Diethylmethylbenzenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between September 10, 1999 and May 3, 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was performed in compliance with Good Laboratory Practice standards and according to OECD Test Guideline 473 Genetic Toxicology: Chromosome Aberration Test and Method B.10 of Commission Directive 92/69/EEC.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Diethylmethylbenzenediamine

EC Number:

270-877-4

EC Name:

Diethylmethylbenzenediamine

Cas Number:

68479-98-1

Molecular formula:

C11H18N2

IUPAC Name:

diethylmethylbenzenediamine

Details on test material:

- Name of test material (as cited in study report): LZ1224.20, DETDA 80, Diethyltoluenediamine
- Physical state: Clear liquid
- Analytical purity: 98.4%
- Impurities (identity and concentrations): 0.4% ethyl toluenediamine (GC, % area); <0.0150% toluenediamine (GC, % area); 0.02 % water (KF, w/w)
- Composition of test material, percentage of components: 77.6% 3,5-diethyltoluene-2,4-diamine (GC, % area); 20.8% 3,5-diethyltoluene-2,6-diamine (GC, % area)
- Purity test date: 2000-08-31
- Lot/batch No.: 2235
- Storage condition of test material: in the dark at approximately 4 degrees Celsius under nitrogen

Method

Target gene:

chromosomal aberrations

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be 16.7 hours under typical experimental exposure conditions.

CELL CULTURE
Cells were grown in Eagle's minimal essential medium, (supplemented with sodium bicarbonate, HEPES buffer, L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum) at 37 degrees Celsius with 5% carbon dioxide in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 micrograms/ml final concentration.

CULTURE CONDITIONS
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 8.05-9.05 ml MEM, 15% (FCS); 0.1 ml Li-heparin; 0.1 ml phytohaemagglutinin; and 0.75 ml heparinised whole blood.

Metabolic activation:

with and without

Metabolic activation system:

Microsomal enzyme fractions were prepared from livers of male Sprague-Dawley rats (bw = ~250 grams). Rats had received 3 daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphtoflavone (100 mg/kg), prior to S9 isolation on day 4.

Test concentrations with justification for top dose:

112.5 to 1800 micrograms/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 750 micrograms/ml for cultures, in the absence of metabolising enzymes in Experiment 1.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: 0.2 micrograms/ml for cultures in the absence of metabolising enzymes in Experiment 2

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: 25 micrograms/ml for cultures where S9 was included

Details on test system and experimental conditions:

PREPARATION OF TEST AND CONTROL MATERIALS
The test material was accurately weighed, dissolved/suspended in MEM and serial dilutions prepared. The molecular weight of the test material was 178.28; therefore, the maximum dose level was 1800 micrograms/ml, which was approximately equivalent to 10 mM. The purity of the test material was 98.4%, therefore, a correction for purity was made during the preparation of the dosing solutions. Initially, to achieve the maximum limit dose level of 10 mM (1800 micrograms/ml) a 9 mg/ml dosing solution was dosed at 20% final volume instead of the normal 10%, this was considered not to affect the integrity of the study. However, in the second and third experiments the 10 mM dose level was achieved by dosing at 10% using an 18 mg/ml dosing solution. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOSM.

Vehicle and positive controls were used in parallel with the test material.

TREATMENT WITH-ACTIVATION
After approximately 48 hours incubation at 37 degrees Celsius with 5% carbon dioxide in humidified air, the cultures were centrifuged, after transfer into tubes, and approximately 9 ml of the culture medium removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 ml of the appropriate solution of vehicle control or test material (2 ml at 1800 micrograms/ml in Experiment 1) was added to each culture. Where appropriate, cultures were dosed with the positive control using a 0.1 ml dose volume. One ml of 10% S9-mix (i.e., 1% final concentration of S9) in standard co-factors was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 ml of 20% S9-mix (i.e., 2% final concentration of S9) in standard co-factors, was added. In a third experiment, two final levels of S9 were used as follows: 1 ml of 26% S9-mix (i.e., 2% final concentration of S9) and 1 ml of 50% S9-mix (i.e., 5% final concentration of S9). All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.

