment period was therefore confirmed (Reference 2).

I-4 Animals

Both sexes of F344/DuCrlCrlj (Fischer) rats (SPF animals), planned for use in the carcinogenicity study

of ETBE, received from Charles River Laboratories Japan Inc. (795 Shimo-furusawa, Atsugi, Kanagawa,

Japan) were employed in this study.

A total of 75 four week old animals of each sex were obtained and allowed one week of quarantine and a

one week acclimation period, and then 60 males and 60 females (body weight range; males: 116-129g,

females: 93-103g) were selected for the study. These animals showed no abnormality in general condition

and body weight gain, and were near to the median in body weight values.

F344/DuCrlCrlj rats (SPF animals) were selected because of their genetic stability, routine use for

carcinogenicity studies and high sensitivity to carcinogenicity of chemicals.


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II Study methods

II-1 Administration

II-1-1 Administration routeInhalation (whole body ) of the test material was selected as the exposure route in this study.

II-1-2 Method of administration of the test material

Air including the test compound adjusted to the set concentration was introduced into inhalation

chambers accommodating the animals for examination, which received whole body exposure.

II-1-3 Study period

The duration of administration by exposure was for 13 weeks (6 hours/day, 5 days/week), for a total of

65 exposures.

II-1-4 Administration concentration

The administration concentrations were set at three levels of 500, 1500 and 5000 ppm (volume ratio v/v).

In addition, a control group was maintained with clean air ventilation.

II-1-5 The reason for the selection of the route and method of test material

Systemic exposure by inhalation was selected as the administration routs as one main human exposure

route of ETBE.

The duration of administration was 13 weeks (5days/week) in order to determine dose levels for the

carcinogenicity test.

The administration concentrations were set in discussion with the sponsor, taking account of the results

of a previous publication (Medinsky et al.; Reference 3).

In their study, both sexes of F344 rats were given 0, 500, 1750 and 5000 ppm ETBE by systemic

inhalation exposure (6 hours/day, 5 day/week) for 13 weeks. No mortality or body weight retardation was

found in any group. However, significant increases of kidney and liver weights were found in males of

the 1750 and 5000 ppm groups, and significant increase of adrenal weights was noted in males at 5000 ppm.

Significant increases of kidney weights were also found in females of the 1750 and 5000 ppm group, with

significant increases of liver, adrenal and heart weights at 5000 ppm. On histopathological examination,

alterations in kidneys were found in the male treated groups, in testes in the 1750 and 5000 ppm groups,and in bone marrows of females at 1750 and 5000 ppm. From these results, it was considered that the

highest dose level should be set as 5000 ppm, at which alterations were found in organ weight and

histopathology, and the lowest level should be set at less than 500 ppm, at which was observed kidney

lesion histopathologically.

Thus, for the present study the highest dose was decided as 5000 ppm (volume ratio; v/v), and the lower

levels were set as 1500, 500, 150 and 50 ppm.


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II-1-6 Vaporization method and adjustment of test material levels

ETBE was placed in the container of an organic solvent vapor generator (model; specially made to order,

Shibata Scientific Technology Ltd., Tokyo, Japan), and vaporized by bubbling of clean air heated with a

thermostated circulator. The air flow containing the saturated vapor was diluted with clean air, cooled

through a thermostated condenser, then warmed in a thermostated circulator, which served to stabilize thevapor concentration. The flow rate of the vapor-air mixture was regulated with a flow meter, for supply to

the line mixer in the inhalation exposure chambers.

Chamber concentrations of the test material were monitored by gas chromatography, and supply volume

of test material to the inhalation chamber was regulated to adjust to the intended concentrations based on

the data obtained.

II-1-7 Measurement of test material concentrations

Chamber concentrations of ETBE were analyzed by gas chromatography with an automatic sampler

(model GC-14B, Shimadzu Corporation, Kyoto, Japan) every 15 minutes during the exposure period.

For each treated group, differences between the found and intended concentrations ((found concentration

intended concentration) / intended concentration100) were less than 0.3 %, and variation indexes

(standard deviation / mean value100) were less than 0.4 %. Thus, it was shown that ETBE

concentrations in the chamber were maintained with high precision (Reference 2).

