2-(propyloxy)ethanol

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:

6 December 2010 to 27 January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to generally valid and/or internationally accepted testing guidelines and followed recognized GLP standards.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(propyloxy)ethanol

EC Number:

220-548-6

EC Name:

2-(propyloxy)ethanol

Cas Number:

2807-30-9

Molecular formula:

C5H12O2

IUPAC Name:

2-(propyloxy)ethanol

Details on test material:

- Name of test material (as cited in study report): ethylene glycol monopropyl ether
- Physical state: clear colorless liquid
- Analytical purity: 99.820 wt%
- Impurities (identity and amount): 0.012 wt%
- Purity test date: 15 November 2010 (COA)
- Lot/batch No.: TXEG
- Receipt date: 25 November 2010
- Expiration date of the lot/batch: 25 November 2011
- Storage condition of test material: room temperature in the dark
- Other: The integrity of supplied data relating to the identity, purity and stability of the test item was the responsibility of the Sponsor.

Method

Target gene:

Salmonella typhimurium:
TA 1535: his G 46; rfa-; uvrB-
TA 98: his D 3052; rfa-; uvrB-; R-factor
TA 1535: his G 46; rfa-; uvrB-
TA 100: his G 46; rfa-; uvrB-; R-factor

Escherichia coli (WP2uvrA/pKM101): trp-; uvrA-
contains the pKM101 plasmid

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate

Experiment 1
50, 150, 500, 1500 and 5000 microg/plate

Experiment 2 - with preincubation
50, 150, 500, 1500 and 5000 microg/plate

Vehicle / solvent:

Sterile distilled water (50 mg/mL)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Pos. Control for TA98 at 5 microg/plate (+S9)

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

Pos. Control for TA100 at 1 microg/plate (+S9); TA1535 and TA1537, 2 microg/plate (+S9); and E. coli WP2uvrA- at 10 microg/plate (+S9)

Controls 3

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Pos. Control for E. coli WP2uvrA- at 2 microg/plate (-S9); TA100 at 3 microg/plate (-S9); TA1535 at 5 microg/plate (-S9)

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Pos. Control for TA1537 at 80 microg/plate (-S9)

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Pos. Control for TA98 at 0.2 microg/plate (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of S9. Ten dose levels and controls were tested up to and including 5,000 microg/plate. The assay was conducted by mixing 0.1 ml the bacterial culture (TA100 or WP2uvrA-), and 0.1 ml of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 ml of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 deg C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Experiment 1:
Five concentrations of the test material were assayed with or without S9-mix against each tester strain using the above described direct plate incorporation method. An additional dose level and expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material.

Experiment 2:
A second experiment was performed at the same concentrations as in Experiment 1 with fresh bacterial cultures, test material and control solutions. Preincubation was employed. Thus, measured aliquots (0.1 mL) of each bacterial culture were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of vehicle or test material formulation and incubated for 20 minutes at 37 deg C with shaking at approximately 130 rpm prior to addition of 2 mL of molten trace histidine or tryptophan supplemented top agar. The contents of the tubes were mixed and poured onto the surface of Vogel-Bonner Minimal agar plates. This procedure was repeated for each bacterial strain either with or without S9.

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Evaluation criteria:

Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- All positive control chemicals should induce marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- The test should include a minimum of four non-toxic dose levels.
- There should be no evidence of excessive contamination.

Evaluation Criteria:

A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain either with or without metabolic activation. Biological relevance of the response is to be considered first, as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing (1989). Statistical methods can be used as an aid to evaluation but may not be the only determining factor for a positive response. Fold increase greater than 2x the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Mean values with standard deviations were reported.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Please refer to Tables 1 to 5 for details of results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic at 5000 microg/plate to tester strains TA100 and WP2uvrA-. The results are summarized in Table 1.

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9-mix	Strain	Dose (mg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	95	86	80	93	76	89	90	99	106	96	88
+	TA100	104	80	99	77	88	90	81	84	74	74	87
-	WP2uvrA-	41	45	40	38	44	37	33	40	33	41	33
+	WP2uvrA-	51	52	43	44	44	54	49	51	53	44	42

 

Table 2: Experiment 1 - without metabolic activation

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	100	24	47	21	11
50	106	26	43	24	12
150	100	22	43	22	13
500	104	20	47	27	12
1500	94	25	43	18	14
5000	99	24	40	19	11
ENNG (3)	403
ENNG (5)		285
ENNG (2)			290
4NQO (0.2)				184
9AA (80)					2993

Abreviations: ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4-nitroquinoline-1-oxide

 

Table 3: Experiment 1 - with metabolic activation

 

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	112	14	50	27	12
50	96	14	46	21	14
150	91	13	53	24	12
500	92	14	49	23	13
1500	97	11	52	22	13
5000	104	12	53	25	12
2AA (1)	472
2AA (2)		247			285
2AA (10)			327
BP (5)				188

 

Table 4: Experiment 2 - without metabolic activation

 

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	113	23	49	22	10
50	118	18	49	19	11
150	101	21	48	15	7
500	102	21	36	20	14
1500	104	21	51	23	12
5000	127	18	48	17	9
ENNG (3)	386
ENNG (5)		921
ENNG (2)			198
4NQO (0.2)				212
9AA (80)					1311

 

Table 5: Experiment 2 - with metabolic activation

 

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	122	12	49	37	13
50	91	15	41	37	12
150	103	16	46	35	13
500	120	13	46	42	14
1500	107	13	58	32	9
5000	130	15	57	33	16
2AA (1)	957
2AA (2)		344			247
2AA (10)			364
BP (5)				297

 

Applicant's summary and conclusion

Conclusions:

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

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains.

The test material was considered to be non-mutagenic under the conditions of this test.