PROJECT ID OF CONTRACTING

INSTITUTE:

RCC PROJECT NUMBER 848629

RCC-CCR STUDY NUMBER 785202

IN VITRO

CHROMOSOME ABERRATION TEST

IN CHINESE HAMSTER V79 CELLS

WITH

DEA/ACID ANHYDRIDE REACTION PRODUCT

FINAL REPORT

Study Completion Date:

August 28, 2003
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1 COPY OF GLP CERTIFICATE
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2 CONTENTS

1 COPY OF GLP CERTIFICATE 2

2 CONTENTS 3

3 PREFACE 4

3.1 General 4

3.2 Responsibilities 4

3.3 Schedule 4

3.4 Project Staff Signatures 5

3.5 Good Laboratory Practice 5

3.6 Guidelines 5

3.7 Archiving 6

3.8 Deviations from the Study Plan 6

4 STATEMENT OF COMPLIANCE 7

5 STATEMENT OF QUALITY ASSURANCE UNIT 8

6 SUMMARY OF RESULTS 9

6.1 Conclusion 9

7 INTRODUCTION 11

7.1 Aims of the Study 11

8 MATERIALS AND METHODS 12

8.1 Test Item 12

8.2 Controls 13

8.3 Test System 13

8.4 Mammalian Microsomal Fraction S9 Mix 14

8.5 Range-finder 15

8.6 Dose Selection 15

8.7 Experimental Performance 16

8.8 Data Recording 18

8.9 Acceptability of the Test 19

8.10 Evaluation of Results 19

9 RESULTS AND DISCUSSION 20

10 DISTRIBUTION OF THE REPORT 21

11 REFERENCES 22

12 ANNEX I: TABLES OF RESULTS 23

12.1 Pre-Test on Toxicity 23

12.2 Experiment I and II: Determination of Toxicity 24

12.3 Experiment I 25

12.4 Experiment II 28

12.5 Biometry 31

13 ANNEX II: 32

13.1 Chromosome Aberrations: Classification and Criteria 32

14 ANNEX III: 33

14.1 Historical laboratory control data 33
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3 PREFACE

3.1 General

Title: In vitro Chromosome Aberration Test

in Chinese Hamster V79 Cells

with DEA/Acid anhydride reaction product

Sponsor: Rockwool International A/S

Hovedgaden 584

DK-2640 Hedehusene

Denmark

Study Monitor: Fleming A. Simonsen, DVM

Danish Toxicology Centre

2 Kogle Allé

DK-2970 Hoersholm

Denmark

Test Facility: RCC

CYTOTEST CELL RESEARCH GMBH

In den Leppsteinswiesen 19

D-64380 Rossdorf

Contracting Institute: RCC Ltd

CH-4452 Itingen

3.2 Responsibilities

Study Director: Dr. Markus Schulz

Deputy Study Director: Dr. Naveed Honarvar

Management: Dr. Wolfgang Völkner

Head of

Quality Assurance Unit: Frauke Hermann

3.3 Schedule

Date of Study Plan: May 05, 2003

Experimental

Starting Date: May 14, 2003

Experimental

Completion Date: July 04, 2003

Date of Draft Report: August 18, 2003

Date of Final Report: August 28, 2003

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3.7 Archiving

RCC Cytotest Cell Research will archive the following data for 15 years:

Raw data, study plan, and a copy of the final report.

A sample of the test item will be archived for at least 2 years following the date on which the

report is audited by the Quality Assurance Unit and also at least until the next inspection of RCC

Cytotest Cell Research by the GLP-authority.

Microscopic slides will be archived for at least 12 years.

No data will be discarded without the sponsor ́s consent.

3.8 Deviations from the Study Plan

a) General

Contracting Institute: RCC Ltd

CH-4452 Itingen

b) Good Laboratory Practice

”Chemikaliengesetz“ (Chemicals Act) of the Federal Republic of Germany, ”Anhang 1“

(Annex 1), dated July 25, 1994 (”BGBl. I 1994“, pp. 1703), last revision dated June 27, 2002.

Reason for the deviations (printed in bold letters): updating.

This deviation has no detrimental impact on the outcome of the study.


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6 SUMMARY OF RESULTS

The test item DEA/Acid anhydride reaction product, dissolved in culture medium (MEM), was

assessed for its potential to induce structural chromosome aberrations in V79 cells of the

Chinese hamster in vitro in two independent experiments. The following study design was

performed:

without S9 mix with S9 mix

exp. I exp. II exp. I exp. II

Exposure period 4 hrs 18 hrs 28 hrs 4 hrs 4 hrs

Recovery 14 hrs - - 14 hrs 24 hrs

Preparation interval 18 hrs 18 hrs 28 hrs 18 hrs 28 hrs

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates

were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (5000 μg/mL) was chosen with

respect to the current OECD Guideline 473. In all experimental parts at least the pH of the

highest test item concentration (5000 μg/mL) was adjusted to physiological values.

Dose selection of the cytogenetic experiments was performed considering the toxicity data. The

chosen treatment concentrations are described in Table 2 (page 16). The evaluated

experimental points and the results are summarised in Table 1 (page 10).

No toxic effects indicated by reduced cell numbers or mitotic indices were observed after

treatment with the test item.

In both independent experiments, no biologically relevant increase in the number of cells

carrying structural chromosomal aberrations was observed after treatment with the test item.

The observed dose-relation in experiment I in the presence of S9 mix and the statistical

significance in two test groups are regarded as being biologically irrelevant.

No increase in the frequencies of polyploid metaphases was found after treatment with the test

item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant

increases (p < 0.05) in cells with structural chromosome aberrations.

