2-hexyloxyethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

GLP studies, conducted according to or equivalent to OECD guidelines 471, 473, 474 and 476, are available for ethylene glycol hexyl ether.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

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.

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

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.

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

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'.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with GLP and similar to OECD guideline 476.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

none

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: CHO cells used in these studies were obtained fromAbraham Hsie at Oak Ridge National Laboratory with the designation CHO-K1-BH4- (subclone Dl) (or simply CHO for report purposes). Cells are thawed periodically from frozen stock cultures and are maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine. For treatment of cells without metabolic activation, F12 medium with 50 units/ml of penicillin, 50 pg/ral streptomycin, and 5% (v/v) of dialyzed bovine serum (F12-D5) is used. For treatments incorporating an S9 metabolic activation system^ Identical mediums but without serum, is employed. For determination of mutant frequencies, F12-D5 medium containing 2.0 pg/ml TG (6~thloguanine) is used as a "selective medium."

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9, rat liver homogenate

Test concentrations with justification for top dose:

0.3 to 0.8 mg/ml (-S9)
0.3 to 1.0 mg/ml (+S9)

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate (-S9), Dimethylnitrosamine (+S9)

Remarks:

none

Details on test system and experimental conditions:

Exposure duration: 5 hours

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
A test substance, for which the results do not meet the above criteria is considered nonmutagenic in this system.
Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. A positive concentrationresponse that is reproducible is most meaningful. Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.

Statistics:

The specific test used to obtain the level of statistical significance is Indicated with a footnote on the table. Data from the CHO tests do not follow a normal distribution according to experience with historical controls. Thus, the data were analyzed after transformation of the mutation frequencies (MF) and SCE values according to the conversion method of Box and Cox (1964). This procedure for CHO data follows procedures described by Snee and Irr; (MF + I) 0 * 1 5 (Snee, R.D. and J.D. Irr, Mutation Research, 85 (1981), 77-93). For CHO mutation studies with a concurrent control frequency of zero mutants, the variance of recent historical controls was used for the statistical analyses. For SCE data, statistical analyses of historical data at BRRC indicate that an exponent of 0.15 is the appropriate value for transformation of SCE values (BRRC Intramural Report 46-64). Positive controls for the CHO mutation test were run concurrently to assess the sensitivity of the assays in comparison to historical experience with the test system. Data for positive control agents were not compared statistically whenever differences were at least 5 times the concurrent negative control value and results were within the historical positive control range.
Rounding of data to either two decimal places or to the appropriate number of significant figures was performed for presentation on tables. Although statistically significant decreases in mutation indices can occur because of cytotoxic responses, only statistically significant increases in responses above control values are indicated on tables for simplicity. All statistical tests were performed to determine whether the treatment with the test agent produced a response statistically different from the value(s) obtained with the concurrent solvent control. The degree of statistical significance at BRRC is denoted by the letters; a: 0.05 > p > 0.01, b: 0.01 > p > 0.001, or c: p <0,001. No superscript (or NS) Indicates p > 0.05.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

hexyl CELLOSOLVE was relatively less cytotoxlc when tested with S9 metabolic activation than without S9. A concentration of 1.5 mg/ml produced complete growth Inhibition and death of CHO cells tested without S9 activation and > 90% growth inhibition with

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill no more than (approximately) 90% of the treated cells. Test results with ethylene glycol hexyl ether indicated that concentrations above 2.0 tag/ml were lethal to CHO cells. The highest suitable concentrations for testing were 1.0 ing/ml without S9 and 1.3 mg/ml with S9 activation for the CHO/HGPRT mutation test.

COMPARISON WITH HISTORICAL CONTROL DATA: was in the range of historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:The highest concentrations evaluated in each assay were the maximum attainable doses for each test. Only slightly greater concentrations of 1.0 mg/ml (-S9) and 1.3 mg/ml (+S9) produced excessive cytotoxicity and killed the duplicate cultures for these doses. Hexyl CELLOSOLVE was judged to lack mutagenic activity In vitro under the conditions of the CHO/HGPRT test system.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

See the attached word doc for tables and figure.

Conclusions:

Interpretation of results (migrated information):
negative

The results indicated that Ethylene glycol hexyl ether did not produce a dose-related or repeatable, significant mutagenic effect in HGPRT gene mutation test.

Executive summary:

Ethylene glycol hexyl ether was evaluated for potential genotoxic activity using the Chinese Hamster Ovary (CHO) gene mutation test (HGPRT). Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill no more than (approximately) 90% of the treated cells.

The results of the study indicated that concentrations above 2.0 mg/ml were lethal to CHO cells. The highest suitable concentrations for testing were 1.0 mg/ml without S9 and 1.3 mg/ml with S9 activation for the CHO gene mutation test. Various dose levels of ethylene glycol hexyl ether for testing were attained by direct addition of various aliquots of the diluted or undiluted test agent into the cell culture medium. Ethanol was used as the diluent when necessary. Duplicate cultures of CHO cells were exposed for 5 hours to a minimum of five concentrations of ethylene glycol hexyl ether in tests both with and without the addition of a rat-liver S9 metabolic activation system. The plating efficiency of these cells was assessed in non-selective medium using 4 plates/dosed culture with 100 cells/plate. For the definitive tests to determine potential effects upon SCEs, a range of doses from 0.1 to 0.8 mg/ml was tested without addition of an S9 metabolic activation system and 0.1 to 1.0 mg/ml was tested with S9 activation, respectively.

