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Genetic toxicity in vitro

Description of key information

WoE, genetic toxicity in vitro -bacterial reverse mutation assay similar to OECD 471, Salmonella typhimurium strains TA 98 and TA 100, both with and without a rat liver enzyme preparation for metabolic activation (S-9 mix), results: 2,5-xylenol showed no mutagenic potential in this test.

WoE_Read-across, genetic toxicity in vitro -bacterial reverse mutation assay similar to OECD 471, Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and in addition TA 1538 with and without addition of S9-mix, results: 2,4-xylenol did not show mutagenic effects, both in the presence and absence of a metabolic activation system

WoE, QSAR prediction with QSAR Toolbox database (v.4.1) on gene mutations in the bacterial reverse muation assay, results: negative

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Method described by Ames et al.: Ames BN, McCann J, Yamasaki E (1975) Methods for detecting carcinogens and mutagens with the Salmonella / mammalian-microsome mutagenicity test. Mutat Res 31, 347.
Deviations:
yes
Remarks:
Only S. typhimurium strains TA 98 and TA 100 have been tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
TA98: hisD3052, uvrB, rfa (frameshift plus R factor)
TA100: his G46, uvrB, rfa (missense plus R factor)
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix prepared from rat livers induced with Aroclor 1254
Test concentrations with justification for top dose:
All shale oil fractions and compounds examined were tested with the plate assay over at least a 1000-fold concentration range with the two tester strains TA98 and TA100. Positive or questionable results were retested with a narrower range of concentrations.
Vehicle / solvent:
Compounds to be tested were suspended in dimethylsulfoxide (DMSO, supplied sterile, spectrophotometric grade from Schwarz/Mann) to concentrations in the range 10-20 mg/ml.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
The strain to be treated with the potential mutagen was added to soft agar containing a low level of histidine and biotin along with varying amounts of the test substance. The suspension containing approximately 2 x 10E8 bacteria was overlaid on minimal agar plates, and revertants to wild type were counted after a 48-hour incubation period. The studies were carried out with parallel series of plates plus and minus a rat liver enzyme preparation for metabolic activation (S9-mix). The potential mutagen was in some cases assayed for general toxicity, which was indicated by bacterial survival, with strain TA1537.
Evaluation criteria:
Revertant colonies were counted after 48 hours of incubation.
Statistics:
The recorded numbers of revertant colonies were plotted versus respective concentrations of 2,5-xylenol, and the slopes of the induction curves were determined.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
2,5-Xylenol showed no mutagenic potential in the bacterial reversion mutation assay using S. typhimurium strains TA 98 and TA 100, both with and without metabolic activation.
Executive summary:

Shale oil fractions and known constituents such as 2,5-xylenol were examined in the in vitro bacterial reversion mutation assay (Ames test) using Salmonella typhimurium strains TA 98 and TA 100, both with and without a rat liver enzyme preparation for metabolic activation (S-9 mix). The test was well documented and the procedure was similar to OECD Guideline 471. Over a 1000-fold concentration range of the test material, no increase of revertant colonies was noted. Thus, 2,5-xylenol showed no mutagenic potential in this test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Bacterial reverse mutation assays did not indicate a variation in properties between members of the category (i.e. no evidence of a gene mutation potential was found). Therefore, the general hypothesis for the category approach that xylenol isomers exert qualitatively and quantitatively similar toxicity effects applies for the endpoint "genetic toxicity in vitro".

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
Ames tests which have been performed using 2,5-xylenol, 4 isomer analogues and xylenol (mixed isomers) did not provide evidence that the chemical structure of any xylenol isomer or a metabolite produced due to metabolic activation with added S9 mix has the potential to induce gene mutations in bacterial test systems. It is further noted that the induction of chromosome aberrations by 2,6-xylenol in the presence of metabolic activation in vitro was not confirmed in a Mammalian Bone Marrow Chromosome Aberration Test comparable to OECD Testing Guideline 475 in vivo (EFSA, 2008; disseminated REACH dossier of 2,6-xylenol). For further information, see attached read-across justification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
S. typhimurium strain TA1538 was used in place of TA102 or E. coli strains.
GLP compliance:
no
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:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix with cofactors (60 mg S-9 protein/10 ml S-9 mix) from Aroclor-pretreated Sprague-Dawley rats.
Test concentrations with justification for top dose:
0, 0.5, 5, 50, 500, 5000 µg per plate - the maximum concentration resulted in toxicity which was apparent as a thinning of the background lawn.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
The test for their reversion to histidine prototrophy was performed using the plate incorporation assay as described by Ames, McCann and Yamasaki (1975). 2,4-xylenol was dissolved in DMSO and tested in five concentrations in quadruplicate in the presence of buffer pH 7.4 or S-9 mix with cofactors (60 mg S-9 protein/10 ml S-9 mix) from Aroclor-pretreated Sprague-Dawley rats. The S-9 protein content was determined using the biuretmethod-kit of Boehringer, Mannheim, Germany. The sensitivity of the S-9 and of the indicator strains was monitored routinely using positive control compounds during each experiment.
Evaluation criteria:
significant increase in the number of histidine revertant colonies
Statistics:
Significance levels for positive dose-response effects were obtained with the Joncheere test (Hollander and Wolfe, 1973).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: at 5000 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: at 5000 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: at 5000 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: at 5000 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: at 5000 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The number of histidine revertants, which was scored in the presence of the test compound, never more than slightly exceeded that number of spontaneously arising revertants. The only dose-related effect was toxicity at the highest tested concentration (5000 µg). This was apparent as a thinning of the bacterial background lawn and as a reduced number of spontaneous revertants.
Conclusions:
2,4-xylenol did not show mutagenic effects, both in the presence and absence of a metabolic activation system, when tested in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100.
Executive summary:

The genotoxic potential of the isomer analogue 2,4-xylenol was assessed in the in vitro bacterial reversion mutation assay (Ames test) using standard Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and in addition TA 1538. The study was well documented and similar to OECD Guideline 471.