6.1 Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did

not induce structural chromosome aberrations as determined by the chromosome aberration

test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, DEA/Acid anhydride reaction product is considered to be non-clastogenic in this

chromosome aberration test with and without S9 mix when tested up to the highest required

concentration.
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Table 1: Summary of results of the chromosome aberration study

with DEA/Acid anhydride reaction product

Exp. Preparation Test item Polyploid Cell numbers Mitotic indices Aberrant cells

interval concentration cells in % in % in %

in μg/mL in % of control of control incl. gaps* excl. gaps* with

exchanges

Exposure period 4 hrs without S9 mix

I 18 hrs negative control1 3.2 100 100 2.5 1.5 0.0

positive control2 2.8 n.t. 91 23.0 20.5S 9.5

1250 3.2 90 143 2.5 2.0 0.5

2500 3.2 116 92 2.5 1.5 0.0

5000 3.7 99 106 1.5 1.0 0.0

Exposure period 18 hrs without S9 mix

II 18 hrs negative control1 2.1 100 100 1.5 0.0 0.0

positive control2 2.0 n.t. 83 14.5 10.0S 3.0

1250 1.3 118 97 2.0 2.0S 0.5

2500 1.5 91 84 2.5 0.5 0.0

5000 2.2 81 67 2.5 1.5 0.0

Exposure period 28 hrs without S9 mix

II 28 hrs negative control1 2.4 100 100 2.0 1.0 0.0

positive control2 0.9 n.t. 90 26.5 26.5S 11.0

5000 1.7 68 94 2.5 1.5 0.0

Exposure period 4 hrs with S9 mix

I 18 hrs negative control1 2.8 100 100 1.0 0.5 0.0

positive control3 2.5 n.t. 72 24.0 23.5S 7.0

1250 2.6 75 96 2.0 1.5 0.5

2500 2.8 97 104 3.0 2.0 0.5

5000 2.8 129 97 3.5 3.0S 1.0

II 28 hrs negative control1 2.8 100 100 3.5 2.5 0.0

positive control4 2.0 n.t. 76 11.0 10.5S 1.5

1250 1.2 79 103 2.5 2.5 0.0

2500 1.5 82 87 2.0 1.5 0.5

5000 0.9 88 96 3.5 3.0 1.5

* inclusive cells carrying exchanges

n.t. not tested

S aberration frequency statistically significant higher than corresponding control values 1 MEM (minimal essential medium) 2 EMS 200 μg/mL 3 CPA 0.7 μg/mL 4 CPA 1.0 μg/mL
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7 INTRODUCTION

According to national and international acts chemicals have to be tested before introduction to

the market for a possible hazard to humans and the environment. Genotoxicity studies provide

important information for the assessment of the mutagenic potential of these substances (1, 2).

The in vitro Chromosome Aberration Test performed in this study is an essential part of

genotoxicity test batteries of substances.

This in vitro test is a test for the detection of structural chromosomal aberrations. Such

aberrations are frequently lethal to the damaged cells (3, 4). However, cytogenetic damage in

somatic cells is an indicator of a potential to induce more subtle chromosomal damage that may

be compatible with cell division. Similar damage induced in germ cells may lead to heritable

cytogenetic abnormalities. Heritable cytogenetic abnormalities are known to have deleterious

effects in man, e.g. induction of neoplastic events or birth defects. Also, chromosome

abnormalities in somatic cells may become one of the reasons why a transformed cell

population develops, possibly leading to neoplastic processes.

Structural chromosomal aberrations are generally evaluated in first post treatment mitoses. With

the majority of chemical mutagens induced structural aberrations are of the chromatid type, but

chromosome type aberrations also occur.

For treatment, cell cultures should be in exponential growth to guarantee that there are cells in

all stages of the cell cycle (i.e. an asynchronous population). Since the normal cell cycle time is

12 hrs (see page 13) and the guidelines require fixation times of about 1.5-fold of the normal

cell cycle, a fixation time of around 18 hours is appropriate. Because there may be chemicals

which induce a very extensive mitotic delay at clastogenic concentrations or may be clastogenic

only when cells have passed through more than one cell cycle after the beginning of treatment

an additional later sampling time (28 hrs) should be included (5). Due to the limited capacity of

the V79 cells for metabolic activation of potential mutagens an exogenous metabolic activation

system is included (6).

The frequency of polyploid/endoreduplicated cells should also be noted so that the level in

control cultures can be monitored and their induction by the test item can be recorded,

especially when a late sampling time is used.

To validate the test reference mutagens were tested in parallel to the test item.

7.1 Aims of the Study

This in vitro test was performed to assess the potential of DEA/Acid anhydride reaction product

to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79

cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of

metabolic activation was performed in two independent experiments at one preparation interval

(18 hrs) in experiment I and at two preparation intervals (18 hrs and 28 hrs) in experiment II.
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8 MATERIALS AND METHODS

8.1 Test Item

Internal RCC-CCR Test Item Number: S 3859 11

The test item and the information concerning the test item were provided by the sponsor.

Identity: DEA/Acid anhydride reaction product

Batch No.: 200501.UN2810

Aggregate State at

Room Temperature: Liquid, highly viscous

Molecular Weight: Mean MW approx. 250-300

Colour: Amber

Purity: Not indicated by the sponsor

Stability in solvent: 180 days in water

Storage: At approx. 4° C

Expiration Date: September 2003

On the day of the experiment (immediately before treatment), the test item was dissolved in

culture medium (minimum essential medium: MEM). The solvent was chosen to its solubility

properties and its relative nontoxicity to the cell cultures. 

In the pre-experiment as well as in experiment I, at 2500 and 5000 μg/mL the pH was adjusted

to physiological values using small amounts of 2 N NaOH. Besides, in experiment II the pH of

the stock solutions (5000 μg/mL) was adjusted prior to the preparation of the dilution series by

using 2 N NaOH.
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8.2 Controls

8.2.1 Negative Controls

Concurrent negative controls (MEM) were performed.

8.2.2 Positive Control Substances

Without metabolic activation

Name: EMS; ethylmethane sulfonate

Supplier: ACROS ORGANICS, B-2440 Geel

Catalogue no.: 820774

Purity: ≥ 98 %

Lot no.: A015053301

Expiration Date: March 2005

Dissolved in: nutrient medium

Final Concentration: 200 μg/mL (1.6 mM)

Solutions were prepared on the day of experiment. The stability of the positive control

substance in solution was proven by the mutagenic response in the expected range.

With metabolic activation

Name: CPA; cyclophosphamide

Supplier: Aldrich Chemie, D-89555 Steinheim

Catalogue no.: 21.870-7

Purity: ≥ 98 %

Lot no.: 91K1176

Expiration Date: March 2005

Dissolved in: nutrient medium

Final Concentration: 0.7 – 1.0 μg/mL (2.5 – 3.5 μM)

Solutions were prepared on the day of experiment. The stability of CPA in solution at room

temperature is good. At 25° C only 3.5 % of its potency is lost after 24 hours (7).