The results indicated that ethylene glycol hexyl ether did not produce a dose-related or repeatable, significant mutagenic effect in gene mutation test at the used experimental conditions.

Reference 3

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

Study period:

1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with GLP and similar to OECD guideline 473.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 (modified) medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum but lacking hypoxanthine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9, rat liver homogenate

Test concentrations with justification for top dose:

0.1 to 0.8 mg/ml (-S9)
0.08 to 0.4 mg/ml (+S9)

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: triethylenemelamine (-S9), cyclophosphamide (+S9)

Remarks:

none

Details on test system and experimental conditions:

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell cycle progression.
A test substance for which the results do not meet the above criteria is considered nonmutagenic in this system.
Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
Positive results from the in vitro chromosome aberration test indicate that the test substance induces structural chromosome aberrations in cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in
cultured mammalian somatic cells.

Statistics:

Analyses of the test data employed the Fisher' s Exact Test (one-tailed) to determine statistical significance of differences between the test and control populations. This statistical test was considered appropriate for the analysis of the data because it is a distribution independent test and
cytogenetic data often vary from a normal distribution required f o r parametric analyses. The levels of s t a t i s t i c a l differences are denoted a t BRRC by the letters : a = 0.05 > p > 0.01; b = 0.01 > p > 0.001; c = p < 0.001. A positive test r e s u l t was interpreted by the attainment of differences from the control a t the p < 0.05 level of significance f o r a t l e a s t one test concentration, together with an indication of a concentration-related effect of exposures or reproducibility between the duplicate cultures . Rounding of data to e i t h e r one or two decimal places, or to the appropriate
number of significant figures, was performed for presentation on tables. Statistical tests were performed t o determine whether the treatment with t e
test agent produced a response t h a t was significantaly increased above the value(s) obtained with the concurrent solvent control cultures.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

concentrations of 0.6 mg/ml and 0.8 mg/ml, tested respectively with and without S9 metabolic activation, produced corresponding levels of 62.5% and 50.0% reduction in the percentages of mitotic cells.

Vehicle controls validity:

other:

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In a preliminary study CHO cells were exposed to a range of concentrations of the test material to determine an appropriate cytotoxic range of doses. A concentration range between 0.1 mg/ml t o 0.8 mg/ml was tested in cytogenetic test without S9, while concentrations between 0.08 mg/ml t o 0.4 mg/ml were tested in the presence of S9. The highest three concentrations i n each t e s t which did not
produce excessive mitotic inhibition were evaluated for chromosome effects .


ADDITIONAL INFORMATION ON CYTOTOXICITY: concentrations of 0.6 mg/ml and 0.8 mg/ml, tested respectively with and without S9 metabolic activation, produced corresponding levels of 62.5% and 50.0% reduction in the percentages of mitotic cells.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

See the attached word doc. for tables and figures:

Conclusions:

Interpretation of results (migrated information):
negative

Ethylene glycol hexyl ether did not produce statistically significant increases in the incidence of chromosome aberrations in CHO cells in tests conducted with and without the addition of a rat-liver homogenate, S9 metabolic activation system.

Executive summary:

Ethylene glycol hexyl ether was evaluated for potential genotoxic activity using the Chinese Hamster Ovary (CHO) in vitro chromosome aberration test system. Test concentrations of ethylene glycol hexyl ether ranged from 0.1 mg/ml to 0.8 mg/ml without S9 activation and from 0.08 mg/ml to 0.4 mg/ml with a rat-liver S9 metabolic activation system. Test concentration ranges were chosen on the basis of a preliminary cytotoxicity test and the highest dose produced approximately 40-60% mitotic inhibition. The highest three doses which did not produce excessive cytotoxic inhibition of mitotic cells were scored for the incidence of chromosome aberrations.

Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would not excessive inhibition of the mitotic index. Test results indicated that concentrations of 0.6 mg/ml and 0.8 mg/ml, tested respectively with and without S9 metabolic activation, produced corresponding levels of 62.5% and 50.0% reduction in the percentages of mitotic cells. Concentrations of 0.4 mg/ml and 0.8 mg/ml were chosen as the maximum test doses to be used in the definitive tests with and without activation, respectively.

Ethylene glycol hexyl ether did not produce statistically significant increases in the incidence of chromosome aberrations in CHO cells in tests conducted with and without the addition of a rat-liver homogenate, S9 metabolic activation system. The positive control agents, cyclophosphamide and triethylenemelamine, both produced highly significant increases in chromosome aberrations indicative of the appropriate sensitivity of the test system. Hence, ethylene glycol hexyl ether was not considered to be a clastogenic agent under the conditions of this in vitro test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ethylene glycol hexyl ether was tested for potential genotoxic activity in the the Salmonella/microsome bacterial mutagenicity assay (Ames test), in the Chinese Hamster Ovary (CHO) gene mutation test (HGPRT), in a chromosome aberration in CHO cells and in SCE assay in CHO cells. All tests were conducted with and without the addition of a rat-liver homogenate, S9 metabolic activation system.

Under the conditions of these in vitro tests, ethylene glycol hexyl ether was not genotoxic, with and without addition of an S9 activation system.

Justification for selection of genetic toxicity endpoint
GLP study conducted according to OECD TG 471

Justification for classification or non-classification

Ethylene glycol hexyl ether was not genotoxic in any of the 4 in vitro tests, with and without addition of an S9 activation system.