2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine)

EC Number:

229-962-1

EC Name:

2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine)

Cas Number:

6864-37-5

Molecular formula:

C15H30N2

IUPAC Name:

4-[(4-amino-3-methylcyclohexyl)methyl]-2-methylcyclohexan-1-amine

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1 BH4

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The Chinese hamster ovary (CHO-K1 BH4) cell line, isolated by Kao and Puck (1967) and cloned by O'Neill et al (1977) was used.

MEDIA USED
Ham's F-12 medium, supplemented with 10% foetal bovine serum and antibiotics, at 37°C with 5% CO2 in air

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
It was prepared from the livers of male Sprague-Dawley rats weighing ca. 200 g. These had received a single i .p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation . The S9 was stored at -196°C.
- concentration or volume of S9 mix and S9 in the final culture medium
10% S9- mix was freshly prepared by mixing 2 mL of S9 with 1 mL of 0.1M NADP, 1 mL of 0 .1M G6P, 2 mL of 330mM KC1/80mM MgCl2 and 14 mL of 0 .1M Phosphate buffer (pH 7 .4) . The S9 mix was stored at 4°C for a maximum of 20 minutes before use .

Test concentrations with justification for top dose:

0, 78.13, 156.25, 312.5 μg/mL (- S-9, 12-hour culture)
0, 156.25, 312.5, 625 μg/mL (+ S-9, 12-hour culture)
0, 156.25, 312.5, 625 μg/mL (+ S-9, 20-hour culture)

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.5 x 10E6 cells per flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Without S9 mix
i) 12 hours exposure to the test material
With S9 mix
ii) 4 hours exposure to the test material and S9 mix (0.5 mL per 4 .5 mL culture medium, of 10% S9 in standard cofactors). A phosphate buffered saline wash and then a further 8 hours in treatment-free media prior to cell harvest .
iii) 4 hours exposure to the test material and S9 mix (0.5 mL per 4 .5 mL culture medium, of 10% S9 in standard cofactors). A phosphate buffered saline wash and then a further 16 hours in treatment-free media prior to cell harvest .

- Spindle inhibitor: Mitosis was arrested by addition of demecolcine (0.1 µg/mL) two hours before the required harvest time .

- Methods of slide preparation and staining technique used including the stain used:
The cells were resuspended in 3.0 mL of fresh fixative if necessary before centrifugation and resuspension in 0.5 mL of fixative. Three or four drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was
permanently labelled with the appropriate identification data. When the slides were dry they were stained in 2% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and mounted in Depex mounting medium.

- Number of cells spread and analysed per concentration:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 18 to 22 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976)
If the cell had more than 22 chromosomes then it was recorded on a separate sheet as an aneuploid or polyploid cell, chromosome aberrations in such cells were not recorded. Endoreduplicated cells are included as polyploid cells but can also be evaluated separately if necessary. All chromosome aberrations were checked by a senior cytogeneticist prior to decoding the slides .

Mitotoc index: A total of 2000 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 .

METHODS FOR MEASUREMENT OF CYTOTOXICITY
A cytotoxicity test was performed on cell cultures using a 4-hour exposure time with metabolic activation followed by an 8 and 16-hour culture period in treatment free media. Treatment without metabolic activation was continuous with cell harvest at 12 hours. Growth inhibition was estimated by couating the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent negative control value .

Evaluation criteria:

A positive response was recorded for a particular treatment if the % diploid cells with aberrations (gaps excluded) exceeded the maximum historical value and gave a statistically significant increase over the concurrent control value. If only the % diploid cells with aberrations (gaps included) exceed
historical values then a ± response was recorded.

Positive responses were also recorded if the % cells with aberrations (gaps excluded) was statistically significantly greater than the concurrent control level, even if it was below historical levels, but only if there was an indication of a dose response. However, consideration is given to a number of factors, such as the frequency of chromosome exchange events which are comparatively rare in control cultures, and the ultimate designation must rely upon experience and sound scientific judgement (UKEMS Guidelines for Mutagenicity Testing, 1983).

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was observed at doses of 313 μg/mL without and 625 μg/mL with S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
The pH of culture medium containing S9 mix and test item at 0, 312.5 and 625 µg/mL was measured and values of 7.05, 7.45 and 7.55 were obtained.

RANGE-FINDING/SCREENING STUDIES:
It was observed that the test item showed evidence of a dose-related increase in cell toxicity at dose levels up to 312.5 µg/mL without S9 and up to 625 µg/mL with S9 .

Chromosome aberration test (CA) in mammalian cells:
The negative control cultures gave values of chromosome aberrations within the expected range. The frequency of aberrations was consistent between the four negative control groups, the highest frequency (4 .0% cells with aberrations without gaps) being seen in the 20-hour culture group with S9.
The positive control cultures gave significant increases in the frequency of aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. Cyclophosphamide (10 µg/mL) gave a stronger response at the 20-hour harvest time point than that seen after 12 hours .

The test item was seen to induce no significant, dose-related increases in the frequency of aberrations in any of the treatment groups .

The test item induced a significant increase in the numbers of polyploid cells at the 625 µg/mL dose-level in the 20-hour treatment with S9. The duplicate slides from cultures A and B were rechecked for the incidence of polyploid cells and values of 26.5 and 14.5% respectively were obtained, thus confirming the original observations. An Increase in the frequency of polyploid cells was observed at one dose level only, but this was considered to be possibly due to the pH change induced by the test item when added to the culture medium .