3,5-xylenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22 April 2004 to 12 May 2004

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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:

Mixed xylenols

IUPAC Name:

Mixed xylenols

Details on test material:

- Name of test material (as cited in study report): mixed xylenols
- Substance type: alcohol
- Physical state: liquid
- Analytical purity: not stated
- Impurities (identity and concentrations):
- Composition of test material, percentage of components: 2,6-xylenol: 15.24%, 2,4-xylenol: 23.15%, 2,5-xylenol: 16.44%, 2,3-xylenol: 18.82%, 3,5-xylenol: 10.71%, 3,4-xylenol: 15.38% and other compounds
- Isomers composition: not applicable
- Purity test date: not stated
- Lot/batch No.: 20NOV2003
- Expiration date of the lot/batch: not stated
- Stability under test conditions: not stated
- Storage condition of test material: room temperature in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from Aroclor 1254 induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate for strains TA98, TA100, TA1535, TA1537 and WP2 uvrA for the preliminary toxicity study (with and without metabolic activation).
75, 200, 600, 1800 and 5000 µg/plate for TA98, TA100, TA1535, TA1537 and WP2 uvrA for the bacterial mutation assay (with and without metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO) used for vehicle and positive control solvent (2-aminoanthracene, 2-nitrofluorene, 9-aminoacridine and methyl methanesulphonate). Water used as a postive control solvent for sodium azide.
- Justification for choice of solvent/vehicle: Not soluble in prefereable solvent (water), therefore DMSO used which is commonly used solvent.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (+S9 for all strains); 2-nitrofluorene (-S9, TA98); Sodium azide (-S9, TA100, TA1535); 9-aminoacridine (-S9, TA1537); methyl methansulfonate (-S9, Wp2uvrA)

Remarks:

2-aminoanthracene for WP2 uvrA in the presence of metabolic activation. 2-nitroluorene for TA98; sodium azide for TA100 and TA1535; 9-aminoacridine for TA1537; methyl methanesulfonate for WP2 uvrA in the absence of metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

DURATION
- Exposure duration: 48 - 72 hours
- Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Evaluation criteria:

All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls. The mean of each positive control must exhibit at least a 3.0 fold increase in the number of revertants over the mean value of the respective control. A minimum of three non toxic dose levels is required for evaluation. A dose level is considered to be toxic if there is a >50% reduction in the mean number of revertants/plate compared to the mean vehicle control value and at least a moderate reduction in the background lawn. (codes 3,4,5 ).

Statistics:

None undertaken, but rather a fold increase was used to interpret the data.

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:

Determined by visual assessment of the background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data were found to support the study outcome.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The results of the genotoxicity study found that mixed xylenols were not genotoxic at any dose level tested. A summary of the results are presented below.

Table 1: Summary of results of the mutagenicity assay

Dose (µg/plate)	Average revertants per plate ± standard deviation
	TA98	TA100	TA1535	TA1537	WP2 uvrA
In the absence of metabolic activation (values in parathenesis refer to background lawn, a measure of cytotoxicity)
Vehicle control	17 ± 2 (1)	153 ± 13 (1)	16 ± 2 (1)	7 ± 2 (1)	24 ± 3 (1)
75	14 ± 5 (1)	126 ± 3 (1)	23 ± 5 (1)	7 ± 1 (1)	24 ± 2 (1)
200	17 ± 2 (1)	126 ± 31 (1)	19 ± 4 (1)	6 ± 2 (1)	20 ± 5 (1)
600	14 ± 5 (1)	133 ± 26 (1)	19 ± 2 (1)	6 ± 1 (1)	15 ± 3 (1)
1800	16 ± 3 (1)	125 ± 11 (1)	21 ± 7 (1)	4 ± 1 (1)	13 ± 3 (1)
5000	3 ± 1 (3)	0 ± 0 (4)	3 ± 1 (3)	0 ± 0 (4)	1 ± 2 (4)
Positive control	115 ± 12 (1)	551 ± 8 (1)	272 ± 6 (1)	644 ± 121 (1)	117 ± 7 (1)
In the presence of metabolic activation
Vehicle control	20 ± 2 (1)	151 ± 15 (1)	17 ± 5 (1)	6 ± 2 (1)	20 ± 6 (1)
75	22 ± 5 (1)	163 ± 20 (1)	17 ± 2 (1)	5 ± 1 (1)	19 ± 6 (1)
200	18 ± 5 (1)	168 ± 6 (1)	19 ± 2 (1)	6 ± 2 (1)	16 ± 2 (1)
600	22 ± 3 (1)	154 ± 6 (1)	21 ± 4 (1)	6 ± 3 (1)	16 ± 3 (1)
1800	22 ± 2 (1)	144 ± 11 (1)	23 ± 2 (1)	6 ± 2 (1)	9 ± 3 (1)
5000	4 ± 2 (1)	0 ± 0 (4)	6 ± 2 (3)	0 ± 0 (4)	0 ± 0 (4)
Positive control	1000 ± 147 (1)	74 ± 74 (1)	108 ± 9 (1)	128 ± 17 (1)	794 ± 95 (1)

Background lawn code

1. Normal

3. Moderately reduced

4. Extremley reduced

Applicant's summary and conclusion

Conclusions:

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

It is concluded that mixed xylenols showed no evidence of mutagenic activity in this bacterial system when tested up to 5000 ug/plate (the maximum concentration in accordance with current guidelines) under the test conditions employed.

Executive summary:

All criteria for a valid study were met. The plate incorporation method was used. Whilst a confirmatory second test was not undertaken, the preliminary toxicity test also determined the number of revertant colonies in both the presence and absence of S9 mix, with all strains. The maximum dose tested in all strains was 5000 ug/plate (the maximum dose in accordance with current guidelines). Toxicity was observed begining at 1800 or 5000 ug/mL. Mixed xylenols were therefore concluded to be negative for genotoxicity.

As 3,5 xylenol was only present in the mixture at 10.71%, the maximum concentration that this isomer was tested up to was equivalent to ~535 µg/plate. In the absence of marked toxicity higher concentrations could have been tolerated in the test system.

 

Four further Ames studies (Deanet al.,1985; Florinet al., 1985; Pool & Lin, 1982 and Curren, 1980) on 2,4; 2,6 and 3,5 xylenol show that similar isomers are indistinguishable in the Ames assay. This provides strong evidence that read across in particular from both 2,4 and 2,6-xylenol can be used to provide support for 3,5-xylenol, confirming that up to a maximum recommended concentration (5 mg/plate) there is no evidence of gene mutation in this assay type. These studies have been included as supporting studies.