II-2 Animal management

II-2-1 Number of animals of each group

Three higher-dose treatment groups in the study entitled 13-Week Toxicity Study of

2-Ethoxy-2-methylpropane in F344 rats (Inhalation Study) [Preliminary Carcinogenicity Study] and one

control group from the original study (Study no. 0666), a total of 4 groups (10 rats/sex/group) were

employed in the present study.

No. of animals (Animal numbers)Group name

Male Female

Control group 10 (1001-1010) 10 (2001-2010)

500 ppm group 10 (1301-1310) 10 (2301-2310)

1500 ppm group 10 (1401-1410) 10 (2401-2410)

5000 ppm group 10 (1501-1510) 10 (2501-2510)


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II-2-2 Allocation and individual identification methods

Allocation was carried out by a method to minimize the deviation of body weights among groups (proper

stratification method) (Reference 4). First, animals, which were normal for general condition and body

weight gain, were allotted in order of heaviest body weight. Second, comparing with the total weight of

each group, heaviest weight animals were, in turn, allotted to the lightest weight groups, this procedure thenbeing repeated.

During the quarantine/acclimation period, individual animals were identified by tail marking with a

felt-pen. During the treatment period, individual animals were identified by ear punch. In addition, a

label carrying individual animal numbers was affixed to each cage.

Animals were housed in an independent room (Room nos. 518 and 516) in a barrier area, and the study

number, animal species and animal numbers were displayed on the door of the room to allow distinction

from other studies and species of animals.

II-2-3 Animal husbandry

(1) Housing conditions

Animals were housed in a quarantine room (room no: 518) during the quarantine period, and in an

inhalation animal room (room no: 516) during the acclimation and administration period.

Environmental conditions of the animal rooms are shown below. The actual values (mean standard

deviation) for temperature and relative humidity of animal room are given in < >. No changes that

could have affected conditions of health of the animals were apparent in the environment for either animal

room (Reference 2).

Temperature:

Quarantine room; 232C

Inhalation room; 222C

Inhalation chamber; 222C < (21.6-22.9C)

Relative humidity:

Quarantine room; 5515%

Inhalation chamber; 5020%

Lighting: artificially illuminated for 12h (8:00 to 20:00) and 12h off (20:00 to 8:00)

Ventilation frequency: Quarantine room; 15-17 times/hour

Inhalation test room; 7-9 times/hour

Inhalation chambers; 131 times /hour

Pressure power: In inhalation chamber; 0- -15x10 Pa

Accommodation method: one animal to a cage

Materials/shape/dimensions of cages:

Quarantine period; two-linked stainless steel mesh cages [170(W)x294(D)x176(H) mm/5 animals]

Acclimation period; six-linked stainless steel mesh cages [125(W)x216(D)x176(H) mm/animal]

Treatment period; five-linked stainless steel mesh cages [150(W)x216(D)x176(H)mm/animal]


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(2) Food

Rats had ad libitumaccess to CRF-1 pellet diet (sterilized by 30 kGy- rays irradiation) obtained from

Oriental Yeast Co., Ltd. (Chiba factory, 8-2 Shinminato, Mihama-ku, Chiba, Japan) placed in feeders for

pellets throughout the experimental period. However, animals were fasted from the evening of the

previous day for scheduled necropsy.Analytical data for nutrients for each lot used in the present study were obtained from Oriental Yeast Co.,

Ltd., and archived. Analytical data for concomitants in diet for each lot used in the present study were

also received from Japan Food Research Laboratories, Foundation (52-1 Motoyoyogi-cho, Shibuya-ku,

Tokyo, Japan), and contaminant levels in diet were confirmed to be within acceptable ranges, and then

archived.

(3) Drinking water

Animals had ad libitumaccess via automatic water suppliers to filtered and ultraviolet irradiated tap

water (Hadano-shi, Kanagawa water service station supply) as drinking water throughout the experimental

period.