8.3 Test System

8.3.1 Reasons for the Choice of the Cell Line V79

The V79 cell line has been used successfully for many years in in vitro experiments. Especially

the high proliferation rate (doubling time of clone V79/D3 in stock cultures: 12 hrs, determined

on January 13, 2003) and a reasonable plating efficiency of untreated cells (as a rule more than

70 %) both necessary for the appropriate performance of the study, recommend the use of this

cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Lacking metabolic activities of cells under in vitro conditions are a disadvantage of tests with cell

cultures as many chemicals only develop a mutagenic potential when they are metabolized by

the mammalian organism. However, metabolic activation of chemicals can be achieved at least

partially by supplementing the cell cultures with liver microsome preparations (S9 mix).
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8.3.2 Cell Cultures

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP,

Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell

bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture

batch in experiments. Before freezing each batch was screened for mycoplasm contamination

and checked for karyotype stability. Consequently, the parameters of the experiments remain

similar because of standardized characteristics of the cells.

Thawed stock cultures were propagated at 37° C in 80 cm2 plastic flasks (GREINER, D-72632

Frickenhausen). About 5 x 105

 cells per flask were seeded into 15 mL of MEM (Minimal

Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS;

PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell

cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 %

air).

8.4 Mammalian Microsomal Fraction S9 Mix

8.4.1 S9 (Preparation by RCC Cytotest Cell Research)

Phenobarbital/β-Naphthoflavone induced rat liver S9 was used as the metabolic activation

system. The S9 was prepared from 8 - 12 weeks old male Wistar HanIbm rats, weight approx.

220 - 320 g (supplied from RCC Ltd; Biotechnology & Animal Breeding Division, CH-4414

Füllinsdorf) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335

Hamburg) and β-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive

days. The livers were prepared 24 hours after the last treatment. The S9 fractions were

produced by dilution of the liver homogenate with a KCl solution (1:3 parts respectively)

followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in

ampoules at -80° C. Small numbers of the ampoules were kept at -20°C for up to one week.

The protein concentration was 26.2 mg/mL (Lot. no. 070303) in the pre-test and in experiment I

and II.

8.4.2 S9 Mix

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to

result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the

S9 mix to reach the following concentrations:

8 mM MgCl2

33 mM KCl

5 mM glucose-6-phosphate

4 mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was

performed according to Ames et al. (1).
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8.5 Range-finder

A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to

determine the toxicity of the test item (8). Cytotoxicity was determined using concentrations

separated by no more than a factor of 2 - √10. The general experimental conditions in this pretest were the same as described below for the cytogenetic main experiment. The following

method was used:

In a quantitative assessment, exponentially growing cell cultures (seeding 45,840 cells/slide,

with regard to the culture time 48 hrs) were treated with the test item for simulating the

conditions of the main experiment. A qualitative evaluation of cell number and cell morphology

was made 4 hrs and 24 hrs after start of treatment. 24 hrs after start of treatment the cells were

stained. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate

defined fields of the slides (2 slides per treatment group). The cell number of the treatment

groups is given as % cells in relation to the control.

8.6 Dose Selection

The highest concentration used in the cytogenetic experiments was chosen with regard to the

current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top

concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control,

whichever is the lowest concentration, and/or the occurrence of precipitation. In case of

nontoxicity the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 mM, whichever is the

lowest, if formulability in an appropriate solvent is possible.

5000 μg/mL of DEA/Acid anhydride reaction product were applied as top concentration for

treatment of the cultures in the pre-test. Test item concentrations between 39.1 and 5000 μg/mL

(with and without S9 mix) were chosen for the evaluation of cytotoxicity.

Using reduced cell numbers as an indicator for toxicity in the pre-test, no toxic effects were

observed after 4 hrs treatment up to the highest test item concentration. Considering the toxicity

data of the pre-test, 5000 μg/mL (with and without S9 mix) were chosen as top concentration in

experiment I.

Dose selection of experiment II was also based on toxicity data. In the range finding experiment

no reduced cell numbers were observed after 24 hrs exposure up to the highest test item

concentration. Therefore, 5000 μg/mL were chosen as top treatment concentration for

continuous exposure in the absence of S9 mix. In the presence of S9 mix 5000 μg/mL were

chosen as top treatment concentration with respect to the results obtained in experiment I. 

The applied concentrations in the cytogenetic experiments are presented in Table 2, page 16.
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Table 2: Doses applied in the Chromosome aberration test with 

DEA/Acid anhydride reaction product

Preparation Exposure Exp. Concentration

interval period in μg/mL

without S9 mix

18 hrs 4 hrs I 156.3 312.5 625.0 1250.0 2500.0 5000.0

18 hrs 18 hrs II 156.3 312.5 625.0 1250.0 2500.0 5000.0

28 hrs 28 hrs II 625.0 1250.0 2500.0 5000.0

with S9 mix

18 hrs 4 hrs I 156.3 312.5 625.0 1250.0 2500.0 5000.0

28 hrs 4 hrs II 156.3 312.5 625.0 1250.0 2500.0 5000.0

evaluated experimental points were printed in bold characters

8.7 Experimental Performance

8.7.1 Schedule

without S9 mix with S9 mix

exp. I exp. II exp. I exp. II

Exposure period 4 hrs 18 hrs 28 hrs 4 hrs 4 hrs

Recovery 14 hrs - - 14 hrs 24 hrs

Preparation interval 18 hrs 18 hrs 28 hrs 18 hrs 28 hrs

8.7.2 Seeding of the Cultures

Exponentially growing stock cultures more than 50 % confluent are treated with trypsin-EDTAsolution at 37° C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding

complete culture medium and a single cell suspension is prepared. The trypsin concentration for

all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution (Invitrogen GIBCO, D-76131

Karlsruhe).
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Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution.

The Ca-Mg-free salt solution is composed as follows (per litre):

NaCl 8000 mg

KCl 200 mg

KH2P04 200 mg

Na2HP04 150 mg

The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained

microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 104

 -

 6 x 104 cells were seeded with regard to the preparation time. The medium was MEM with 10 %

FCS (complete medium).

8.7.3 Treatment

Exposure period 4 hours

The culture medium of exponentially growing cell cultures was replaced with serum-free

medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with

10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 μL

S9 mix per mL culture medium were added. Concurrent negative and positive controls were

performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were

cultured in complete medium for the remaining culture time.

