2,3-xylenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered chemical i.e.3-dimethylphenol (CAS 526-75-0) was tested non-mutagenic (negative) up to 0.625 mg/plate with and without S9 metabolic activation in a bacterial reverse mutation test (OECD 471) using Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study contains experimental data of the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: 21st July 1997, Corrected: 26th June 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 98.06%

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Cofactors complemented S9 homogenate obtained from phenobarbitone and β-naphthoflavone-induced rat liver

Test concentrations with justification for top dose:

0 mg/plate (negative control)
0mg/plate (vehicle control)
0.0390625 mg/plate
0.078125 mg/plate
0.15625 mg/plate
0.3125 mg/plate
0.625 mg/plate

Justification for the top dose: A preliminary cytotoxicity test was conducted with test concentrations of 0 (NC), 0 (VC, DMSO), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate and with and without S9 metabolic activation using S. typhimurium TA 98 and TA 100. No revertant colony and complete inhibition of the background lawn were observed at 5 and 2.5 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation, compared to the vehicle and negative control data. A substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 1.25 mg/plate. A moderate decrease in the revertant count and no inhibition in the background lawn was noted at 0.625 mg/plate both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. No reduction in the revertant count and/or inhibition in the background lawn was noted at concentrations from 0.3125 to 0.039625 mg/plate, neither in the presence (10% v/v S9 mix) nor the absence of metabolic activation when compared to the vehicle control data. Based on the results of the pre-test, 0.625 mg/plate was selected as the top concentration for the main test.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Nitrofluoren: TA98 (-S9) Sodium azide: TA100, TA1535 (-S9) 9-Aminoacridine: TA1537 (-S9) Mitomycin C: TA102 (-S9) Benzo[a]pyrene: TA98, TA100, TA1535, TA1537, TA102 (+S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: Two
Trial I: plate incorporation assay
Trial II: pre-incubation assay

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added in medium; in agar (plate incorporation); pre-incubation; in suspension; as impregnation on paper disk: In medium (plate incorporation, in suspension (pre-incubation assay)

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period, if applicable: 20 min at 37 ± 2°C
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times):48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g., background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: cytotoxicity was determined by the reduction in revertant colony count and/or inhibition of the background lawn
- Any supplementary information relevant to cytotoxicity: NA

Rationale for test conditions:

Test Item Solubility and Precipitation:

The test item was insoluble in distilled water (50 mg/ml) and was soluble in dimethyl sulfoxide (DMSO). The precipitation test was performed by adding 100 µl of the highest test item concentration (50 mg/ml) to 2 ml of top agar and plated on to minimal glucose agar plate.
Test Item Dilution Preparation for Cytotoxicity Test
250.4 mg of the test item was dissolved in 5.008 ml of DMSO to get a stock concentration of 50 mg/ml. The stock solution was subsequently diluted using a twofold spacing factor to achieve the concentrations of 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5 and 25 mg/ml. All stock solution preparations and subsequent serial dilutions were performed inside the laminar airflow unit freshly before treatment.

Test Item Dilution Preparation of Trial I and Trial II:

In Trial I, 31.4 mg of the test item was dissolved in 5.024 ml of DMSO to get a stock concentration of 6.25 mg/ml. The stock solution was subsequently diluted with DMSO using a twofold spacing factor to achieve the concentrations of 0.390625.0.78125, 1.5625 and 3.125 mg/ml.
In Trial II, 62.5 mg of the test item was dissolved in 10 ml of DMSO to get a stock concentration of 6.25 mg/ml. The stock solution was subsequently diluted with DMSO using a twofold spacing factor to achieve the concentrations of 0.390625.0.78125, 1.5625 and 3.125 mg/ml.
All the stock solution preparations and subsequent serial dilutions of test item concentrations were performed inside the laminar airflow unit before treatment.

Sterility Check:
The sterility of the top agar, minimal glucose agar plates, S9 cofactor mix, phosphate buffer, Test Item and the vehicle were confirmed in all the study phases.

Evaluation criteria:

A test item was considered a mutagen if a biologically relevant increase in the mean number of revertants exceeded the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control was observed.
A dose-dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
A dose-dependent increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent experiment.
A dose-dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent experiment.

