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Toxicological information

Genetic toxicity: in vitro

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 index1

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 index1

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.