The drinking water was routinely sampled and shipped to Food and Drug Safety Center, Hatano

Research Institute, Japan (729-5 Ochiai, Hadano, Kanagawa, Japan) for analyses performed in accordance

with Water Supply Act. Analytical results were received, and contaminant levels in drinking water were

confirmed to be within acceptable ranges, and then archived.

II-3 Observation and laboratory investigations, and methods

II-3-1 Observation of status and general conditions

Animals were observed for their status (alive, dead and in extremis) daily, and also for detailed general

condition once a week.

No treatment related mortalities and changes in general condition were found in any group.

II-3-2 Measurement of body weights

Animals were weighed weekly for the 13 weeks.

Retardation of body weight gain was noted in the male 5000 ppm ETBE group throughout the treatment

period. Tendencies for lowering of body weights were noted in other treated groups. However, it was

considered that these changes might not be related to the treatment, because of the lack of clear

dose-related trends (Ref. 2).


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II-4 Micronucleus test

The micronucleus test was conducted using rats which were killed on the scheduled date in the study

entitled 13-Week toxicity study of 2-ethoxy-2-methylpropane in F344 rats (Inhalation study) [Preliminary

carcinogenicity study] (Study No. 0666). Femurs were excised from rats of the control group and the

three treated groups (500, 1500 and 5000 ETBE groups). Bone marrow cells were collected from the

femurs, smeared on slides, stained with Acridine Orange, and then observed by fluorescent microscopy.

II-4-1 Preparation of specimens and microscopic observation (Ref. 5)

(1) Preparation of specimens

Bone marrow cells were harvested from the left femur. Namely, its contents were flushed through a

syringe by 1 mL fetal bovine serum to a centrifuge tube (10 mL). Cell suspensions were centrifuged at

1000 rpm (KS5200 and ST-480, Kubota Co., Ltd., Japan) for 5 minutes, and the supernatant was discarded,

and then marrow cell suspensions were made by adding a small amount of fetal bovine serum. A small

drop of suspension was placed on each slide, and smears were prepared (2 slides/ animal). Slides were air

dried at room temperature, and fixed with methanol for 5 minutes. Observation was performed just after

staining with 20 L of Acridine Orange (40 g/mL), mounting the coverslips onto glass slides, using a

fluorescent microscope.

(2) Microscopic observation of specimens

1) Microscopic observation

Just after staining, bone marrow smears were microscopically examined in two areas which

demonstrated well smeared conditions, on each slide. One thousand polychromatic erythrocytes (PCE) in

each area were counted and the number of micronucleated polychromatic erythrocytes (MNPCE) was also

counted, to calculate the ratio (MNPCE/PCE). In addition, 500 erythrocytes (polychromatic and

normochromatic erythrocytes) were examined, and the number of polychromatic erythrocytes (PCE) wasalso counted, to calculate the ratio (PCE/E).

Observation was carried out by a blinded method using high magnification (x 400) under a fluorescent

microscope (BX51, Olympus Co., Ltd., Japan)

2) Distinction of polychromatic erythrocytes from normochromatic erythrocytes

Anucleate cells emitting red fluorescence were counted as polychromatic erythrocytes and

those did not were diagnosed as normochromatic erythrocytes.

3) Identification (discrimination) of micronuclei

Size: The upper limit was half of a diameter of an normochromatic erythrocyte, and the lower limit was

the size that could be distinguished.

Shape: A circle is the main form, but the following forms are also found: oval, crescent, doughnut, and

hemicycle etc.

Color: Should be same as the nucleus of an adjacent nucleated cell.

Outline: Should be clear because the micronucleus has the nucleus membrane.

Focus: Granules suggestive of micronuclei were confirmed by moving the focus up and down


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II-5 Numerical value processing and statistical methods

II-5-1 The handling of numerical values, and their display

All numerical data are presented depending on the precision of measuring apparatus.Body weights (grams as the unit) were measured to 1 digit of integer value and shown as such.

The frequency (% as the unit) of micronucleated polychromatic erythrocytes (MNPCE) was rounded off

to three decimal places, and shown as two decimal places.

The ratio (PCE/E) (% as the unit) of the number of polychromatic erythrocytes (PCE) to the total number

of erythrocytes (E) was rounded off to two decimal places, and shown as one decimal place.

