2-hexyloxyethanol

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

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD Guideline 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-hexyloxyethanol

EC Number:

203-951-1

EC Name:

2-hexyloxyethanol

Cas Number:

112-25-4

Molecular formula:

C6H13OCH2CH2OH

IUPAC Name:

2-hexyloxyethanol

Details on test material:

- Name of test material: n-Hexylglycol
- Physical state: liquid, colorless, clear
- Analytical purity: > 99.2%
- Lot/batch No.: Tank: B711 (December 1st, 2007)
- Expiration date of the lot/batch: May 31th, 2008
- Storage condition of test material: Room temperature (N2 conditions)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

20; 100; 500; 2 500 and 5 000 μg/plate (Standard Plate Test)
312.5; 625; 1 250; 2 500 and 5 000 μg/plate (Preincubation Test)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

without tester strains

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9 mix: 2-aminoanthracene (2.5 µg/plate in DMSO). Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate in DMSO; TA 1535 & TA 100), 4-nitro-o-phenylenediamine (10 µg/plate in DMSO; TA 98), 9-aminoacridine (100 µg/plate in DMSO; TA 1537)

Remarks:

S. typhimurium strains

Controls 2

Untreated negative controls:

yes

Remarks:

without tester strains

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9 mix: 2-aminoanthracene (60 µg/plate in DMSO). Without S9 mix: 4-nitroquinoline-N-oxide (5 µg/plate in DMSO)

Remarks:

E. coli WP2 uvrA

Details on test system and experimental conditions:

STANDARD PLATE TEST:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. (Mut. Res. 31:347-364, 1975; Mut. Res. 113:173-215, 1983).

- Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] 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 about 42-45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48–72 h in the dark, the bacterial colonies (his+ revertants) are counted.

- Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42-45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 sec. The composition of the minimal agar (SA1 selective agar) is based on the description of Green MHL and Muriel WJ (Mut. Res. 38:3-32, 1976), with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48-72 h in the dark, the bacterial colonies (trp+ revertants) are counted.

PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48:121-130, 1977) and Matsushima et al. (In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York (1980)).
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 min using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 sec.
After incubation at 37°C for 48-72 h in the dark, the bacterial colonies are counted.

TITER DETERMINATION:
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. Test tubes containing 2-mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42-45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10E-6)
0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 min using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 sec. After incubation at 37°C for 48-72 h in the dark, the bacterial colonies are counted.

Evaluation criteria:

Evaluation criteria are mutagenicity of the test substance, bacterial titer, toxicity and solubilty of the test substance.
Acceptance criteria are as follows:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was > 10E8/mL.
Assessment criteria are as follows:
- The test chemical is considered positive in this assay if a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system, is observed.
- A test substance is generally considered non-mutagenic in this test if the number of revertants for all tester strains are within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

> 2500 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (decrease in the number of his+ revertants, reduction in the titer) was occasionally observed in the standard plate test depending on the strain and test conditions from about 2 500 μg/plate onward. In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed from about 2 500 μg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of the Standard Plate Test:

Dose/plate (µg)	Without S9 mix		With S9 mix (1:9)
	Mean	SD	Mean	SD
Salmonella typhimurium TA 1535
0 (DMSO)	16	1	15	4
20	16	5	12	2
100	13	3	14	4
500	14	1	12	2
2500	13	4	11	4
5000	8	1	4	1
MNNG 5.0 µg	559	28
2-AA 2.5 µg			163	20
Salmonella typhimurium TA 100
0 (DMSO)	111	3	112	7
20	96	7	115	8
100	110	3	109	13
500	98	11	117	6
2500	91	10	116	7
5000	71	10	82	3
MNNG 5.0 µg	586	58
2-AA 2.5 µg			913	141
Salmonella typhimurium TA 1537
0 (DMSO)	9	3	9	2
20	9	2	11	1
100	8	3	10	1
500	8	2	8	2
2500	8	2	6	2
5000	3	2	4	2
AAC 100.0 µg	467	25
2-AA 2.5 µg			154	15
Salmonella typhimurium TA 98
0 (DMSO)	29	6	36	9
20	25	7	36	8
100	29	3	57	14
500	29	7	41	3
2500	26	2	28	4
5000	15	6	27	7
NOPD 10.0 µg	433	41
2-AA 2.5 µg			914	60
Escherichia coli WP2 uvrA
0 (DMSO)	35	3	38	6
20	33	9	53	4
100	35	4	46	5
500	39	12	44	12
2500	33	2	49	9
5000	29	7	32	11
4-NQO 5.0 µg	792	39
2-AA 60 µg			277	7

Table 2: Results of the Preincubation Test:

Dose/plate (µg)	Without S9 mix		With S9 mix (1:9)
	Mean	SD	Mean	SD
Salmonella typhimurium TA 1535
0 (DMSO)	16	1	15	3
312.5	18	7	15	4
625	14	1	14	1
1250	14	3	14	2
2500	11	2	11	2
5000	0B	0B	0B	0B
MNNG 5.0 µg	539	18
2-AA 2.5 µg			129	18
Salmonella typhimurium TA 100
0 (DMSO)	103	13	96	6
312.5	99	13	102	7
625	98	8	92	9
1250	104	9	106	6
2500	78	12	78	7
5000	0B	0B	0B	0B
MNNG 5.0 µg	723	48
2-AA 2.5 µg			768	28
Salmonella typhimurium TA 1537
0 (DMSO)	8	2	9	3
312.5	8	2	5	2
625	8	3	6	3
1250	7	2	7	3
2500	7	1	5	1
5000	0B	0B	0B	0B
AAC 100.0 µg	487	57
2-AA 2.5 µg			130	18
Salmonella typhimurium TA 98
0 (DMSO)	29	5	33	6
312.5	26	5	33	3
625	28	5	34	3
1250	27	6	34	4
2500	23	4	31	6
5000	0B	0B	0B	0B
NOPD 10.0 µg	581	57
2-AA 2.5 µg			583	34
Escherichia coli WP2 uvrA
0 (DMSO)	34	2	42	3
312.5	45	10	39	11
625	41	2	38	7
1250	38	5	37	10
2500	38	3	29	3
5000	0B	0B	0B	0B
4-NQO 5.0 µg	587	39
2-AA 60 µg			254	34

B: Reduced background growth

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The study was performed according to OECD Guideline 471 under GLP conditions.

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The used strains were S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvr. The applied dose ranges were 20-5000 μg/plate for the standard plate test (SPT) and 312.5 -5000 μg/plate for the preincubation test (PIT), respectively. SPT and PIT were performed both with and without metabolic activation (induced rat liver S9 mix). No precipitation of the test substance was found. A bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 μg/plate onward. An increase in the number of his+ (S. typhimurium) or trp+ (E. coli) revertants was not observed in the SPT or in the PIT either without S9 mix or after the addition of a metabolizing system.

Conclusion: The test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.