After 4 hours at 37 degrees Celsius, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37 degrees Celsius in 5% carbon dioxide in humidified air.

TREATMENT WITHOUT METABOLIC ACTIVATION
In Experiment 1, after approximately 48 hours incubation at 37 degrees Celsius with 5% carbon dioxide in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 ml of the appropriate vehicle control, test material solution (2 ml at 1800 micrograms/ml) or 0.1 ml of positive control. The total volume for each culture was a nominal 10 ml.

After 4 hours at 37 degrees Celsius, the cultures were centrifuged; the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, the exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established, the culture volume was 9.0 ml (nominal). After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 ml of vehicle control, test material dose solution, or 0.1 ml of positive control. The nominal final volume of each tube was 10 ml. The cultures were then incubated at 37 degrees Celsius for 24 hours.

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 7 to 1800 micrograms/ml. Parallel flasks, containing culture medium without whole blood, were established for all three culture conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of the slides prepared from the treatment cultures, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and select dose levels for the main study.

MAIN STUDY
Experiment 1
4-hour exposures to the test material were conducted with or without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. After mitotic index determination, the dose levels selected for metaphase analysis were: 450, 900, and 1800 micrograms/ml.

Experiment 2
24-hour continuous exposure to the test material was conducted prior to cell harvest. After mitotic index determination, the dose levels selected for metaphase analysis were: 112.5, 225, and 337.5 micrograms/ml.

4-hour exposure to the test material with S9-mix was conducted, followed by 20-hour culture in treatment-free media prior to cell harvest. After mitotic index determination, the dose levels selected for metaphase analysis were: 450, 900, 1350 and 1800 micrograms/ml.

Experiment 3
4-hour exposures to the test material with S9-mix (either 2% or 5% final concentration) were conducted, followed by 20-hour culture in treatment-free media prior to harvest. After mitotic index determination, the dose levels selected for metaphase analysis were: 1400 and 1800 micrograms/ml.

CELL HARVEST
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 microgram/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCl. After approximately fifteen minutes (including five minutes centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KC1 cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4 degrees Celsius for at least four hours to ensure complete fixation.

PREPARATION OF METAPHASE SPREADS
The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

STAINING
When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

QUANTITATIVE SLIDE ASSESSMENT
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

CODING
The slides were coded using a computerized random number generator.

MITOTIC INDEX
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

SCORING OF CHROMOSOME DAMAGE
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1 976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. In some instances where the response was lopsided between duplicate slides, or slide quality was less than optimum, another 100 metaphase cells were scored.

Savage, J.R.K. (1976) “Annotation: Classification and relationships of induced chromosomal structural changes”, JOURNAL OF MEDICAL GENETICS, Vol. 13, pp. 103-122.

Evaluation criteria:

A positive response was recorded for a particular treatment if the percentage of cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose relationship. For modest increases in aberration frequency, a dose-response relationship was required and an appropriate statistical test was applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact and Chi-squared tests.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: Chromosome Aberration Test - Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with S9 (1%) or without

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: Chromosome Aberration Test - Experiment 1

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Significant increase in polyploid cells in the absence of S9 at 1800 micrograms/ml and in the presence of S9 (1%) at 900 micrograms/ml

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes: Chromosome Aberration Test - Experiment 2

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Statistically significant dose-related increase in the frequency of cells with chromosome aberrations in the presence of S9 (2%)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

lymphocytes: Chromosome Aberration Test - Experiment 2

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Significant increase in polyploid cells in the absence of S9 at 1800 micrograms/ml and in the presence of S9 (2%) at 900 micrograms/ml

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

lymphocytes: Chromosome Aberration Test - Experiment 3

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with S9 (2% or 5%)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

lymphocytes: Chromosome Aberration Test - Experiment 3

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Significant increase in polyploid cells in the presence of S9 (2%) at 1800 micrograms/ml

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test material induced toxicity in all three treatment groups (i.e., #1: 4-hour treatment, 20-hour recovery –S9; #2: 4-hour treatment, 20-hour recovery +S9; #3: 24-hour treatment –S9). The response was more pronounced in the continuous exposure cultures without metabolic activation. No precipitate of the test material was observed. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to the 10 mM limit dose of test material (1800 micrograms/ml) in the pulse treatments with and without S9-mix, and up to 450 micrograms/ml in the 24-hour continuous treatment without S9-mix.