The "Saline G" solution was composed as follows (per litre):

NaCl 8000 mg

KCl 400 mg

Glucose 1100 mg

Na2HP04x7H20 290 mg

KH2P04 150 mg

pH was adjusted to 7.2

Exposure period 18 and 28 hours

The culture medium of exponentially growing cell cultures was replaced with complete medium

(with 10 % FCS) containing different concentrations of the test item without S9 mix. The

medium was not changed until preparation of the cells.

All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
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8.7.4 Preparation of the Cultures

15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added

(0.2 μg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in

the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the

hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid

(3:1 parts respectively). Per experiment two slides per group were prepared. After preparation

the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).

8.7.5 Evaluation of Cell Numbers

For evaluation of cytotoxicity indicated by reduced cell numbers additional two cultures per test

item and negative control group, not treated with colcemid, were set up in parallel. These

cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically

the cell number within 10 defined fields per coded slide. The cell number of the treatment

groups is given in percentage compared to the respective negative control.

8.7.6 Analysis of Metaphase Cells

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe

der Industrie, Cytogenetik" [9]) using NIKON microscopes with 100x oil immersion objectives.

Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as

structural chromosome aberrations. Gaps were recorded as well but not included in the

calculation of the aberration rates. 100 well spread metaphase plates per culture were scored

for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome

numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index

(% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase

cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line

polyploid means a near tetraploid karyotype).

8.8 Data Recording

The data generated were recorded in the raw data file. The results are presented in tabular

form, including experimental groups with the test item, negative, and positive controls.
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8.9 Acceptability of the Test

The chromosome aberration test performed in our laboratory is considered acceptable if it

meets the following criteria:

a) The number of structural aberrations found in the negative and/or solvent controls falls within

the range of our historical laboratory control data: 0.0 - 4.0 %.

b) The positive control substances should produce significant increases in the number of cells

with structural chromosome aberrations, which are within the range of the laboratories

historical control data:

Test group

Final concentration

Aberrant cells in %

(excl. gaps)

range

Test group

Final concentration

Aberrant cells in %

(excl. gaps)

range

without S9 mix with S9 mix

EMS 100 – 1000 μg/mL 8.0 - 100.0 CPA 0.7 – 1.0 μg/mL 7.5 - 95.5

8.10 Evaluation of Results

A test item is classified as non-clastogenic if:

− the number of induced structural chromosome aberrations in all evaluated dose groups is in

the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).

and/or

− no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:

− the number of induced structural chromosome aberrations is not in the range of our historical

control data (0.0 - 4.0 % aberrant cells, exclusive gaps).

and

− either a concentration-related or a significant increase of the number of structural

chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher ́s exact test (10) (p < 0.05).

However, both biological and statistical significance should be considered together. If the

criteria mentioned above for the test item are not clearly met, the classification with regard to

the historical data and the biological relevance is discussed and/or a confirmatory experiment is

performed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it

is important to include the polyploids and endoreduplications. The following criteria is valid:

A test item can be classified as aneugenic if:

− the number of induced numerical aberrations is not in the range of our historical control data

(0.0 - 8.5 % polyploid cells).
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9 RESULTS AND DISCUSSION

The test item DEA/Acid anhydride reaction product, dissolved in culture medium (MEM), was

assessed for its potential to induce structural chromosome aberrations in V79 cells of the

Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In experiment I, the exposure period was 4 hrs

with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9

mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and

II) and 28 hrs (exp. II) after start of treatment with the test item.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates

were scored for structural chromosome aberrations.

In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as

an indicator for cytotoxicity. Concentrations between 39.1 and 5000 μg/mL were applied. No

clear toxic effects were observed after treatment up to the highest test item concentration (see

Table 4, page 23).

In the pre-experiment, neither precipitation nor relevant influence of the test item on the

osmolarity was observed (negative control 320 mOsm versus 348 mOsm at 5000 μg/mL). Due

to a slight pH shift observed the top concentrations 2500 and 5000 μg/mL were adjusted with

2 N NaOH to physiological values (negative control pH 7.4 versus pH 7.3 at 5000 μg/mL).

Table 3: Evaluated experimental points after treatment with

DEA/Acid anhydride reaction product

Exp. Preparation

interval

Exposure

period

Concentration

in μg/mL

Preparation

interval

Exposure

period

Concentration

in μg/mL

without S9 mix with S9 mix

I 18 hrs 4 hrs 1250 2500 5000 18 hrs 4 hrs 1250 2500 5000

II 18 hrs 18 hrs 1250 2500 5000

II 28 hrs 28 hrs 5000 28 hrs 4 hrs 1250 2500 5000

Similar to the pre-experiment, in both main experiments with and without metabolic activation

the pH of at least the stock solutions was adjusted with 2 N NaOH to physiological values.

No cytotoxicity indicated by reduced cell numbers or mitotic indices were observed after

treatment with the test item (see Table 5, 6, and 9, page 24, 25, and 28).

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in

the number of cells carrying structural chromosome aberrations was observed (see Tabel 7 – 8

and Table 10 - 11, page 26, 27, 29, and 30). The aberration rates of the cells after treatment

with the test item (0.5 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the

negative control values (0.0 - 2.5 % aberrant cells, exclusive gaps) and within the range of our

historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

Significant (p < 0.05; page 31) increases were observed in experiment I at preparation interval

18 hrs after treatment with 5000 μg/mL in the presence of S9 mix and in experiment II after

18 hrs continuous treatment with 1250 μg/mL in the absence of S9 mix. Although these

increases of 3.0 % and 2.0 % aberrant cells, respectively, were statistically significant compared

to the low response (0.0 % and 0.5 % aberrant cells, respectively) in the negative control data,
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the responses are within the historical control data range (0.0 - 4.0 % aberrant cells).

Additionally, in experiment I in the presence of S9 mix at preparation interval 18 hrs a doserelated increase in the number of cells carrying structural chromosome aberrations (1.5, 2.0,

and 3.0 %) was observed in the concentration range evaluated (1250 to 5000 μg/mL). However,

these values were within the historical control data range and, additionally, the dose-related

increase could not be confirmed in experiment II in the presence of S9 mix. Therefore, the doserelation and the statistical significances have to be regarded as being biologically irrelevant.