Statistics:

No statistics was used.

Key result

Species / strain:

other:

Remarks:

TA98, TA100, TA1535, TA1537, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 0.625 mg/plate, moderate reductions in revertant colony counts were noted in TA98, TA100, TA1535, TA1537 and TA102 with no inhibition of the background lawn.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity test:
A preliminary cytotoxicity test was conducted with test concentrations of 0 (NC), 0 (VC, DMSO), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate in triplicates along with the negative, vehicle and positive controls using S. typhimurium TA 98 and TA 100 tester strains. The test was performed both in the presence and absence of S9 metabolic activation (liver S9 microsomal fraction was obtained from phenobarbitone and β-naphthoflavone-induced rats). No revertant colony and complete inhibition of the background lawn were observed at 5 and 2.5 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation, compared to the vehicle and negative control data. A substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 1.25 mg/plate. A moderate decrease in the revertant count and no inhibition in the background lawn was noted at 0.625 mg/plate both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. No reduction in the revertant count and/or inhibition in the background lawn was noted at concentrations from 0.3125 to 0.039625 mg/plate, neither in the presence (10% v/v S9 mix) nor the absence of metabolic activation when compared to the vehicle control data. Based on the results of the pre-test, 0.625 mg/plate was selected as the top concentration for the main test.

Remarks on result:

other: No mutagenic potential

Mean Revertant Colony Count – Preliminary Cytotoxicity Assay

Test Item Concentration (mg/plate)	TA 98				TA 100
	- S9		+ S9		- S9		+ S9
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	21.67 (NI)	1.53	22.33 (NI)	1.53	102.67 (NI)	1.15	103.67 (NI)	5.86
VC (Dimethyl sulfoxide)	21.67 (NI)	2.52	22.00 (NI)	1.00	105.33 (NI)	3.06	100.33 (NI)	2.08
T1 (0.0390625)	23.00 (NI)	2.00	20.67 (NI)	3.06	98.33 (NI)	4.04	98.33 (NI)	4.04
T2 (0.078125)	21.33 (NI)	1.53	21.00 (NI)	2.00	96.00 (NI)	6.24	100.33 (NI)	4.16
T3 (0.15625)	22.67 (NI)	1.53	21.67 (NI)	1.53	97.33 (NI)	2.89	97.67 (NI)	4.04
T4 (0.3125)	22.33 (NI)	1.15	19.33 (NI)	1.15	98.33 (NI)	3.21	96.67 (NI)	2.52
T5 (0.625)	17.33 (NI)	1.53	17.33 (NI)	1.15	87.33 (NI)	6.03	81.67 (NI)	2.52
T6 (1.25)	9.00 (MI)	1.73	9.67 (MI)	1.53	21.00 (MI)	4.36	22.00 (MI)	3.61
T7 (2.5)	0.00 (CI)	0.00	0.00 (CI)	0.00	0.00 (CI)	0.00	0.00 (CI)	0.00
T8 (5.0)	0.00 (CI)	0.00	0.00 (CI)	0.00	0.00 (CI)	0.00	0.00 (CI)	0.00
PC	343.00	9.85	333.00	10.15	715.67	17.62	714.00	7.94

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T8 = Test Item concentration from lower to higher.

Mean Revertant Colony Count in Trial I (Plate Incorporation Method)

Absence of metabolic activation (-S9)							Presence of metabolic activation (+S9 10 % v/v S9 Mix)
Test Item Concentration (mg/plate)	TA 1535		TA 1537		TA 102		TA 1535		TA 1537		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	16.00	1.00	7.00	1.00	238.67	5.03	15.67	1.15	6.67	2.31	236.67	4.04
VC (Dimethyl sulfoxide)	17.33	1.15	6.67	1.15	236.67	7.23	15.33	0.58	7.00	1.73	237.33	6.66
T1 (0.0390625)	17.00	1.73	6.00	1.73	239.00	3.61	14.67	2.08	6.33	1.15	237.00	2.65
T2 (0.078125)	15.33	1.15	5.33	1.53	233.00	6.00	14.67	2.52	6.67	1.53	233.67	3.06
T3 (0.15625)	14.67	1.15	6.33	0.58	237.00	5.00	14.33	1.15	6.00	0.00	236.00	6.00
T4 (0.3125)	13.67	1.15	5.33	0.58	238.67	2.52	13.33	1.15	6.00	1.73	233.33	2.08
T5 (0.625)	10.67	0.58	3.33	1.15	216.00	7.21	11.33	1.53	2.67	0.58	212.33	5.86
PC	332.67	11.59	200.33	12.06	1667.33	8.62	327.67	8.02	193.67	4.51	1717.00	27.62

