Glutaral

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:

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:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Glutaral

EC Number:

203-856-5

EC Name:

Glutaral

Cas Number:

111-30-8

Molecular formula:

C5H8O2

IUPAC Name:

glutaraldehyde

Test material form:

liquid

Specific details on test material used for the study:

- Physical state: colourless liquid
- Analytical purity: ca. 50% (analytical report data)
- Impurities (identity and concentrations): methanol 0.2%
- Composition of test material, percentage of components: 50.3% glutaraldehyde, 46% water, 0.2% methanol
- Lot/batch No.: 50-4402
- Stability under test conditions: the stability of the test substance at room temperature in water over a period of 14 days had been verified analytically (Bioanalytical Laboratory of the Experimental Toxicology and Ecology, BASF AG)
- Storage condition of test material: refrigerator, under N2 conditions

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was prepared according to Ames et al. (Mut. Res. 31:347-364, 1975) from the S9 liver fraction of Aroclor 1254-treated male Sprague-Dawley rats, mixed with appropriate cofactors.

Test concentrations with justification for top dose:

A range-finding cytotoxicity test was performed prior to the main test the test concentrations for the main assay were selected on the basis of the results of the pre-test:
Without S9 mix: 0.25, 0.5 and 1 µg/mL
With S9 mix: 2.5, 5 and 10 µg/mL

Vehicle / solvent:

The cell culture medium MEM medium with Earle´s salt was used as vehicle.

Controls

Untreated negative controls:

yes

Remarks:

the untreated negative controls were performed with and without S9 mix and consisted of cells in culture medium (MEM medium with Earle´s salt).

Negative solvent / vehicle controls:

yes

Remarks:

the vehicle control was identical to the untreated control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

CYTOTOXICITY
A range-finding cytotoxicity test was performed prior to the main test. The test concentrations in the absence of S9 mix were 0.25, 0.5, 1.0, 2.0 and 4.0 µg/mL; the test concentrations in presence of S9 mix were 1.25, 2.5, 5.0, 10 and 20 µg/mL.

MAIN ASSAY
The test substance was dissolved in 1mL serum-free culture medium and was then added to the culture medium with or without 1 mL of S9 mix.
The cells were incubated in the test solutions over a period of 4 hours.
Following exposure of the cells to the test material, the culture medium was replaced by fresh medium supplemented with 10% fetal calf serum and the cells were incubated again for further 14 hours until harvesting. Approximatively 2 to 3 hours prior harvesting, 0.2 µg/mL Colcemid in culture medium was added to the cells in order to stop mitosis in the metaphase. The cells were harvested after a total period of 18 hours, corresponding to 1 - 1.5 x the normal cycle time of the cells.
For harvesting, the culture medium was completely removed and the cells were subjected to an hypotonic treatment (0.4% KCl, 37 °C, 20 minutes); thereafter they were fixed (methanol: glacial acetic acid, 3:1), dried and stained on the slides with a solution of Giemsa and Titrisol. The cell preparations were mounted using Corbit-Balsam.
In each culture, 200 (100 for positive controls) well-spread metaphases were counted and cells displaying 20 to 22 chromosomes were examined for structural and numerical chromosome aberrations.

Evaluation criteria:

- Cells displaying 20 to 22 chromosomes were examined for structural chromosome aberrations, such as gaps, breaks, fragments, segment loss, multiple aberrations, pulverization (break down of chromosomes into irregular particles) and/or exchanges.
- Numerical aberrations such as e.g. hyperploidy (metaphase with additional chromosomes) also were considered.
- The mitotic index was determined and the cells were counted for determination of cytotoxicity using additional cultures treated as those of the main test.
The definitions used within the chromosomal analysis were based on following references:
(1)- Evans HJ, O'Riordan ML (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutat Res. 31(3): 135-48 (published)
(2)- Savage JRK (1975) Classification and relationships of induced chromosomal structural changes. J. Med. Genet. 12: 103-122 (published)
(3)- Standard-Protokoll zur cytogenetischen Auswertung von Mitose- und Meiose-chromosomen bei der Routineuntersuchung; ausgearbeitet von der Arbeitsgruppe der Industrie, Cytogenetik“, 1987.