Table 6 and 9, page 25 and 28, show the occurrence of polyploid metaphases. In both

experiments, no biologically relevant increase in the rate of polyploid metaphases was found

after treatment with the test item (0.9 - 3.7 %) as compared to the rates of the negative controls

(2.1 - 3.2 %).

In both experiments, EMS (200 μg/mL) and CPA (0.7 and 1.0 μg/mL, respectively) were used

as positive controls and showed distinct increases in cells with structural chromosome

aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item

DEA/Acid anhydride reaction product did not induce structural chromosome aberrations in V79

cells (Chinese hamster cell line) when tested up to the highest required concentration.

10 DISTRIBUTION OF THE REPORT

Sponsor 2x (1x original, 1x copy)

Study Director 1x (copy)
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11 REFERENCES

1. Ames, B.N., McCann, J. and Yamasaki, E. (1977)

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome

mutagenicity test. Kilbey, B.J. et al. (eds.). "Handbook of Mutagenicity Test Procedures",

Elsevier, Amsterdam, 1-17

2. Bradley, M.O., Bhuyan, B., Francis, M.C., Langenbach, R., Peterson, A. and Huberman, E. (1981)

Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature.

A report of the gene-tox program

Mutation Res., 87, 81-142

3. Preston, R.J.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.H., McFee, A.F., Wolff

S. and Wassom, J.S. (1981)

Mammalian in vivo and in vitro cytogenetic tests. A report of the U.S. EPA`s gene-tox program.

Mutation Res., 87, 143-188

4. Swierenga, S.H.H., Heddle, J.A., Sigal, E.A., Gilman, J.P.W., Brillinger, R.L., Douglas G.R. and

Nestmann, E.R. (1991)

Recommended protocols based on the survey of current practice in genotoxicity testing laboratories,

IV: Chromosome aberration and sister chromatid exchange in Chinese hamster ovary, V79 Chinese

hamster lung and human lymphocyte cultures.

Mutation Res., 246, 301-322

5. Bean, C.L., Armstrong, M.J. and Galloway, S.M. (1992)

Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary

cells.

Mutation Res., 265, 31-44

6. Glatt, H.R. (1993)

Fremdstoffmetabolismus und Mutagenitätsprüfung, in: Fahrig, R. (ed.): Mutationsforschung und

genetische Toxikologie, Wiss. Buchgesellschaft, Darmstadt, 169-185

7. Gallelli, J.F. (1967)

Am. J. Hosp. Pharm., 24, 425. Cited from: The Extra Pharmacopeas (29th edition)

8. Armstrong, M.J., Bean, C.L. and Galloway, S.M. (1992)

A quantitative assessment of the cytotoxicity associated with chromosome aberration detection in

Chinese hamster ovary cells.

Mutation Res., 265, 45-60

9. Engelhardt, G. (1987)

"Arbeitsgruppe der Industrie, Cytogenetik". Standard-Protokoll zur cytogenetischen Auswertung von

Mitose- und Meiosechromosomen bei der Routineuntersuchung.

10. Richardson, C., et al (1989)

Analysis of data from in vitro cytogenetic tests

Kirkland, D.J. (ed.). ”Statistical evaluation of mutagenicity test data“, Cambridge University Press,

Cambridge, 141-154

Supplementary References:

Kirkland, D.J. (1992)

Chromosome aberration tests in vitro: problems with protocol design and interpretation of results.

Mutagenesis, 2, 95-106
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12 ANNEX I: TABLES OF RESULTS

12.1 Pre-Test on Toxicity

In the pre-test the toxicity of the test item was examined using the determination of the cell

number. Cell numbers of two cultures (10 coordinate defined fields per culture) were determined

for each experimental group.

Table 4: Cytotoxicity of DEA/Acid anhydride reaction product to cultures of Chinese hamster cell

line V79.

without S9 mix, 4 hrs exposure with S9 mix, 4 hrs exposure

Concentration

in μg/mL 

Number of

cells

% of

negative control

Concentration

in μg/mL 

Number of

cells

% of

negative control

Negative control 942 100 Negative control 809 100

39.1 747 79 39.1 849 105

78.1 960 102 78.1 771 95

156.3 705 75 156.3 835 103

312.5 827 88 312.5 738 91

625.0 805 85 625.0 635 79

1250.0 667 71 1250.0 692 86

2500.0 652 69 2500.0 489 60

5000.0 780 83 5000.0 639 79

without S9 mix; 24 hrs exposure

Concentration

in μg/mL 

Number of

cells

% of

negative control

Negative control 1051 100

39.1 854 81

78.1 648 62

156.3 889 85

312.5 712 68

625.0 853 81

1250.0 671 64

2500.0 729 69

5000.0 715 68
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12.2 Experiment I and II: Determination of Toxicity

The toxicity of the test item was examined using the determination of the cell number. Cell

numbers of two cultures (10 coordinate defined fields per culture) were determined for each

experimental group, except the positive control.

Table 5: Number of cells in % of negative control

without metabolic activation (S9 mix)

Experiment I: 4 hrs exposure Experiment II: continuous exposure

Preparation

interval

Concentration

in μg/mL

Cells in % of

negative control

Preparation

interval

Concentration

in μg/mL

Cells in % of

negative

control

18 hrs 156.3 76 18 hrs 156.3 136

" 312.5 99 " 312.5 129

" 625.0 92 " 625.0 147

" 1250.0 90 " 1250.0 118

" 2500.0 116 " 2500.0 91

" 5000.0 99 " 5000.0 81

28 hrs 625.0 98

" 1250.0 72

" 2500.0 75

" 5000.0 68

with metabolic activation (S9 mix)

Experiment I: 4 hrs exposure Experiment II: 4 hrs exposure

Preparation

interval

Concentration

in μg/mL

Cells in % of

negative control

Preparation

interval

Concentration

in μg/mL

Cells in % of

negative

control

18 hrs 156.3 73 28 hrs 156.3 101

" 312.5 96 " 312.5 103

" 625.0 85 " 625.0 107

" 1250.0 75 " 1250.0 79

" 2500.0 97 " 2500.0 82

" 5000.0 129 " 5000.0 88

experimental groups evaluated for cytogenetic damage were printed in bold characters
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12.3 Experiment I

