Reaction mass of 2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol and 4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March to April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

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:

4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Molecular formula:

C27H44N4O2

IUPAC Name:

4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Constituent 2

Reference substance name:

2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}- 1-methylethyl)phenol

Molecular formula:

C24H37N3O2

IUPAC Name:

2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}- 1-methylethyl)phenol

Constituent 3

Reference substance name:

2-[(Dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol

Molecular formula:

C21H30N2O2

IUPAC Name:

2-[(Dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

impurity 2

Reference substance name:

Xylene

EC Number:

215-535-7

EC Name:

Xylene

Cas Number:

1330-20-7

Molecular formula:

C8H10

IUPAC Name:

Benzene, dimethyl-

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 4

Reference substance name:

Sulphated ash

IUPAC Name:

Sulphated ash

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): HPP 12879-1

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in ethanol (0.01-423 mg/ml) for 24hrs at room temperature (Currenta File No.: 2014/0048/08; date: 2015-03-24)


FORM AS APPLIED IN THE TEST (if different from that of starting material): On the day of the experiment, the test item was dissolved in ethanol. All formulations were prepared freshly before treatment and used within two hours of preparation.

Method

Target gene:

mutant histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

plate incorporation assay (experiment I):
3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix
preincubation assay (experiment II):
33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix

The test was performed up to and including the limit dose of 5000 µg/plate. In both experiments no precipitation of the test item in the overlay agar in the test tubes occurred up to and including the highest investigated dose. The test item precipitated in the overlay agar on the incubated agar plates in experiment I at 5000 μg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording.
Toxic effects, evident as reduced background growth, were described for all test strains in experiment II without and with S9 mix at 5000 µg/plate.
Toxic effects, evident as a reduction in the number of revertants below the induction factor of 0.5) were observed in experiment I for TA 100 without S9 mix at 5000 µg/plate. In experiment II at 5000 µg/plate without S9 mix in TA 1535, TA 98 and TA 100 and with S9 mix in TA 100.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF TOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, performed according to OECD TG 471. In both experiments no precipitation of the test item in the overlay agar in the test tubes occurred up to and including the highest investigated dose. The test item precipitated in the overlay agar on the incubated agar plates in experiment I at 5000 μg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test item at 5000 µg/plate showed reduced background growth at 5000 μg/plate in experiment II (+/- S9 mix). Toxic effects, evident as a reduction in the number of revertants, occurred in some test groups with and without metabolic activation at 5000 µg/plate in both experiments. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.