# 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 vitroChromosome 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 datedJune 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 vitroin 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 significanthigher 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 andthe environment. Genotoxicity studies provide important information for the assessment of the mutagenic potential of these substances (1, 2). The in vitroChromosome Aberration Test performed in this study is an essential part of genotoxicity test batteries of substances. Thisin vitrotest 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 atclastogenic 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
Thisin vitrotest 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 preparationintervals (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 of2 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 inin vitroexperiments. 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 underin vitroconditions 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 cultureswith 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 5mg/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 basedon 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 wereprinted 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 CulturesExponentially 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 KH2

### P04 

### 200 mg Na2

### HP04 

### 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 Na2

### HP04

### x7H2

### 0 290 mg KH2

### P04 

### 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 thetest 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 vitroin 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 320mOsm 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-relatedincrease 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)

### RCC Project Number 848629 Final Report - page 22 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product 

# 11REFERENCES 1. Ames, B.N., McCann, J. and Yamasaki, E. (1977) Methods for detecting carcinogens and mutagenswith 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 currentpractice 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 aberrationyield 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 thecytotoxicity 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 fromin vitrocytogenetic tests Kirkland, D.J. (ed.). ”Statistical evaluation of mutagenicity testdata“, Cambridge University Press, Cambridge, 141-154 

### Supplementary References: Kirkland, D.J. (1992) Chromosome aberration testsin vitro: problems with protocol design and interpretation of results. Mutagenesis, 2, 95-106

### RCC Project Number 848629 Final Report - page 23 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product 

# 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

### RCC Project Number 848629 Final Report - page 24 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


# 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 evaluatedfor cytogenetic damage were printed in bold characters

### RCC Project Number 848629 Final Report - page 25 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


# 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 eachtest 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

### RCC Project Number 848629 Final Report - page 26 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


### 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)

### RCC Project Number 848629 Final Report - page 27 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


### 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)

### RCC Project Number 848629 Final Report - page 28 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


# 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 eachtest 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

### RCC Project Number 848629 Final Report - page 29 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


### 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)

### RCC Project Number 848629 Final Report - page 30 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


### 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)

### RCC Project Number 848629 Final Report - page 31 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product


# 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 rateis equal or lower than the control rate S 

### aberration rate is statistically significant higher than the control rate

### RCC Project Number 848629 Final Report - page 32 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product 

# 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 chromosomeof 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 itwas 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 isrecorded and the cell classified as an aneuploid cell. If multiple copies of the haploid chromosomenumber (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 of2, the cell is scored as endoreduplicated (e).

### RCC Project Number 848629 Final Report - page 33 of 33 - RCC-CCR Study Number 785202 DEA/Acid anhydride reaction product 

# 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