II-5-2 Statistical processing

Statistical comparisons between control and treated groups of numerical data obtained for body weights

were conducted using the Bartletts test. If homogenous, the data were analyzed with respect to one-way

layout variance, and if significant differences were found the Dunnetts multiple comparison test was

applied. If not homogenous, the data were ranked through each group, and analyzed by Kruskal-Walliss

test, followed by a multiple comparison test of Dunnetts type when a significant difference was found

between groups.

For the frequency of micronucleated PCE (MNPCE/PCE), the data was determined by the method of

Kastenbaum and Bowman at a significance level of 1% (Re. 6). The ratio (PCE/E) of the number of

polychromatic erythrocytes (PCE) to the total number of erythrocytes (E) was analyzed with the Wilcoxon

rank sum test at a significance level of 5% (Ref. 7).

III. Results and discussion

The micronucleus test was conducted using rats killed on schedule in the study entitled 13-Week

Toxicity Study of 2-Ethoxy-2-methylpropane in F344 rats (Inhalation Study) [Preliminary Carcinogenicity

Study] (Study No. 0666). The rats were systemically exposed to air containing the test chemical ETBE

for 13 weeks (6 hours/day, 5 days/week). Animal husbandry and administration of the test chemical were

appropriately managed.

Although no mortality was found during the treatment period of 13 weeks, retardation of body weight

gain was noted in males exposed to 5000 ppm ETBE.

The micronucleus test was conducted for both sexes of controls and three treated groups exposed to 500,

1500 and 5000 ppm ETBE. Each group consisted of 10 rats of either sex. At sacrifice, the bone marrowcells were collected from the femur, smears were prepared, and then the ratio (MNPCE/PCE) of

micronucleated polychromatic erythrocytes (MNPCE) in polychromatic erythrocytes (PCE) and the ratio

(PCE/E) of PCE in the total number of erythrocytes (E) were calculated.

The results of micronucleus test are shown in Tables 1 and 2, and Figure 1.


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The frequencies of MNPCE/PCE in males were 0.140.06% in the control group, 0.160.07% in the 500

ppm ETBE group, 0.170.07% in the 1500 ppm ETBE group, and 0.180.10% in the 5000 ppm ETBE

group. Those in females were 0.160.13% in the control group, 0.180.08% in the 500 ppm ETBE group,

0.160.09% in the 1500 ppm ETBE group, and 0.160.08% in the 5000 ppm ETBE group. No

statistically significant increases as compared to control values, or dose-dependent tendencies for increase,in the frequencies of MNPCE/PCE were noted in either sex of the treated groups. In addition, no

significant changes in the PCE/E ratio were found in either sex of the treated groups.

IV Judgement of the results

It was considered that the highest level of ETBE was appropriately selected for this study, since

retardation of body weight gain was observed in males exposed to 5000 ppm ETBE.

As the values of the controls were within the background data (0.150.09%, N=97) for F344 rats in our

facility, it was evident that the study was well conducted. No statistically significant differences in the

ratios of MNPCE to PCE were observed in either sex of the treated groups, as compared to the control

values. In addition, no dose-dependent increased tendencies for MNPCE/PCE were found in either sex of

the treated groups.

Thus, from these results, ETBE was assessed to be negative on micronucleus induction for bone marrow

in rats exposed to ETBE (inhalation) for 13 weeks.

V Unforeseeable circumstances that might have affected the reliability of the study and

deviations from the protocol

No particulars require mention which might have affected the reliability of the study. In addition, no

particulars require mention which deviated from the protocol.

References

1) McLafferty FW, ed. 1994. Wiley Registry of Mass Spectral Data. 6th ed. New York, NY : John Wiley

and Sones.

2) Japan Bioassay Research Center. 2007. 13-Week toxicity study of 2-ethoxy-2-methylpropane in F344

rats (Drinking water study) [Preliminary carcinogenicity study]. Final Report. Kanagawa, Japan: Japan

Industrial Safety & Health Association, Japan Bioassay Research Center. (in Japanese)


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Study No. 7047

11

3) Japan Petroleum Energy Center (JPEC): Report on the Study of the Safety Tests of Ethyl tertiary

Butyl Ether (ETBE), fiscal 2006.