0, 0.5, 5, 50, 500, or 5000 µg of the test material, which had been dissolved in DMSO, was added to the culture medium of a test plate. Each concentration was tested in quadruplicate, with and without addition of S9-mix. The maximum concentration resulted in toxicity which was apparent as a thinning of the background lawn and a reduced number of spontaneous revertants. Lower test concentrations did not induce a significant increase in the number of histidine revertant colonies. In conclusion 2,4-xylenol did not show mutagenic effects, both in the presence and absence of a metabolic activation system, when tested in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100.

This result can be transferred to the target test item 2,5-xylenol based on high structural similarities in the read-across category of xylenol isomers (see 'Read-across statement' in section 13.2).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to "Attached justification".
Qualifier:
no guideline followed
Principles of method if other than guideline:
QSAR Toolbox prediction for single chemical:
Predicted endpoint: Gene mutation; No effect specified; Escherichia coli, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, Salmonella typhimurium, TA 98 and TA 100; No duration specified; No guideline specified
Data gap filling method: Read-across analysis
Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 7 neighbours
GLP compliance:
no
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined

The experimental values for the following 7 nearest neighbours in the database were negative: 3,4-xylenol (CAS No 95-65-8), 2,3-xylenol (CAS No 526-75-0), 2,6-xylenol (CAS No 576-26-1), 2,4-xylenol (CAS No. 105-67-9), 4-ethylphenol (CAS No 123-07-9), trimethylhydroquinone (CAS No 700-13-0), and 2-ethylphenol (CAS No 90-00-6)

Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):

The prediction is based on 68 values, 68 of them (100%) equal to predicted value

Prediction confidence is measured by the p-value: 3,6E-33

Conclusions:
2,5-xylenol is predicted by read-across analysis, which is supported by QSAR Toolbox v.4.1, to be non-mutagenic in the bacterial reverse mutation assay. Regarding the high confidence of the prediction (p < 3,6E-33), the result is considered to be reliable.
Executive summary:

Based on the QSAR Toolbox database (v.4.1), 2,5-xylenol was predicted not to induce gene mutations in the bacterial reverse mutation assay. The read-across analysis refers to the highest mode value from the following 7 nearest neighbours with test results obtained in different Salmonella typhimurium strains: 3,4-xylenol (CAS No 95-65-8), 2,3-xylenol (CAS No 526-75-0), 2,6-xylenol (CAS No 576-26-1), 2,4-xylenol (CAS No. 105-67-9), 4-ethylphenol (CAS No 123-07-9), trimethylhydroquinone (CAS No 700-13-0), and 2-ethylphenol (CAS No 90-00-6). In total, the prediction relies on 68 values, with all of them equal to the predicted value (i.e. negative). The p-value of 3,6E-33 reflects the high confidence of the prediction and indicates that the prediction is reliable.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro - bacterial reverse mutation assay

Shale oil fractions and known constituents such as 2,5-xylenol were examined by Epler and coworkers (1979) in the in vitro bacterial reversion mutation assay (Ames test) using Salmonella typhimurium strains TA 98 and TA 100, both with and without a rat liver enzyme preparation for metabolic activation (S-9 mix). The test was well documented and the procedure was similar to OECD Guideline 471. Over a 1000-fold concentration range of the test material, no increase of revertant colonies was noted. Thus, 2,5-xylenol showed no mutagenic potential in this test.

In a read-across approach the genotoxic potential of the isomer analogue 2,4-xylenol was assessed in the in vitro bacterial reversion mutation assay (Ames test) using standard Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and in addition TA 1538 (Pool and Lin 1982). The study was well documented and similar to OECD Guideline 471. 0, 0.5, 5, 50, 500, or 5000 µg of the test material, which had been dissolved in DMSO, was added to the culture medium of a test plate. Each concentration was tested in quadruplicate, with and without addition of S9-mix. The maximum concentration resulted in toxicity which was apparent as a thinning of the background lawn and a reduced number of spontaneous revertants. Lower test concentrations did not induce a significant increase in the number of histidine revertant colonies. In conclusion, 2,4-xylenol did not show mutagenic effects, both in the presence and absence of a metabolic activation system, when tested in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100.

Based on the QSAR Toolbox database (v.4.1), 2,5-xylenol was predicted not to induce gene mutations in the bacterial reverse muation assay. The read-across analysis refers to the highest mode value from the following 7 nearest neighbours with test results obtained in different Salmonella typhimurium strains: 3,4-xylenol (CAS No 95-65-8), 2,3-xylenol (CAS No 526-75-0), 2,6-xylenol (CAS No 576-26-1), 2,4-xylenol (CAS No. 105-67-9), 4-ethylphenol (CAS No 123-07-9), trimethylhydroquinone (CAS No 700-13-0), and 2-ethylphenol (CAS No 90-00-6). In total, the prediction relies on 68 values, with all of them equal to the predicted value (i.e. negative). The p-value of 3,6E-33 reflects the high confidence of the prediction and indicates that the prediction is reliable.

Justification for classification or non-classification

The weight of evidence from bacterial reversion mutation assays (Ames test), which have been performed using 2,5-xylenol or isomer analogues, suggests that the substance does not induce gene mutations in bacterial test systems. The information requirements according to Annex VII of Regulation (EC) No 1272/2008 are met, and classification for genotoxicity is not required.