Table 6: Number of polyploid cells and mitotic index;

preparation interval 18 hrs with and without S9 mix

Treatment Conc. S9 Exposure Polyploid cells* Mitotic indices**

group per mL mix period/ culture total % absolute mean %***

Recovery 1 2 1 2

Neg. control# - 4 / 14 hrs 17 15 32 3.2 11.5 13.7 12.6 100.0

Pos. control## 200 μg - 4 / 14 hrs 17 11 28 2.8 12.9 10.0 11.5 90.9

Test item 1250 μg - 4 / 14 hrs 20 12 32 3.2 17.0 19.0 18.0 142.9

′′ 2500 μg - 4 / 14 hrs 16 16 32 3.2 14.0 9.2 11.6 92.1

′′ 5000 μg - 4 / 14 hrs 18 19 37 3.7 12.6 14.2 13.4 106.3

Neg. control# + 4 / 14 hrs 14 14 28 2.8 15.7 16.4 16.1 100.0

Pos. control### 0.7 μg + 4 / 14 hrs 8 17 25 2.5 9.8 13.3 11.6 72.0

Test item 1250 μg + 4 / 14 hrs 13 13 26 2.6 14.0 16.9 15.5 96.3

′′ 2500 μg + 4 / 14 hrs 16 12 28 2.8 16.2 17.3 16.8 104.4

′′ 5000 μg + 4 / 14 hrs 18 10 28 2.8 12.7 18.4 15.6 96.9

* The number of polyploid cells was determined of each test group in a sample of 500 cells per culture

** The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

*** For the positive control groups and the test item groups, the relative values of the mitotic index are

related to the negative controls

# MEM (minimal essential medium)

## EMS

### CPA
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Table 7: Structural chromosome aberrations Experiment I;

preparation interval 18 hrs without S9 mix: exposure period 4 hrs

Slide Cells % Aberrant cells Aberrations

no. scored incl. excl. with ex- gaps chromatid type chromosome type other

gaps gaps* changes g ig b f d ex ib if id cx ma cd

without S9 mix

Negative control

1 100 2 0 1 0 0 0 0 0 0 0 0 0

2 100 0 0 0 2 0 0 0 0 0 0 0 0

1 + 2 200 2.5 1.5 0.0 2 0 1 2 0 0 0 0 0 0 0 0

Positive control: EMS 200 μg / mL

1 100 5 0 14 2 1 11 6 4 0 0 0 0

2 100 5 0 14 0 0 9 2 0 0 0 0 0

1 + 2 200 23.0 20.5 9.5 10 0 28 2 1 20 8 4 0 0 0 0

Test item: 1250 μg / mL

1 100 0 1 0 0 0 0 0 0 0 0 0 0

2 100 0 0 2 0 0 0 1 0 0 1 0 0

1 + 2 200 2.5 2.0 0.5 0 1 2 0 0 0 1 0 0 1 0 0

Test item: 2500 μg / mL

1 100 1 0 1 2 0 0 0 0 0 0 0 0

2 100 1 0 0 0 0 0 0 0 0 0 0 0

1 + 2 200 2.5 1.5 0.0 2 0 1 2 0 0 0 0 0 0 0 0

Test item: 5000 μg / mL

1 100 1 0 0 0 0 0 1 0 0 0 0 0

2 100 0 0 1 0 0 0 0 0 0 0 0 0

1 + 2 200 1.5 1.0 0.0 1 0 1 0 0 0 1 0 0 0 0 0

* inclusive cells carrying exchanges

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a

minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = isofragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell

[excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal

disintegration (= pulverization)
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Table 8: Structural chromosome aberrations Experiment I;

preparation interval 18 hrs with S9 mix: exposure period 4 hrs

Slide Cells % Aberrant cells Aberrations

no. scored incl. excl. with ex- gaps chromatid type chromosome type other

gaps gaps* changes g ig b f d ex ib if id cx ma cd

with S9 mix

Negative control

1 100 0 0 2 0 0 0 0 0 0 0 0 0

2 100 1 0 0 0 0 0 0 0 0 0 0 0

1 + 2 200 1.0 0.5 0.0 1 0 2 0 0 0 0 0 0 0 0 0

Positive control: CPA 0.7 μg / mL

1 100 3 0 17 4 0 6 16 1 0 0 0 0

2 100 0 0 9 1 0 10 6 2 0 0 0 0

1 + 2 200 24.0 23.5 7.0 3 0 26 5 0 16 22 3 0 0 0 0

Test item: 1250 μg / mL

1 100 0 0 0 0 0 1 1 1 0 0 0 0

2 100 1 0 0 0 0 0 0 0 0 0 0 0

1 + 2 200 2.0 1.5 0.5 1 0 0 0 0 1 1 1 0 0 0 0

Test item: 2500 μg / mL

1 100 3 0 0 1 0 1 1 1 0 0 1 0

2 100 1 0 0 0 0 0 0 0 0 0 0 0

1 + 2 200 3.0 2.0 0.5 4 0 0 1 0 1 1 1 0 0 1 0

Test item: 5000 μg / mL

1 100 1 0 2 0 0 0 0 0 0 0 1 0

2 100 0 0 2 0 0 2 0 0 0 0 0 0

1 + 2 200 3.5 3.0 1.0 1 0 4 0 0 2 0 0 0 0 1 0

* inclusive cells carrying exchanges

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a

minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = isofragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell

[excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal

disintegration (= pulverization)
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12.4 Experiment II

Table 9: Number of polyploid cells and mitotic index;

preparation interval 18 and 28 hrs without S9 mix;

preparation interval 28 hrs with S9 mix

Treatment Conc. S9 Exposure Polyploid cells* Mitotic indices**

group per mL mix period/ culture total % absolute mean %***

Recovery 1 2 1 2

Neg. control# - 18 / - hrs 10 11 21 2.1 15.0 14.6 14.8 100.0

Pos. control## 200 μg - 18 / - hrs 9 11 20 2.0 13.7 11.0 12.4 83.4

Test item 1250 μg - 18 / - hrs 4 9 13 1.3 12.7 16.1 14.4 97.3

′′ 2500 μg - 18 / - hrs 8 7 15 1.5 9.8 15.0 12.4 83.8

′′ 5000 μg - 18 / - hrs 9 13 22 2.2 9.9 10.0 10.0 67.2

Neg. control# - 28 / - hrs 5 19 24 2.4 17.7 16.0 16.9 100.0

Pos. control## 200 μg - 28 / - hrs 7 2 9 0.9 17.2 13.2 15.2 90.2

Test item 5000 μg - 28 / - hrs 6 11 17 1.7 16.5 15.1 15.8 93.8

Neg. control# + 4 / 24 hrs 15 13 28 2.8 17.5 20.4 19.0 100.0

Pos. control### 1 μg + 4 / 24 hrs 12 8 20 2.0 14.5 14.3 14.4 76.0

Test item 1250 μg + 4 / 24 hrs 4 8 12 1.2 19.9 19.0 19.5 102.6

′′ 2500 μg + 4 / 24 hrs 8 7 15 1.5 17.6 15.5 16.6 87.3

′′ 5000 μg + 4 / 24 hrs 5 4 9 0.9 18.6 17.8 18.2 96.0

* The number of polyploid cells was determined of each test group in a sample of 500 cells per culture