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T5 = Test Item concentration from lower to higher.

Mean Revertant Colony Count in Trial II (Preincubation Method)

	Absence of metabolic activation										Presence of metabolic activation (+S9 10% v/v S9 Mix)
Test Item Concentration (mg/plate)	TA 1535		TA 1537		TA 102		TA 98		TA 100		TA 1535		TA 1537		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	16.33	1.53	6.33	1.15	238.00	6.56	20.7	1.5	102.67	3.21	15.67	1.53	6.00	1.73	238.33	4.04	23.00	1.73	100.33	2.08
VC (Dimethyl sulfoxide)	16.33	1.15	7.00	1.73	236.00	4.36	21.0	1.0	100.67	3.06	16.33	0.58	5.67	0.58	228.33	2.52	21.00	1.00	100.33	1.53
T1(0.0390625)	15.67	0.58	7.00	1.73	227.00	2.00	20.7	1.5	99.00	2.00	15.00	1.00	5.67	2.31	236.33	0.58	22.33	1.53	97.00	2.08
T2 (0.078125)	17.00	1.00	5.67	1.53	235.00	4.36	19.3	2.3	100.00	5.57	14.33	0.58	6.00	1.73	231.33	6.11	20.33	0.58	96.33	1.53
T3 (0.15625)	16.67	1.15	6.00	1.73	232.67	5.13	21.3	3.1	99.00	3.61	14.00	1.73	5.67	0.58	233.67	8.02	22.00	2.65	95.00	2.00
T4 (0.3125)	16.33	1.53	6.67	1.53	229.33	6.66	20.0	1.7	99.33	2.31	14.67	0.58	5.00	1.73	235.00	7.21	21.00	1.00	94.33	1.15
T5 (0.625)	12.33	1.53	3.67	1.66	213.00	4.00	16.7	0.6	83.67	3.79	10.33	0.58	3.67	0.58	212.00	2.65	16.00	1.73	83.00	1.73
PC	339.33	9.07	224.00	4.58	1714.33	33.17	346.7	7.8	732.00	7.55	332.67	8.96	217.67	8.02	1691.00	22.87	342.00	4.58	733.67	2.08

Key:     NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation,T1-T5 = Test Item concentration from lower to higher.

Fold increase

	Trial I - Plate Incorporation Method										Trial II – Preincubation Method
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 102		TA 98		TA 100		TA 1535		TA 1537		TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
NC (Distilled water)	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
VC (Dimethyl sulfoxide)	1.00	0.99	1.03	0.97	1.08	0.98	0.95	1.05	0.99	1.00	1.02	0.91	0.98	1.00	1.00	1.04	1.11	0.94	0.99	0.96
T1 (0.0390625)	1.06	0.94	0.93	0.98	0.98	0.96	0.90	0.90	1.01	1.00	0.98	1.06	0.98	0.97	0.96	0.92	1.00	1.00	0.96	1.04
T2 (0.078125)	0.98	0.95	0.91	1.00	0.88	0.96	0.80	0.95	0.98	0.98	0.92	0.97	0.99	0.96	1.04	0.88	0.81	1.06	1.00	1.01
T3  (0.15625)	1.05	0.98	0.92	0.97	0.85	0.93	0.95	0.86	1.00	0.99	1.02	1.05	0.98	0.95	1.02	0.86	0.86	1.00	0.99	1.02
T4 (0.3125)	1.03	0.88	0.93	0.96	0.79	0.87	0.80	0.86	1.01	0.98	0.95	1.00	0.99	0.94	1.00	0.90	0.95	0.88	0.97	1.03
T5 (0.625)	0.80	0.79	0.83	0.81	0.62	0.74	0.50	0.38	0.91	0.89	0.79	0.76	0.83	0.83	0.76	0.63	0.52	0.65	0.90	0.93
PC	15.83	15.14	6.79	7.12	19.19	21.37	30.05	27.67	7.05	7.23	16.51	16.29	7.27	7.31	20.78	20.37	32.00	38.41	7.26	7.41