4) Abe M. 1986. Establishment of an "adequate stratification method" by analysis of changes in body

weight gain of rats and mice. Jpn. Pharmacol. Ther. 14: 7285-7302. (in Japanese)

5) The Micronucleus Test (ed. Makoto Hayashi), Scientist Inc., Tokyo (1991). (in Japanese)

6) Kastenbaum MA, Bowman KO. 1970. Table for determining the statistical significance of mutation

frequencies, Mutation Res. 9: 527-549.

7) Statistical Analysis of Toxicity and Pharmacology (ed. Kou Yoshimura), Scientist Inc., Tokyo (1987).

(in Japanese)


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E)

Study No.7047

Table 1 Results of Micronucleus test (Male) Inhalation study for 13 weeks

Test substance : 2-Ethoxy-2-methylpropane

GROUP NAME ANIMALID NO.

SLIDEID NO.

PCE/500E MNPCE/1000PC PCE/E MNPCE/PCE

a

a

a

a () (

0666- 1001 7047- 12 124 119 1 1 24.3 0.100666- 1002 7047- 80 121 123 3 1 24.4 0.200666- 1003 7047- 19 91 93 2 0 18.4 0.100666- 1004 7047- 16 113 97 1 0 21.0 0.05

Control Group 0666- 1005 7047- 66 131 104 0 1 23.5 0.050666- 1006 7047- 51 139 130 2 2 26.9 0.200666- 1007 7047- 75 114 118 3 0 23.2 0.150666- 1008 7047- 40 115 124 1 3 23.9 0.200666- 1009 7047- 58 130 137 1 2 26.7 0.150666- 1010 7047- 67 106 105 1 3 21.1 0.20

0.05 0.2 28 23.32.6e 0.140.06f

0666- 1301 7047- 28 144 145 2 2 28.9 0.200666- 1302 7047- 3 93 101 1 1 19.4 0.100666- 1303 7047- 43 130 148 1 1 27.8 0.100666- 1304 7047- 50 124 127 1 4 25.1 0.25

500 ppm Group 0666- 1305 7047- 56 117 117 2 1 23.4 0.150666- 1306 7047- 73 117 115 2 1 23.2 0.150666- 1307 7047- 49 153 131 2 3 28.4 0.250666- 1308 7047- 6 108 112 1 2 22.0 0.150666- 1309 7047- 17 101 114 2 2 21.5 0.200666- 1310 7047- 53 107 131 0 1 23.8 0.05

0.05 0.25 32 24.43.2 0.160.070666- 1401 7047- 55 122 107 2 2 22.9 0.200666- 1402 7047- 18 106 94 1 3 20.0 0.200666- 1403 7047- 35 161 141 2 1 30.2 0.150666- 1404 7047- 8 108 115 1 0 22.3 0.05

1500 ppm Group 0666- 1405 7047- 68 120 129 0 1 24.9 0.050666- 1406 7047- 48 100 96 2 2 19.6 0.200666- 1407 7047- 26 84 91 3 3 17.5 0.300666- 1408 7047- 38 136 151 2 1 28.7 0.150666- 1409 7047- 60 133 146 1 2 27.9 0.150666- 1410 7047- 15 114 100 3 1 21.4 0.20

0.05 0.30 33 23.54.3 0.170.070666- 1501 7047- 63 93 94 1 1 18.7 0.100666- 1502 7047- 54 106 101 1 3 20.7 0.200666- 1503 7047- 36 75 89 3 5 16.4 0.40

0666- 1504 7047- 41 122 124 2 2 24.6 0.205000 ppm Group 0666- 1505 7047- 20 103 94 2 1 19.7 0.150666- 1506 7047- 22 129 110 1 1 23.9 0.100666- 1507 7047- 1 146 160 0 3 30.6 0.150666- 1508 7047- 64 103 123 1 0 22.6 0.050666- 1509 7047- 72 124 123 3 3 24.7 0.300666- 1510 7047- 30 162 171 1 2 33.3 0.15

0.05 0.40 36 23.55.2 0.180.10

a Observation region on the slide.b) Minimum value of MNPCE/PCE (%) in each group.c) Maximum value of MNPCE/PCE (%) in each group.d) Total number of MNPCE observed 10000PCE in each group.e) AverageSD of PCE/E (%) in each group.f) AverageSD of MNPCE/PCE (%) in each group.