** The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

*** For the positive control groups and the test item groups, the relative values of the mitotic index are

related to the negative controls

# MEM (minimal essential medium)

## EMS

### CPA
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Table 10: Structural chromosome aberrations Experiment II;

preparation interval 18 hrs without S9 mix: exposure period 18 hrs

Slide Cells % Aberrant cells Aberrations

no. scored incl. excl. with ex- gaps chromatid type chromosome type other

gaps gaps* changes g ig b f d ex ib if id cx ma cd

without S9 mix

Negative control

1 100 4 0 0 0 0 0 0 0 0 0 0 0

2 100 0 0 0 0 0 0 0 0 0 0 0 0

1 + 2 200 1.5 0.0 0.0 4 0 0 0 0 0 0 0 0 0 0 0

Positive control: EMS 200 μg / mL

1 100 11 1 12 0 0 1 1 1 0 0 0 0

2 100 4 0 3 0 0 5 1 0 0 0 0 0

1 + 2 200 14.5 10.0 3.0 15 1 15 0 0 6 2 1 0 0 0 0

Test item: 1250 μg / mL

1 100 0 0 2 0 0 0 0 0 0 0 0 0

2 100 0 0 0 0 0 1 1 0 0 0 0 0

1 + 2 200 2.0 2.0 0.5 0 0 2 0 0 1 1 0 0 0 0 0

Test item: 2500 μg / mL

1 100 2 1 1 0 0 0 0 0 0 0 0 0

2 100 1 0 0 0 0 0 0 0 0 0 0 0

1 + 2 200 2.5 0.5 0.0 3 1 1 0 0 0 0 0 0 0 0 0

Test item: 5000 μg / mL

1 100 1 0 1 0 0 0 1 0 0 0 0 0

2 100 1 0 0 0 0 0 1 0 0 0 0 0

1 + 2 200 2.5 1.5 0.0 2 0 1 0 0 0 2 0 0 0 0 0

* inclusive cells carrying exchanges

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a

minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = isofragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell

[excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal

disintegration (= pulverization)
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Table 11: Structural chromosome aberrations Experiment II;

preparation interval 28 hrs without S9 mix: exposure period 28 hrs;

preparation interval 28 hrs with S9 mix: exposure period 4 hrs

Slide Cells % Aberrant cells Aberrations

no. scored incl. excl. with ex- gaps chromatid type chromosome type other

gaps gaps* changes g ig b f d ex ib if id cx ma cd

without S9 mix

Negative control

1 100 1 0 1 0 0 0 0 0 0 0 0 0

2 100 1 0 0 1 0 0 0 0 0 0 0 0

1 + 2 200 2.0 1.0 0.0 2 0 1 1 0 0 0 0 0 0 0 0

Positive control: EMS 200 μg / mL

1 100 0 0 6 3 0 14 4 2 0 0 5 0

2 100 3 0 11 5 0 14 4 2 0 0 3 0

1 + 2 200 26.5 26.5 11.0 3 0 17 8 0 28 8 4 0 0 8 0

Test item: 5000 μg / mL

1 100 1 0 1 1 0 0 0 1 0 0 0 0

2 100 0 1 0 0 0 0 0 0 0 0 0 0

1 + 2 200 2.5 1.5 0.0 1 1 1 1 0 0 0 1 0 0 0 0

with S9 mix

Negative control

1 100 1 0 1 0 0 0 0 0 0 0 0 0

2 100 2 0 2 0 0 0 0 1 1 0 0 0

1 + 2 200 3.5 2.5 0.0 3 0 3 0 0 0 0 1 1 0 0 0

Positive control: CPA 1 μg / mL

1 100 0 0 3 1 0 1 1 5 0 0 0 0

2 100 1 0 3 1 0 2 4 4 0 0 0 0

1 + 2 200 11.0 10.5 1.5 1 0 6 2 0 3 5 9 0 0 0 0

Test item: 1250 μg / mL

1 100 0 0 0 1 0 0 1 2 0 0 0 0

2 100 0 0 0 0 0 0 0 0 0 0 1 0

1 + 2 200 2.5 2.5 0.0 0 0 0 1 0 0 1 2 0 0 1 0

Test item: 2500 μg / mL

1 100 1 2 1 0 0 0 0 1 0 0 0 0

2 100 0 0 0 0 0 1 0 0 0 0 0 0

1 + 2 200 2.0 1.5 0.5 1 2 1 0 0 1 0 1 0 0 0 0

Test item: 5000 μg / mL

1 100 1 0 1 0 0 0 0 6 0 1 0 0

2 100 0 0 0 0 0 2 0 0 0 0 0 0

1 + 2 200 3.5 3.0 1.5 1 0 1 0 0 2 0 6 0 1 0 0

* inclusive cells carrying exchanges

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a

minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = isofragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell

[excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal

disintegration (= pulverization)
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12.5 Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the

Fisher ́s exact test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Table 12: Biometry of Experiment I

Test group versus

negative control

Preparation

interval

Exposure

period

S9 mix p-value

Test group 1250 μg/mL 18 hrs 4 hrs - 0.362

" 2500 μg/mL 18 hrs 4 hrs - n.c.

" 5000 μg/mL 18 hrs 4 hrs - n.c.

