Tris(2-hydroxyethyl)ammonium acetate

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1997-06-11 to 1997-10-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is the result of a structural analogue substance used as read-across substance. Study is conducted according to Guidelines in a GLP certified laboratory.

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)

Deviations:

no

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:

-

EC Number:

427-360-5

EC Name:

-

IUPAC Name:

427-360-5

Details on test material:

Description: Yellow Solid
Stability of test compound: Stable

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

These cells have a stable karyotype with a modal chromosome number of 22. Before freezing, each batch was screened for mycoplasm contamination and checked for karyotype stability

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Doses applied in the Chromosome aberration assay with Read Across substance 2 (RA2)
Preparation
interval Experiment Concentration in µg/ml
Without S9-Mix
18 h I 300.0 500.0 1000.0 2000.0 3000.0 4000.0
18 h II 250.0 500.0 1000.0 2000.0 4000.0 5000.0
28 h II 1000.0 2000.0 4000.0 5000.0
With S9-Mix
18 h I 100.0 300.0 500.0 1000.0 3000.0 5000.0
18 h II 100.0 300.0 500.0 1000.0 3000.0 5000.0
28 h II 500.0 1000.0 3000.0 5000.0

Vehicle / solvent:

None

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulphonate (EMS) for positive controls without metabolic activation and cyclophosphamide (CPA) for positive controls with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In both independent experiments, the culture medium of exponentially growing cell cultures was replaced with serum free medium containing different concentrations of the test article and 50μl/ml S9 mix. After 4 hours the cultures were washed twice with "Saline G" and then cells were cultures in complete medium for the remaining culture time.

DURATION
- Exposure duration: Exposure time 4 hours (with S9 mix). Exposure time 18 and 28 hours (without S9 mix).

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 200 cells per treatment

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No

Evaluation criteria:

A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points.

A test article showing a lack of both a reproducible significant concentration-related increase in the number of structural chromosome aberrations and a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance was confirmed by means of the Fischer’s exact test (10)

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test article RA2, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberration in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h (exp. I); 18 h and 28 h (exp. II) after the start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h (exp. I) and 28 h (exp. II) without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

 

The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for cytotoxicity. In the absence of S9 mix toxic effects indicated by reduced cell numbers below 50 % of control were observed after treatment with 3000 µg/ml and above. In the presence of S9 mix no clear dose related reductions of the cell numbers was observed after treatment with test article concentrations up to 5000 µg/ml.

 

In experiment I, test article concentrations within the range of 300 – 400 µg/ml (without S9 mix) and 100 -5000 µg/ml (with S9 mix) were applied for the investigation of the potential to induce cytogemetic damage. In experiment II, the applied concentration ranges were 250 – 5000 µg/ml in the absence of S9 mix and 100 – 5000 µg/ml in the presence of S9 mix.

 

No test article precipitation and no influence of the test article on the pH value or osmolarity was observed. The evaluated experimental points and the results are summarised in Table 2.

 

In both experiments, in the absence of S9 mix, the mitotic indices were reduced to ≤ 65 % of control after treatment with 2000  µg/ml and above. In addition cultures after treatment with 4000 µg/ml showed distinctly reduced cell numbers. In the presence of S9 mix in experiment II the mitotic index was slightly reduced after treatment with 5000 µg/ml at the 28 h preparation interval.

 

In both experiments, neither a biologically relevant nor a significant increase in cells carrying structural chromosome aberrations was observed.

 

In both experiments, no biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test article as compared to the frequencies of the controls.

 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.005) in cells carrying structural chromosome aberrations.

 

Table 2: Summary of the chromosomal aberration study with RA2

 

Exp.	Preparation Interval	S9 mix	Concentration of RA2 in µg/ml	Polyploid cells in %	Mitotic index in % of control	incl. gaps	Aberrant cells in % excel. gaps*	exchanges
I	18 h	-	Solvent control	4.0	100.0	3.0	1.0	0.5
		-	500.0	3.5	102.5	1.5	1.0	0.0
		-	2000.0	2.5	63.9	1.0	1.0	0.0
		-	4000.0	2.5	59.3	2.5	1.5	0.5
II	18 h	-	Solvent control	3.5	100.0	1.5	0.5	0.0
		-	500.0	3.5	93.7	1.5	1.0	0.0
		-	2000.0	1.0	61.7	2.0	1.5	0.0
		-	4000.0	4.0	42.9	3.5	2.0	0.5
II	28 h	-	Solvent control	3.0	100.0	2.0	0.5	0.0
		-	2000.0	4.5	60.1	1.5	1.5	0.0
I	18 h	+	Solvent control	3.5	100.0	1.5	0.5	0.0
		+	500.0	3.0	78.9	4.0	2.5	0.0
		+	2000.0	4.5	85.1	2.0	1.5	0.0
		+	5000.0	4.5	83.2	3.0	2.0	0.0
II	18 h	+	Solvent control	3.5	100.0	0.5	0.5	0.0
		+	500.0	3.0	80.0	1.0	0.5	0.5
		+	2000.0	1.5	88.6	1.5	1.5	0.0
		+	5000.0	4.5	80.0	0.5	0.0	0.0
II	28 h	+	Solvent control	2.5	100.0	3.0	1.5	0.5
		+	5000.0	4.5	68.3	1.5	1.0	0.0

*Inclusive cells carrying exchanges

  Aberrant cells in positive control groups: 21.0 % - 25.5 %

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative The Read Across test article (RA2) did not induce structural chromosome aberrations in V79 cells (Chinease hamster Cell line)

Based on the results presented in this study, it can be stated that under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster Cell line) in vitro. Therefore, Read Across substance 2 (RA2) is considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

The test article RA2, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberration in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h (exp. I); 18 h and 28 h (exp. II) after the start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h (exp. I) and 28 h (exp. II) without metabolic activation. In each experimental group two parallel cultures were set up. Per cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

 

The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for cytotoxicity. In the absence of S9 mix toxic effects indicated by reduced cell numbers below 50 % of control were observed after treatment with 3000 µg/ml and above. In the presence of S9 mix no clear dose related reductions of the cell numbers was observed after treatment with test article concentrations up to 5000 µg/ml. Based on these results, the concentrations were chosen for the two main experiments (experiment I and experiment II).

 

In the absence of S9 mix, in both experiments the mitotic indices were reduced to ≤ 65 % of control after treatment with 2000  µg/ml and above. In addition cultures after treatment with 4000 µg/ml showed distinctly reduced cell numbers. In the presence of S9 mix in experiment II the mitotic index was slightly reduced after treatment with 5000 µg/ml at the 28 h preparation interval.

 

In both experiments, neither a biologically relevant nor a significant increase in cells carrying structural chromosome aberrations was observed.

 

In both experiments, no biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test article as compared to the frequencies of the controls.

 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.005) in cells carrying structural chromosome aberrations.

 

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item did not structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line)in vitro.

 

Therefore, RA2 is considered to be non-clastogenic in this chromosome aberration test.