Statistics:

The statistical evaluation of the findings was carried out using the MUCHAN program system (BASF AG). Fisher´s exact test for the hypothesis of equal proportions was used for the comparison of each test dose with the control. The test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Glutaraldehdyde caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases (with/without gaps).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell growth inhibition indicative of cytotoxicity was observed from 1 µg/mL in the absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Glutaraldehyde caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases (with/without gaps).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell growth inhibition indicative of cytotoxicity was observed at the highest tested concentration of 20 µg/mL in the presence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mitotic index (based on 1000 cells/culture):
A weak suppression of the mitotic activity was observed under all experimental conditions, as shown in following table 3.

Cell attachment to the slides and quality of the metaphases:
The treatment of the cells with the different doses of test substance did not disturb cell attachment on the slides. Within the dose-range that was tested, the quality of the metaphases was sufficiently good to allow chromosomal evaluation.

Cytotoxicity, cell count:
A slight growth inhibition of the cells was observed under all experimental conditions, as shown in table 4.

Any other information on results incl. tables

Table 1. Glutaraldehyde-induced chromosome aberration, without metabolic activation:

Dose (µg/ml)	Metaphases with aberrations^a
	Incl. Gaps (%)	Excl. Gaps (%)	EX (%)	MA (%)	CD (%)	AP (%)	PP (%)	EP (%)
Neg. Cont.	3.0	2.0	1.0	0.0	0.0	0.0	0.5	0.0
0.25	4.0	4.0	2.5	0.0	0.0	0.0	0.0	0.0
0.50	5.0	4.0	3.0	0.0	0.0	0.0	0.5	0.0
1.00	12**	11**	11**	0.0	0.0	0.0	0.0	0.0
Pos. Cont.(EMS)	19**	19**	12**	0.0	0.0	0.0	0.0	0.0

^a, Total number of metaphases: 200 (except for positive control, 100)

EX, exchanges

MA, multiple aberrations

CD, chromosomal disintegration

AP, aneuploidy

PP, polyploidy

EP, endoploidy

*: p<=0.05; **: p<=0.01

Table 2. Glutaraldehyde-induced chromosome aberration, with metabolic activation:

Dose (µg/ml)	Metaphases with aberrations^a
	Incl. Gaps (%)	Excl. Gaps (%)	EX (%)	MA (%)	CD (%)	AP (%)	PP (%)	EP (%)
Negative Control	3.0	1.5	0.0	0.0	0.0	0.0	0.0	0.0
2.50	3.5	2.5	2.5*	0.0	0.0	0.0	0.0	1.0
5.00	6.5	6.5*	5.0**	0.0	0.0	0.0	0.5	0.5
10.00	8.0	7.5**	6.5**	0.0	0.0	0.0	0.0	0.0
Positive Control (CPP)	19**	17**	12**	0.0	0.0	0.0	0.0	0.0

^a, Total number of metaphases: 200 (except for positive control, 100)

EX, exchanges

MA, multiple aberrations

CD, chromosomal disintegration

AP, aneuploidy

PP, polyploidy

EP, endoploidy

*: p<=0.05; **: p<=0.01

Table 3. Mitotic index data:

Without S9 mix
Test groups	Relative percentage of mitotic cells (i.e. versus 100% for negative control)
Negative control	100%
0.25 µg/ml	69.2%
0.50 µg/ml	77.1%
1.00 µg/ml	71.8%
Positive control ()	74.0%
With S9 mix
Negative control	100%
2.50 µg/ml	65.9%
5.00 µg/ml	75%
10.0 µg/ml	54.1%
Positive control (CPP)	56.3%

Table 4. Cytotoxicity, cell count data:

Without S9 mix
Test groups	Cell count as percentage of negative control
Negative control	100%
0.25 µg/ml	106.6%
0.50 µg/ml	107.6%
1.00 µg/ml	64.0%
2.00 µg/ml	73.1%
4.00 µg/ml	57.9%
With S9 mix
Negative control	100%
1.25 µg/ml	101.5%
2.50 µg/ml	91.3%
5.00 µg/ml	98.0%
10.0 µg/ml	82.1%
20.0 µg/ml	55.9%

Applicant's summary and conclusion

Conclusions:

Interpretation of results: positive