reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate

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:

1997

Report date:

1997

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)

Version / remarks:

Official Journal of European Communities No L383A/148, 29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Toxicology of BASF SE, Ludwigshafen/Rhein, Germany

Type of assay:

other: chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate

Molecular formula:

C5H11N2O2Na and C8H14N2O4Na2

IUPAC Name:

reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 34-0725

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

1st experiment (with/without S9 mix, 18 h harvest):
962.5, 1925, 3850 µg/mL (based on the aqueous test substance)
[385.0, 770.0, 1540.0 µg/mL (based on the active ingredient)]
2nd experiment (without S9 mix, 18 harvest):
1925, 2887.5, 3850 µg/mL (based on the aqueous test substance)
[770.0, 1155.0, 1540.0 µg/mL (based on the active ingredient)]
3rd experiment (with/without S9 mix):
18 h harvest: 1925, 2887, 3850 µg/mL (based on the aqueous test substance)
[770.0, 1155.0, 1540.0 µg/mL (based on the active ingredient)]
28 h harvest: 3850 µg/mL (based on the aqueous test substance)
[1540.0 µg/mL (based on the active ingredient)]

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Minimal essential medium (MEM including glutamine)
Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation, 0.350 mg/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation, 0.0005 mg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 4 h with S9 mix
- Exposure duration: 18 and 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 30 hours

SPINDLE INHIBITOR: Two - three hours before each preparation time (18 and 28 hours after the beginning of treatment) 0.2 µg colcemide/mL culture medium was added to each well to arrest mitosis in the metaphases. The medium was removed and replaced with 5 mL of a hypotonic solution (0.4 % KCI in dist . water) pre-warmed to 37°C for about 20 min. Thereafter the cells were fixed with 5 mL of ice-cold fixative (methanol : glacial acetic acid = 3 : 1).

STAIN: 10 min in a solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol, pH 7.2)

NUMBER OF REPLICATIONS: Two independent experiments were performed.

NUMBER OF CELLS EVALUATED: first 100 consecutive well spread metaphases of each culture (20 - 22 chromosomes).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (1000 ceels/culture)

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

The test chemical is to be considered positive in this assay if the following criteria are met :
- a dose-related and reproducible significant increase in the number of structural chromosomal aberrations .
- the proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range .

A test substance is generally considered nonclastogenic in this test system if :
- there was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies .
- the aberration frequencies were within the historical control range .

Statistics:

The proportion of metaphases with aberrations was calculated for each group. A comparison of each dose group with the vehicle control group was carried outusing Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

1st experiment:
With 7.5% there was a slight increase in the number of aberrant cells at the highest dose of 3850 µg/mL in the experimental part without metabolic activation after a harvest time of 18 hours . However, this figure which slightly exceeded the upper value of the historical control range, i .e .5.5% was influenced by only one out of the two cultures.
With S9 mix an increase in the number of aberrant metaphases was not observed.

2nd experiment:
For confirmation/substantiation of the findings of the 1st experiment the 2nd experiment was limited to the experimental part without S9 mix only but selecting closer doses to demonstrate a possible dose-response relationship. However, the results of the 1st experiment could not be confirmed, i. e. the number of chromosomally damaged cells was within the range of the concurrent negative control and within the range of the historical control data.

3rd experiment:
In the 3rd experiment including an additionally harvest time of 28 hours a clastogenic activity could not be observed after any of the experimental conditions.

Any other information on results incl. tables

Table 1: Experiment 1, harvest time 18 hours, treatment 18 h (-S9 mix) or 4 h (+S9 mix), mean of two cultures

			Metaphases with aberrations
conc. [µg/mL]	S9 mix	Mitotic index % (mean)	Incl. gap	Excl.gap	Exchanges	Mul. Aber.	Chr. Dis.	Aneupl.	Polypl.
Vehicle control (MEM)	-	6.8	18	6	1	0	0	0	0
962.5#	-	5.6	20	7	2	0	0	0	0
1925.0	-	6.1	13	8	3	0	0	0	0
3850.0	-	6.9	16	13	10	0	0	0	0
Positive control (EMS) 350.0	-	5.7	16	13	9	1	0	0	0
Vehicle control (MEM)	+	11.5	12	3	3	0	0	0	0
962.5	+	12.8	17	6	3	0	0	0	0
1925.0	+	10.6	9	6	2	0	0	0	0
3850.0	+	12.8	15	5	5	0	0	0	0
Positive control (CPP) 0.5	+	7.0	21	18	9	0	0	0	0
# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy

 

Table 2: Experiment 2, harvest time 18 hours, treatment 18 h (-S9 mix), mean of two cultures

			Metaphases with aberrations
conc. [µg/mL]	S9 mix	Mitotic index % (mean)	Incl. gap	Excl.gap	Exchanges	Mul. Aber.	Chr. Dis.	Aneupl.	Polypl.
Vehicle control (MEM)	-	5.2	14	7	1	0	0	0	1
1925#	-	4.9	17	4	1	0	0	1	1
2887.5	-	7.2	11	2	0	0	0	0	0
3850.0	-	6.0	22	6	1	0	0	1	0
Positive control (EMS) 350.0	-	3.8	25	13	4	1	0	0	0
# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy

 

Table 3: Experiment 3, harvest time 18 hours, treatment 18 h (-S9 mix) or 4 h (+S9 mix), mean of two cultures

			Metaphases with aberrations
conc. [µg/mL]	S9 mix	Mitotic index % (mean)	Incl. gap	Excl.gap	Exchanges	Mul. Aber.	Chr. Dis.	Aneupl.	Polypl.
Vehicle control (MEM)	-	6.5	15	6	1	0	0	1	0
1925#	-	4.6	15	8	4	0	0	0	0
2887.5	-	4.2	15	9	2	0	0	0	0
3850.0	-	5.7	13	7	2	1	0	0	0
Positive control (EMS) 350.0	-	4.7	14	14	11	0	0	0	0
Vehicle control (MEM)	+	8.3	11	4	0	0	0	0	0
1925#	+	13.5	20	9	2	0	0	0	1
2887.5	+	7.5	11	2	0	0	0	0	0
3850.0	+	7.7	8	6	0	0	0	0	0
Positive control (CPP) 0.5	+	5.6	18	16	7	1	0	0	0
# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy

 

Table 4: Experiment 3, harvest time 28 hours, treatment 18 h (-S9 mix) or 4 h (+S9 mix), mean of two cultures

			Metaphases with aberrations
conc. [µg/mL]	S9 mix	Mitotic index % (mean)	Incl. gap	Excl.gap	Exchanges	Mul. Aber.	Chr. Dis.	Aneupl.	Polypl.
Vehicle control (MEM)	-	9.8	6	3	2	0	0	0	0
3850.0#	-	9.1	14	8	5	0	0	0	0
Vehicle control (MEM)	+	8.9	14	6	1	0	0	0	0
3850.0	+	14.5	12	2	1	0	0	0	0
# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy