[(dimethylamino)methyl]phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation (LPT, 2019).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-01 to 2019-04-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

in vitro gene mutation study in bacteria

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotropy

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strains were obtained from Trinova Biochem GmbH

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary tests
- Plate incorporation test (cytotoxicity test): 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160, 5000 µg/plate N,N-dimethylaminomethylphenol
- Preincubation method

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Strains: 5 strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535, TA1537)
Concentrations: 10.0, 31.6, 100, 316, 1000 and 3160 µg N,N-Dimethylaminomethylphenol per plate
Plates: 3 per concentration and experiment
Experiments: 2 independent experiments, each with and without metabolic activation
Metabolic activation system: aroclor included rat liver S9

Main test procedure
1st independent experiment - Plate Incorporation Method
Sterile top agar containing 0.6% agar and 0.5% NaCl was molten on the day of the test. 10 mL of a sterile solution of 0.5 mM L-histidine HCl/0.5 mM Biotin were added to 100 mL of molten agar. 2 mL of this top agar were distributed into culture tubes held at 45°C in a heating block. 0.1 mL of Salmonella cell suspension (containing approximately 108 viable cells in the late exponential or early stationary phase), 0.1 mL of test item solution (or 0.1 mL vehicle or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer mentioned above. 
The test components were mixed by vortexing the soft agar for 3 sec at low speed and then poured onto a coded 27.5 mL minimal glucose agar plate (Minimal Glucose Agar medium E). To achieve a uniform distribution of the top agar on the surface of the plate, the uncovered plate was quickly tilted and rotated and then placed on a level surface with the cover on and finally allowed to harden.
Immediately, the plates were inverted and placed in a dark 37°C incubator for 48 h and could be stored after incubation for up to 24 hours at 4°C. The revertant colonies on the test plates and on the control plates were counted with a colony counter , and the presence of the background lawn on all plates was confirmed. A lawn that was thin compared with the lawn on the negative control plate was evidence of bacterial toxicity.
Routine examination of the background lawn of bacterial growth resulting from the trace of histidine added to the top agar can be an aid in determining the presence of toxic effects. If massive cell death has occurred, the background lawn on the test plates will be sparse compared with control plates.
In this case more histidine is available to the individual surviving bacteria and they undergo more cell divisions, consequently appearing as small colonies which can be mistaken for revertants if the absence of a normal background lawn is not noted.

2nd independent experiment - Preincubation Method
The test item was preincubated with the test strain (containing approximately 108 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of the test item solution (or 0.1 mL vehicle or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.

Evaluation criteria:

Bacteria colonies were counted employing the Biosys Biocount 5000 system. Print outs of the colony counts were filed with the raw data. Occurrence of test item precipitation would have been documented after visual inspection of the cultures with the unaided eye. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.

Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by LPT.
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20

Statistics:

Biological relevance of the results should be considered first.
A test item is considered to show a positive response if

- the number of revertants is significantly increased (p <= 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537
and 1.5-fold of the solvent control for TA102 in both independent experiments.

- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.

Key result

Species / strain:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at top concentration of 3160 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary test
N,N-Dimethylaminomethylphenol was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of 3160 and 5000 µg N,N Dimethylaminomethylphenol/plate in both experiments.
Hence, 3160 µg N,N-Dimethylaminomethylphenol per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study
Six concentrations ranging from 10.0 to 3160 µg N,N-Dimethylaminomethyl¬phenol/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity
Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg N,N-Dimethylaminomethylphenol/plate in both experiments, each carried out without and with metabolic activation (plate incorporation test and preincubation test) in all test strains.

Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed for N,N-Dimethylaminomethylphenol, tested up to a concentration of 3160 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the historical control range generated by LPT. Hence, all acceptance criteria are met.

no remarks

Conclusions:

In conclusion, under the present test conditions, N,N-Dimethylaminomethylphenol tested up to a cytotoxic concentration of 3160 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The potential of N,N-Dimethylaminomethylphenol to induce gene mutations was examined in 5 Salmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the secondas a preincubation test.

N,N-Dimethylaminomethylphenol was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO was employed as the negative control.

Preliminary test

N,N-Dimethylaminomethylphenol was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of 3160 and 5000 µg N,N-Dimethylaminomethylphenol/plate in both experiments.

Hence, 3160 µg N,N-Dimethylaminomethylphenol per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 10.0 to 3160 µg N,N-Dimethylaminomethyl­phenol/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg N,N-Dimethylaminomethylphenol/plate in both experiments, each carried out without and with metabolic activation (plate incorporation test and preincubation test) in all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for N,N-Dimethylaminomethylphenol, tested up to a concentration of 3160 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.The results of the negative and positive control cultures are withinthe historical control rangegenerated byLPT.Hence, all acceptance criteria are met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available in vitro genotoxicity data the test item must not be classified according to the criteria of EC Regulation 1272/2008.