5-hexyldihydro-5-methylfuran-2(3H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation assay (Ames test) was conducted in five different bacterial strains and concluded that the test substance was non mutagenic with and without metabolic activation but cytotoxic to the bacteria in high concentrations.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-06-28 to 2002-10-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

S9 addition was not done at all concentration levels with all test strains.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

Deviations:

yes

Remarks:

S9 addition was not done at all concentration levels with all test strains.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium strains had mutations in the histidine locus (histidine deficiency), rfa-minus (deficient lipopolysaccharide envelope), uvrB-minus (deficient excision repair; not TA102) and TA 98, TA 100 and TA 102 carried R-factor plasmid pKM 101 (ampicillin resistance marker). The histidine mutation of TA 102 was unrevertable but a revertable histidine mutation in the pAQ1 plasmid. This allowed the detection of A-T base mutations in contrast to G-C base mutations detected by TA 1535 and TA 100. TA 1537 and TA 98 detected frameshift mutations.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for TA98 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA1537 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for TA100 and TA1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

for TA102 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for TA98, TA100, TA102, TA1535 und TA1537 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant colonies

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity

Statistics:

X2-test

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

COMPARISON WITH HISTORICAL CONTROL DATA: data was stated but no direct comparison was made

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without metabolic activation cytotoxicity was observed for all test strains at a concentration of 1500 µg/plate and for TA102 at 500 µg/plate. With metabolic activation cytotoxicity was detected for 1500 µg/plate in TA 102. The other test strains showed cytotoxicity with metabolic activation at 5000 µg/plate.

Experiment 1 (plate incorporation)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA98	TA100	TA102	TA1535	TA1537
	Results without S9
Untreated	44 ± 4	110 ± 16	269 ± 14	11 ± 5	9 ± 4
DMSO	38 ± 5	101 ± 5	270 ± 8	16 ± 5	8 ± 2
1.5			249 ± 9
5	39 ± 5	101 ± 7	272 ± 10	12 ± 1	8 ± 2
15	42 ± 5	108 ± 13	242 ± 7	10 ± 3	8 ± 1
50	36 ± 6	160 ± 5	282 ± 12	17 ± 0	7 ± 2
150	37 ± 9	100 ± 12	224 ± 10	12 ± 3	7 ± 4
500	33 ± 7	101 ± 8		13 ± 5	4 ± 1
NaN3 (0.7)		491 ± 29		411 ± 9
2-NF (2.5)	463 ± 107
9-AA (50)					454 ± 49
Mitomycin C (0.15)			788 ± 28
	Results with S9
Untreated	38 ± 5	130 ± 16	251 ± 17	12 ± 5	16 ± 2
DMSO	43 ± 2	112 ± 2	260 ± 5	11 ± 0	17 ± 5
50	39 ± 5	98 ± 17	238 ± 33	9 ± 3	16 ± 7
150	37 ± 4	118 ± 7	248 ± 14	11 ± 2	17 ± 5
500	42 ± 10	116 ± 10	225 ± 10	8 ± 5	16 ± 2
1500	47 ± 4	81 ± 5	126 ± 18	8 ± 2	12 ± 5
5000	21 ± 5	0 ± 0	1 ± 2	0 ± 0	2 ± 2
2-AA (0.8)	749 ± 81	613 ± 85
2-AA (0.9)			378 ± 32	132 ± 30
2-AA (1.7)					177 ± 8

Experiment 2 (plate incorporation)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA98	TA100	TA102	TA1535	TA1537
	Results without S9
Untreated	52 ± 8	92 ± 8	214 ± 21	12 ± 3	13 ± 3
DMSO	44 ± 8	91 ± 4	219 ± 10	12 ± 2	11 ± 4
5			223 ± 7
15	49 ± 6	76 ± 7	234 ± 14	10 ± 2	8 ± 2
50	53 ± 7	94 ± 9	237 ± 10	8 ± 4	9 ± 2
150	59 ± 7	86 ± 3	219 ± 41	8 ± 3	10 ± 2
500	48 ± 7	72 ± 4	185 ± 7	10 ± 4	8 ± 3
1500	35 ± 3	51 ± 3		8 ± 2	5 ± 3
NaN3 (0.7)		409 ± 18		322 ± 16
2-NF (2.5)	431 ± 18
9-AA (50)					228 ± 22
Mitomycin C (0.15)			605 ± 65
	Results with S9
Untreated	48 ± 11	88 ± 15	259 ± 11	19 ± 5	11 ± 2
DMSO	44 ± 5	93 ± 6	261 ± 16	16 ± 2	13 ± 0
15	47 ± 2	85 ± 6	230 ± 23	20 ± 5	11 ± 1
50	41 ± 2	83 ± 5	238 ± 4	15 ± 4	11 ± 6
150	38 ± 7	90 ± 5	223 ± 13	16 ± 5	10 ± 2
500	45 ± 8	93 ± 9	228 ± 2	11 ± 3	12 ± 5
1500	39 ± 6	79 ± 9	152 ± 13	14 ± 3	8 ± 3
2-AA (0.8)	707 ± 23	619 ± 43
2-AA (0.9)			377 ± 20	133 ± 21
2-AA (1.7)					194 ± 9

Conclusions:

A bacterial reverse mutation assay (Ames test) was conducted in five different bacterial strains. The test substance was found to be non mutagenic with and without metabolic activation but cytotoxic to the bacteria in high concentrations.

Executive summary:

The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations of 1.5 to 5000 µg per plate in the presence and absence of S9. In the absence of S9-mix the test substance was bacteriotoxic towards all strains at a concentration of 1500 µg/plate and for TA102 at 500 µg/plate. In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 1500 µg /plate and towards the strains TA1535, TA1537, TA98, and TA100 at 5000 μg/plate. Precipitation of the test compound on the plates was not observed. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations of 1.5 to 5000 µg per plate in the presence and absence of S9. In the absence of S9-mix the test substance was bacteriotoxic towards all strains at a concentration of 1500 µg/plate and for TA102 at 500 µg/plate. In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 1500 µg /plate and towards the strains TA1535, TA1537, TA98, and TA100 at 5000 μg/plate. Precipitation of the test compound on the plates was not observed.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.