Isovaleric acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

alkaline comet assay as described by Sing et al, Exp. Cell Res. 175 (1988) 184-191

GLP compliance:

not specified

Type of assay:

comet assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbutan-1-ol

EC Number:

204-633-5

EC Name:

3-methylbutan-1-ol

Cas Number:

123-51-3

IUPAC Name:

3-methylbutan-1-ol

Details on test material:

- Name of test material (as cited in study report): 3-methylbutan-1-ol; obtained from Fluka
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Storage condition of test material: no data

Method

Species / strain

Species / strain 1

Species / strain / cell type:

primary culture, other: human lung carcinoma epithelial A549 cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/mL penicillin and streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data

Species / strain 2

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM Eagle medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/mL penicillin and streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data

Species / strain 3

Species / strain / cell type:

other: peripheral human blood (pB) cells

Metabolic activation:

without

Test concentrations with justification for top dose:

23, 46, and 91 mM (2.03, 4.05, 8.02 mg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): immediately after exposure

NUMBER OF CELLS USED FOR COMET ASSAY: 10,000

DETERMINATION OF CYTOTOXICITY (only for A549 cells)
- Method: Colony forming ability (CFA) after treatment wit test substance; determination of colony numbers from triplicate plates; five to seven concentrations of test substance tested in three to five separate experiments, IC50 value is the concentration resulting in 50% inhibition of colony formation

EXAMINATION:
- alkaline comet assay: immediately after termination of treatment; cells were lysed over night and exposed to alkali for 60 min prior to electrophoresis.

Evaluation criteria:

DNA damage expressed by Tail moment (TM) calculated according to Olive et al Radiat. Res. 122 (1990) 86-94 using 54 randomly selected cells

Statistics:

Non linear regression analysis of concentration-response curves (IC 50 determination) using the GraphPad Prism computer program (version 3.0).
Student's t-test for determination of significance between non-exposed control and exposed cells.

Results and discussion

Test results

Test results 1

Species / strain:

primary culture, other: human lung carcinoma epithelial A549 cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

other: peripheral human blood (pB) cells

Metabolic activation:

without

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Cytotoxic and Genotoxic effects of 3-methylbutanol in the Comet assay

Substance	Cytotoxicity assay A549 cells (colony assay IC50), (mM)^a	Concentration (mM)	Comet Assay - Tail moment
			A549 cells	V79 cells	pB cells^b
Control non-exposed	-	-	1.55 ± 0.3	1.50± 0.54
MMS	0.2	0.025	4.34 ±1.4^c		10.55 ±4.05^c
		0.05	12.03 ± 4.90^c	3.47 ±0.34^c	29.91 ± 3.2^c
3-methyl-butanol	28	23	1.53 ± 0.28		1.62 ± 0.29
		46	1.62 ± 0.79	1.43 ± 0.91
		91	2.38 ± 1.01^c	2.96 ± 0.81	Toxic

a   Inhibitory concentration resulting in 50% reduction of colony formation

b Peripheral human blood cells

c   Significant different to control (t-test; P < 0.05)

The effect seen in A549 cells is considered to be caused by the toxicity of the test substance as in V79 and pB cells (test concentration 25-fold over IC50 value)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

3-methylbutanol was not genotoxic in the Comet Assay using human (A549, peripheral blood cells) Chinese hamster cell lines (V79).

Executive summary:

A459 human lung carcinoma epithelial cells, V79 Chinese hamster lung fibroblasts, and human peripheral blood cells were exposed to 3-methylbutanol at concentrations of 23, 46, and 91 mM (2.03, 4.05, 8.02 mg/mL) without mammalian metabolic activation. After treatment an alkaline comet assay was performed.

The negative and positive controls (0.025 and 0.05 mM methylmethansulfonate) did induce the appropriate response. With 3 -methylbutanol, cytotoxicity was seen at the highest dose level, but there was no evidence of specific genotoxic effects. Hence, 3-methylbutanol was not genotoxic in the Comet Assay (Kreja and Seidel, 2002).

 

There is no guideline for this kind of assay. The study is considered to be valid and suitable for the assessment of 3-methylbutanol. As this substance is rapidly oxidised to isovaleric acid the result providence evidence that isovaleric acid is not genotoxic.