PCE : Polychromatic erythrocytes E : Erythrocytes (Polychromatic and Normochromatic erythrocytes) MNPCE : Micronucleated polychromatic erythrocytes

12


22/25

E)

Study No.7047

Table 1 Results of Micronucleus test (Female) -Continued- Inhalation study for 13 weeks

Test substance : 2-Ethoxy-2-methylpropane

GROUP NAME ANIMALID NO.

SLIDEID NO.

PCE/500E MNPCE/1000PC PCE/E MNPCE/PCE

a

a

a

a () (

0666- 2001 7047- 42 119 122 0 1 24.1 0.050666- 2002 7047- 57 90 101 0 3 19.1 0.150666- 2003 7047- 69 125 152 1 1 27.7 0.100666- 2004 7047- 44 121 124 0 4 24.5 0.20

Control Group 0666- 2005 7047- 76 121 118 1 1 23.9 0.100666- 2006 7047- 2 106 110 0 1 21.6 0.050666- 2007 7047- 79 119 127 5 3 24.6 0.400666- 2008 7047- 47 116 115 1 1 23.1 0.100666- 2009 7047- 10 135 132 3 4 26.7 0.350666- 2010 7047- 32 138 132 0 1 27.0 0.05

0.05 0.4c 31 24.22.6e 0.160.13

0666- 2301 7047- 70 112 118 1 1 23.0 0.100666- 2302 7047- 37 127 144 2 1 27.1 0.150666- 2303 7047- 77 118 111 0 2 22.9 0.100666- 2304 7047- 21 123 125 1 1 24.8 0.10

500 ppm Group 0666- 2305 7047- 61 122 146 1 3 26.8 0.200666- 2306 7047- 13 94 103 1 3 19.7 0.200666- 2307 7047- 46 125 106 4 3 23.1 0.350666- 2308 7047- 27 112 121 2 1 23.3 0.150666- 2309 7047- 24 102 98 1 2 20.0 0.150666- 2310 7047- 52 102 126 4 1 22.8 0.25

0.10 0.35 35 23.42.4 0.180.080666- 2401 7047- 34 124 123 3 2 24.7 0.250666- 2402 7047- 45 115 140 0 1 25.5 0.05

0666- 2403 7047- 31 149 139 2 1 28.8 0.150666- 2404 7047- 78 131 117 2 1 24.8 0.15

1500 ppm Group 0666- 2405 7047- 59 153 146 3 1 29.9 0.200666- 2406 7047- 39 67 78 2 4 14.5 0.300666- 2407 7047- 4 159 185 2 3 34.4 0.250666- 2408 7047- 65 137 120 1 2 25.7 0.150666- 2409 7047- 7 119 104 0 0 22.3 0.000666- 2410 7047- 71 97 110 2 0 20.7 0.10

0.00 0.30 32 25.15.4 0.160.090666- 2501 7047- 29 128 120 0 0 24.8 0.000666- 2502 7047- 11 111 129 3 2 24.0 0.250666- 2503 7047- 33 117 129 1 2 24.6 0.150666- 2504 7047- 9 116 128 2 2 24.4 0.20

5000 ppm Group 0666- 2505 7047- 14 107 123 1 0 23.0 0.050666- 2506 7047- 25 107 117 1 1 22.4 0.100666- 2507 7047- 5 133 128 1 3 26.1 0.200666- 2508 7047- 62 141 140 1 3 28.1 0.200666- 2509 7047- 74 115 112 3 1 22.7 0.200666- 2510 7047- 23 147 150 3 2 29.7 0.25

0.00 0.25 32 25.02.4 0.160.08

a Observation region on the slide.b) Minimum value of MNPCE/PCE (%) in each group.c) Maximum value of MNPCE/PCE (%) in each group.d) Total number of MNPCE observed 10000PCE in each group.e) AverageSD of PCE/E (%) in each group.f) AverageSD of MNPCE/PCE (%) in each group.

