Amines, N-C12–14 (even numbered)-alkyltrimethylenedi-, reaction products with chloroacetic acid and diethylenetriamine, N-C12–14 (even numbered)-alkyl derivs.

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:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

no

Remarks:

Nevertheless, the study was conducted in compliance with Good Laboratory Practice Regulations, albeit prior to implementation of GLP in the EU.

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-C12–C14(even numbered)-alkyltrimethylenedi-, reaction products with chloroacetic acid

Molecular formula:

n.a. (UVCB substance)

IUPAC Name:

Amines, N-C12–C14(even numbered)-alkyltrimethylenedi-, reaction products with chloroacetic acid

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid - liquid: aqueous solution

Details on test material:

- Name of test material: DOPA-Glycinate

Method

Species / strain

Species / strain / cell type:

other: Human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 1.11, 3.33, 10.0 and 30.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Way of application
100 µL test solutions were added to the blood cell culture. In the presence of S9 mix, the exposure was reduced to 2 hours, only. After washing and supplying with new medium the cells were incubated for a further 22 hours including a 2 hour colcemid treatment. In the absence of S9 mix, cells were continuously exposed to tissue culture medium for 24 hours including a 2 hour colcemid treatment. The cultures were incubated at 37°C in humidified air containing 5% CO2.

Pre-incubation time
Not applicable

Other modifications
None

Examinations
Evaluation of chromosome and chromatid aberrations

Number of cells evaluated
2 × 1000 lymphocytes were examined to determine the mitotic index
2 × 100 well-spread metaphases (each containing 46 chromosomes) were scored for each dose group

Results and discussion

Test results

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Genotoxicity
Without metabolic activation: Negative
The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used when compared with the vehicle control values.
The positive control compound, Mitomycin C, showed the expected statistically significant increase in the number of cells with structural chromosome aberrations.

With metabolic activation: Negative
The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used when compared with the vehicle control values.
The positive control compound, Cyclophosphamide, showed the expected statistically significant increase in the number of cells with structural chromosome aberrations.
The results are summarised in Table A6.6.2- 2.

Cytotoxicity
Yes
In a first preliminary cytotoxicity test, the test substance was clearly cytotoxic at concentrations in a range of 82.3–20000 µg/mL with and without metabolic activation. In a second preliminary cytotoxicity test the mitotic index was reduced to about 50% at 27.4 µg/mL both with and without S9 mix.
In the chromosome aberration test a reduction of the mitotic index to about 60 % or 54 % of the vehicle control without or with S9 mix, respectively, was observed at the highest test concentration (30 µg/mL).

Any other information on results incl. tables

Table A6.6.2-1:Metaphase chromosome analysis of human lymphocytes after exposure to the test item or mitomycin C in the absence of S9-mix.

	Control	1.11 µg/mL	3.33 µg/mL	10 µg/mL	30 µg/mL	Mitomycin C
Cytotoxicity	An inhibition of growth of 50 % was judged to lie at a concentration of 27.4 µg/mL of the test item
No. of cells analysed	200	200	200	200	200	200
ABERRATIONS
No. of cells with aberrations
Gaps	0	1	2	1	1	11**
Breaks	0	0	1	1	0	28***
Exchanges	0	0	0	0	0	22***
Multiple	0	0	0	0	0	0
% cells with aberrations
Incl. gaps	0	0.5	1.5	1.0	0.5	26.0***
Excl. gaps	0	0	0.5	0.5	0	22.5***
Mitotic index^1	9.9	8.9	8.1	8.9	5.9	4.2
**) p<0.01 Fisher’s exact probability test (two sided) ***) p<0.001 Fisher’s exact probability test (two-sided) 1) percentage of metaphases determined in 1000 nuclei (mean value of duplicate cultures)

 

Table A6.6.2-2:Metaphase chromosome analysis of human lymphocytes after exposure to the test item or cyclophosphamide in the presence of S9-mix.

	Control	1.11 µg/mL	3.33 µg/mL	10 µg/mL	30 µg/mL	Cyclophosphamide
Cytotoxicity	An inhibition of growth of 50 % was judged to lie at a concentration of 27.4 µg/mL of the test item
No. of cells analysed	200	200	200	200	200	200
ABERRATIONS
No. of cells with aberrations
Gaps	1	0	0	0	0	30***
Breaks	2	0	1	2	1	71***
Exchanges	0	0	0	0	0	44***
Multiple	0	0	0	0	0	0
% cells with aberrations
Incl. gaps	1.5	0	0.5	1.0	0.5	51.0***
Excl. gaps	1.0	0	0.5	1.0	0.5	46.5***
Mitotic index^1	9.9	7.1	8.3	8.2	5.3	1.7
***) p<0.001 Fisher’s exact probability test (two-sided) 1) percentage of metaphases determined in 1000 nuclei (mean value of duplicate cultures)

 

Applicant's summary and conclusion

Conclusions:

DOPA-Glycinate (20% a.i.) did not induce structural chromosome damage in cultured human lymphocytes either in the presence or in the absence of metabolic activation (S9 mix) when tested up to cytotoxic concentrations.

Executive summary:

The in vitro genotoxicity of DOPA-Glycinate (20% a.i.) was tested in human lymphocytes. The study was carried out according to OECD guideline 473 (1983). No relevant deviations from the prescribed test guideline were reported.The test was performed at concentrations of 0, 1.11, 3.33, 10.0 and 30.0 µg/mL in the presence and absence of metabolic activation (S9 mix). A reduction of the mitotic index to about 60 % or 54 % of the vehicle control without or with S9 mix, respectively, was observed at the highest test concentration (30 µg/mL) indicating cytotoxicity. The positive controls resulted in the appropriate responses.

The test item did not induce structural chromosome damage in cultured human lymphocytes either in the presence or in the absence of S9-mix.