" 1250 μg/mL 18 hrs 4 hrs + 0.187

" 2500 μg/mL 18 hrs 4 hrs + 0.108

" 5000 μg/mL 18 hrs 4 hrs + 0.034S

Positive control versus negative

control

EMS 200 μg/mL 18 hrs 4 hrs - < 0.001S

CPA 0.7 μg/mL 18 hrs 4 hrs + < 0.001S

Table 13: Biometry of Experiment II

Test group versus

negative control

Preparation

interval

Exposure

period

S9 mix p-value

Test group 1250 μg/mL 18 hrs 18 hrs - 0.031S

" 2500 μg/mL 18 hrs 18 hrs - 0.250

" 5000 μg/mL 18 hrs 18 hrs - 0.062

" 5000 μg/mL 28 hrs 28 hrs - 0.343

" 1250 μg/mL 28 hrs 4 hrs + n.c.

" 2500 μg/mL 28 hrs 4 hrs + n.c.

" 5000 μg/mL 28 hrs 4 hrs + 0.386

Positive control versus

negative control

EMS 200 μg/mL 18 hrs 18 hrs - < 0.001S

EMS 200 μg/mL 28 hrs 28 hrs - < 0.001S

CPA 1.0 μg/mL 28 hrs 4 hrs + 0.001S

n.c. not calculated as the aberration rate is equal or lower than the control rate

S aberration rate is statistically significant higher than the control rate
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13 ANNEX II:

13.1 Chromosome Aberrations: Classification and Criteria

1. Gaps

Gaps are small areas of the chromosome which are unstained. The chromatids remain aligned

as normal and the gap does not extend along the chromatid for a distance greater than the width

of a chromatid. If the gap occurs on one chromatid only it is a chromatid gap (g).

2. Chromatid Breaks

Chromatid breaks (b) vary in appearance. The chromatid may remain aligned but show a gap

which is too large to classify as a gap. Alternatively, the chromatid may be broken so that the

broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid

fragment (f) should be scored if the chromosome of origin cannot be identified. In addition,

deletions can occur as a result of a break. The missing terminal end of a chromatid in the

assessed metaphase is classified as deletion (d).

3. Chromosome breaks

Chromosome breaks (ib) are breaks in both chromatids of the chromosome. A fragment with two

chromatids is formed and this may be displaced by varying degrees. Breaks are distinguished

from gaps by the size of the unstained region. A chromosome break is scored if the fragment is

associated with a chromosome from which it was probably derived. However, fragments are often

seen in isolation and are then scored as chromatid fragments (if). In addition, isodeletions can

occur as a result of a isobreak. The missing terminal end of a chromosome in the assessed

metaphase is classified as isodeletion (id).

4. Exchanges

Exchanges are formed by faulty rejoining of broken chromosomes and may be of the

chromosome or chromatid type. Chromatid exchanges (ex) have numerous different forms but

are generally not further classified. Where multiple exchanges have occurred each exchange

point is counted as one chromatid exchange. Chromosome exchanges (cx) generally appear as

either a dicentric or a ring form, either of which can be associated with a fragment, which if

possible should be scored as part of the exchange.

5. Multiple Aberrations

If many aberrations are present in one metaphase, the exact details may not be scorable. This is

particularly the case when chromosome pulverisation (cd) occurs. If the number of aberrations is

greater than 4 then the cell is classified as multiple aberrant (ma).

6. Chromosome Number

If the chromosome (centromere) number is 22 ± 1 then it is classified as a diploid cell and scored

for aberrations. If less than 22 ± 1 chromosomes are counted then the cell is ignored under the

assumption that some chromosomes may have been lost for technical reasons. If greater than 22

± 1 chromosomes are scored then the count is recorded and the cell classified as an aneuploid

cell. If multiple copies of the haploid chromosome number (other than diploid) are scored then the

count is recorded and the cell classified as polyploid. If the chromosomes are arranged in closely

apposed pairs, i.e. 4 chromatids instead of 2, the cell is scored as endoreduplicated (e).
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14 ANNEX III:

14.1 Historical laboratory control data

Chinese hamster V79 cell cultures (July 2001 to December 2002)

Without S9 mix

Aberrant cells (%)

inclusive gaps exclusive gaps with exchanges

Test group

Concentration

cells

scored range mean

calculated

range* range mean

calculated.

range* range mean

calculated

range*

Negative control

Culture medium

MEM

54000 0.0-5.0 1.6 0.7-2.4 0.0-4.0 0.9 0.3-1.5 0.0-2.0 0.1 0.0-0.4

Deionised water

10 % (v/v)

7200 0.5-4.5 2.1 1.3-2.9 0.0-3.0 1.1 0.5-1.7 0.0-1.5 0.2 0.0-0.5

Org. solvents**

0.5 % (v/v)

32000 0.0-5.5 1.8 0.8-2.8 0.0-3.0 1.0 0.3-1.6 0.0-1.5 0.2 0.0-0.4

Total 105200 0.0-5.5 1.7 0.8-2.6 0.0-4.0 0.9 0.3-1.6 0.0-2.0 0.2 0.0-0.4

Positive control

EMS

100–1000 μg/mL

54000 8.0-100.0 23.1 12.0-34.3 8.0-100.0 21.7 10.7-32.8 0.0-40.0 6.7 3.0-10.4

With S9 mix

Aberrant cells (%)

inclusive gaps exclusive gaps with exchanges

Test group

Concentration

cells

scored range mean

calculated

range* range mean

calculated.

range* range mean

calculated

range*

Negative control

Culture medium

MEM

37600 0.0-7.0 1.8 0.9-2.8 0.0-4.0 1.2 0.5-1.8 0.0-2.0 0.3 0.0-0.6

Deionised water

10 % (v/v)

12800 0.0-3.5 1.6 0.8-2.4 0.0-2.5 1.1 0.4-1.7 0.0-1.5 0.2 0.0-0.6

Org. solvents**

0.5 % (v/v)

22600 0.0-5.5 1.9 0.9-2.9 0.0-3.5 1.2 0.5-2.0 0.0-2.0 0.3 0.0-0.7

Total 73200 0.0-7.0 1.8 0.9-2.8 0.0-4.0 1.2 0.5-1.8 0.0-2.0 0.3 0.0-0.6

Positive control

CPA

0.7–1.0 μg/mL

37600 8.0-95.5 18.9 13.0-24.9 7.5-95.5 17.2 11.5-22.9 0.0-23.0 6.0 3.4-8.7

* mean ± standard deviation

** organic solvents: acetone, DMSO, ethanol, and tetrahydrofurane