PCE : Polychromatic erythrocytes E : Erythrocytes (Polychromatic and Normochromatic erythrocytes) MNPCE : Micronucleated polychromatic erythrocytes

13


23/25

Table2

Resu

ltsofMicronucleustest(M

ale)

Inha

lationstudyfor13weeks

Testsubstance:2-Ethoxy-2-methylpropane

GROUPNAME

N

UMBER

OF

A

NIMALS

NUMBER

OF

OBSERVED

CELLS

(Cells/Animal)

Ratioof

PCE/E

(%)

Frequencies

of

MNPCE/PC

E

(%)

ControlGroup

10

2000

23

.32.6a

(18.4b26.9c)

0.140.06d

(0.05e

0.20f

28g)

500ppm

Group

10

2000

24

.43.2

(19.428.9

)

0.160.07

(0.05

0.25

32)

1500ppm

Group

10

2000

23

.54.3

(17.530.2)

0.170.07

(0.05

0.30

33)

5000ppm

Group

10

2000

23

.55.2

(16.433.3)

0.180.10

(0.05

0.40

36)

14

a)AverageSDofPCE/E

(%)

b)Minimum

valueofPC

E/E(%)

c)MaximumvalueofPC

E/E(%)

d)AverageSDofMNPC

E/PCE(%)

e)MinimumvalueofMN

PCE/PCE(%)

f)MaximumvalueofMN

PCE/PCE(%)

g)TotalnumberofMNP

CEobserved20000PCEineach

group

PCE:Polychroma

ticerythrocytes

E:Erythrocytes(PolychromaticandNormochrom

aticerythrocytytes)

MNPCE:Micronucleatedpolychromaticerythrocytes

Stud No.7047


24/25

Table2

ResultsofMicronucleustest(Female)

-Continued-

Inhalationstudyfor13weeks

Testsubstance:2-Ethoxy-2-methylpropane

GROUPNAME

N

UMBER

OF

A

NIMALS

NUMBER

OF

OBSERVED

CELLS

(Cells/Animal)

Ratioof

PCE/E

(%)

Frequencies

of

MNPCE/PC

E

(%)

ControlGroup

10

2000

24

.22.6a

(19.1b27.7c)

0.160.13d

(0.05e0

.40f

31g)

500ppm

Group

10

2000

23

.42.4

(19.7

27.1)

0.180.08

(0.100.35

35)

1500ppm

Group

10

2000

25

.15.4

(14.5

34.4)

0.160.09

(0

0

.30

32

)

5000ppm

Group

10

2000

25

.02.4

(22.4

29.7)

0.160.08

(0

0

.25

32

)

15

a)AverageSDofPCE/E

(%)

b)Minimum

valueofPC

E/E(%)

c)MaximumvalueofPC

E/E(%)

d)AverageSDofMNPC

E/PCE(%)

e)MinimumvalueofMN

PCE/PCE(%)

f)MaximumvalueofMN

PCE/PCE(%)

g)TotalnumberofMNP

CEobserved20000PCEineach

group

PCE:Polychroma

ticerythrocytes

E:Erythrocytes(PolychromaticandNormochrom

aticerythrocytytes)

MNPCE:Micronucleatedpolychromaticerythrocytes

Stud No.7047


25/25

Study No.7047

Fig. 1 Results of Micronucleus test

Inhalation study for 13 weeks

Test substance: 2-Ethoxy-2-methylpropane

Frequencies of Micronucleated PCE

0.0

0.2

0.4

0.6

0.8

1.0

0 1000 2000 3000 4000 5000

Dose (ppm)

Micronu

cleatedPCE(%)

Male

Female

Ratio of PCE

0

10

20

30

40

0 1000 2000 3000 4000 5000

Dose (ppm)

Ratioof

PCE

(%)

Male

Female

micronucleus test of etbe using bone marrow of rats.pdf

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