α-hydroxy-β,β-dimethyl-γ-butyrolactone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific principles with acceptable deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

; only 4 bacterial strains were used for testing (5 are recommended)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α-hydroxy-β,β-dimethyl-γ-butyrolactone

EC Number:

209-963-3

EC Name:

α-hydroxy-β,β-dimethyl-γ-butyrolactone

Cas Number:

599-04-2

Molecular formula:

C6H10O3

IUPAC Name:

(3R)-dihydro-3-hydroxy-4,4-dimethyl-2(3H)-furanone

Details on test material:

- Name of test material (as cited in study report): D-Pentolacton kristallin
- Analytical purity: >99 %
- Storage condition of test material: room temperature

Method

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH 7.4).

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 µg per plate, in standard plate test and preincubation test, with and without S9-mix

Vehicle / solvent:

Aqua dest.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "Deatils on test system and conditions"

Details on test system and experimental conditions:

Standard plate test:
The experimental procedure is based on the method of Ames et al.
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test:
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37 °C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Controls:
- Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) is carried out for each tester strain in order to determine the spontaneous mutation rate.
- Positive controls:
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix: 10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535
without S-9 mix: 5 µg N-methyl-N´-nitro-N-nitrosoguanidine (MNNG), (dissolved in DMSO) for the strains TA 100 and TA 1535 and 10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98; 100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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:

No increase in the number of his revertants was seen in the standard plate test and in the preincubation test, with or without the addition of S9-mix.
No bacteriotoxic effect (reduced his background growth) was observed. The test material was completely soluble in aqua dest.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Maximum revertants/plate and corresponding test concentrations in the standard plate test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	Aqua dest	16 ± 4	13 ± 1
	Test substance	24 ± 5 [5000]	21 ± 1 [20]
	Positive Control	1927 ± 64	114 ± 17
S. typhimurium TA100	Aqua dest	105 ± 6	112 ± 13
	Test substance	118 ± 21 [20]	126 ± 13 [500]
	Positive Control	2083 ± 161	1913 ± 42
S. typhimurium TA1537	Aqua dest	8 ± 3	12 ± 2
	Test substance	12 ± 6 [500]	17 ± 3 [500]
	Positive Control	527 ± 34	185 ± 19
S. typhimurium TA98	Aqua dest	30 ± 3	44 ± 1
	Test substance	38 ± 6 [2500]	57 ± 11 [100]
	Positive Control	815 ± 19	1447 ± 160
Table 2: Maximum revertants/plate and corresponding test concentrations in the preincubation test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	Aqua dest	12 ± 2	13 ± 1
	Test substance	19 ± 1 [2500]	14 ± 4 [500]
	Positive Control	1024 ± 29	142 ± 2
S. typhimurium TA100	Aqua dest	104 ± 7	118 ± 4
	Test substance	123 ± 3 [20]	132 ± 19 [100]
	Positive Control	1203 ± 45	1260 ± 70
S. typhimurium TA1537	Aqua dest	8 ± 1	12 ± 1
	Test substance	11 ± 9 [2500]	12 ± 2 [100]
	Positive Control	495 ± 161	162 ± 9
S. typhimurium TA98	Aqua dest	26 ± 6	42 ± 3
	Test substance	37 ± 5 [5000]	48 ± 11 [500]
	Positive Control	1034 ± 106	995 ± 205