CHROMOSOME ABERRATION TEST - EXPERIMENT 1
Mitotic Index
A dose-related inhibition of mitotic index was observed such that 42% mitotic inhibition was achieved at 1800 micrograms/ml in the absence of S9. In the presence of S9, the dose-related response was not pronounced and only 23% mitotic inhibition was achieved at the maximum dose level tested of 1800 micrograms/ml.

Chromosome Aberration
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. However, a dose-related trend was observed when gap-type aberrations were taken into account.

Polyploid Cell Frequency
The test material induced a significant increase in the numbers of polyploid cells in the absence of S9-mix at 1800 micrograms/ml, and in the presence of S9-mix at 900 micrograms/ml.

CHROMOSOME ABERRATION TEST – EXPERIMENT 2
Mitotic Index
The test material induced a dose-related inhibition of mitotic index such that a near optimum of, 52% mitotic inhibition was achieved at 337.5 micrograms/ml in the absence of S9. The 87% inhibition observed at 450 micrograms/ml was considered to be too excessive for that dose level to be included in the metaphase analysis. In the presence of S9, the dose-related response was less clear, however, in the dose levels that were assessed, mitotic inhibition was induced, to a maximum of 41% of the control value at 1800 micrograms/ml.

Chromosome Aberration
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material induced a statistically significant dose-related increase in the frequency of cells with chromosome aberrations in the presence of metabolic activation. A similar response was not observed in Experiment 1. However, the final S9 concentration was increased from 1% to 2% and the difference in S9 concentration may explain the different qualitative responses seen in the two experiments.

Polyploid Cell Frequency
The test material induced a significant increase in the numbers of polyploid cells at 900 micrograms/ml in the presence of S9 mix, confirming the response seen in Experiment 1.

CHROMOSOME ABERRATION TEST – EXPERIMENT 3
Mitotic Index
The test material induced a dose-related reduction in mitotic index using both S9 concentrations, such that 45% mitotic inhibition was achieved at 1800 micrograms/ml in 2% S9 cultures and 52% mitotic inhibition was seen at 1800 micrograms in the 5% S9 cultures.

Chromosome Aberration
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations excluding gap-type aberrations either in the 2 or 5% final S9 concentration treatment groups. However, a significant increase was seen when gap-type aberrations were included at 1800 micrograms/ml in the 5% S9 treatment group. The response seen in Experiment 2 was not reproduced in the 2% final concentration treatment group of this experiment, and only a gap-type response was observed in the 5% final S9 concentration group. However, an unavoidable difference between Experiment 2 and Experiment 3 was that a different batch of S9 was used with a slightly reduced protein content.

Polyploid Cell Frequency
The test material induced a significant increase in the numbers of polyploid cells at the 1800 microgram/ml dose level in the 2% final S9 concentration treatment case; confirming the responses seen in both Experiment 1 and 2. It should be noted that the polyploid cells observed were primarily endoreduplicated cells in all three experiments.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous

The test material induced no statistically significant increase in the frequency of cells with aberrations (excluding gaps) in the first experiment. However, in the second experiment, an increase was observed with metabolic activation (2% S9), which was not clearly dose related. This response was not reproduced in the third experiment using 2% or 5% S9. However, a small statistically significant increase in the frequency of cells with aberrations including gaps was observed in the 5% S9 group. Small increases in polyploid cell frequency, both in the absence and presence of S9, were also noted. Therefore, there was insufficient evidence for a clastogenic effect on human lymphocytes of the test material.