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, T1-T5 = Test Item concentration from lower to higher.

Conclusions:

The registered chemical 3-dimethylphenol (CAS 526-75-0) did not induce gene mutations by base pair changes or frameshifts in the His gene of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102, neither the presence nor in the absence of S9 metabolic activation system in a study performed according to OECD TG 471.

Executive summary:

A bacterial reverse mutation assay (OECD TG 471) was performed to investigate the potential of the registered chemical i. e. 3-dimethylphenol (CAS 526-75-0), to induce gene mutations in comparison to vehicle control following the plate incorporation (Trial I) and the pre-incubation methods (Trial II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments with and without the S9 metabolic activation system (S9 liver microsomal fraction obtained fromphenobarbitone and β-naphthoflavone-induced rats).The test concentrations were selected based on the results of the preliminary cytotoxicity tests: 0.625 mg/plate was selected as the highest test concentration that produced moderate decreases in revertant colony counts in TA98 and TA100. The mutagenicity test was performed with the following test substance concentrations: 0 (NC), 0 (VC), 0.0390625, 0.078125, 0.15625,0.3125 and 0.625 mg/plate in both trials and the presence (+S9) and absence of metabolic activation (-S9). Results: No significant increase in the number of revertant colonies in the presence and absence of metabolic activation was observed at any concentrations tested compared to the vehicle control. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle controls were within the historical range. Each strain-specific positive control in both trials showed a significant increase in the number of revertant colonies. Conclusion: The registered chemical, i.e. 3-dimethylphenol (CAS 526-75-0), did not induce gene mutations by base pair changes or frameshifts in the His gene of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 neither in the presence nor in the absence of S9 metabolic activation in a study performed according to OECD TG 471.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test:

A bacterial reverse mutation assay (OECD TG 471) was performed to investigate the potential of the registered chemical i. e. 3-dimethylphenol (CAS 526-75-0), to induce gene mutations in comparison to vehicle control following the plate incorporation (Trial I) and the pre-incubation methods (Trial II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments with and without the S9 metabolic activation system (S9 liver microsomal fraction obtained fromphenobarbitone and β-naphthoflavone-induced rats).The test concentrations were selected based on the results of the preliminary cytotoxicity tests: 0.625 mg/plate was selected as the highest test concentration that produced moderate decreases in revertant colony counts in TA98 and TA100. The mutagenicity test was performed with the following test substance concentrations: 0 (NC), 0 (VC), 0.0390625, 0.078125, 0.15625,0.3125 and 0.625 mg/plate in both trials and the presence (+S9) and absence of metabolic activation (-S9).Results: No significant increase in the number of revertant colonies in the presence and absence of metabolic activation was observed at any concentrations tested compared to the vehicle control. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle controls were within the historical range. Each strain-specific positive control in both trials showed a significant increase in the number of revertant colonies.Conclusion:The registered chemical, i.e. 3-dimethylphenol (CAS 526-75-0), did not induce gene mutations by base pair changes or frameshifts in the His gene of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 neither in the presence nor in the absence of S9 metabolic activation in a study performed according to OECD TG 471.

Justification for classification or non-classification

The registered substance, i.e. 3-dimethylphenol (CAS 526-75-0), tested non-mutagenic (negative) in fiveSalmonella typhimurium strains in a GLP-compliant and OECD guideline study performed according to OECD TG 471. Hence, the registered substance, i.e. 3-dimethylphenol (CAS 526-75-0), is regarded to be classified as Not Classified for genetic toxicity according to Regulation EC 1272/2008.