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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-August 2021-16-September 2021
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guideline 471. OECD Guideline for Testing of Chemicals - Bacterial Reverse Mutation Test, adopted on 21 July 1997 and corrected on 26 June 2020.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
tris[4-(dimethylamino)phenyl]methano
Cas Number:
467-63-0
Molecular formula:
C25H31N3O
IUPAC Name:
tris[4-(dimethylamino)phenyl]methano
Test material form:
solid
Details on test material:
- Name of test material: tris[4-(dimethylamino)phenyl]methano
- Molecular formula: C25H31N3O
- Molecular weight: 389.54g/mol
- Smiles notation: OC(c1ccc(N(C)C)cc1)(c1ccc(N(C)C)cc1)c1ccc(N(C)C)cc1
- InChl : 1S/C25H31N3O/c1-26(2)22-13-7-19(8-14-22)25(29,20-9-15-23(16-10-20)27(3)4)21-11-17-24(18-12-21)28(5)6/h7-18,29H,1-6H3
- Substance type: Organic
- Physical state: Solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under storage conditions:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Preparation of a nanomaterial dispersion (incl. dilution):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:

Method

Target gene:
Histidine Operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
Salmonella typhimurium is a commonly used test system for bacterial reverse mutation studies, and it is recommended by international guidelines ( OECD Guideline 471), and it meets the regulatory requirement of most regulatory agencies
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 was prepared in-housed rom a combination of phenobarbitone and Beta-naphthoflavone-induced rat liver microsomal enzymes (S9 homogenate).
- method of preparation of S9 mix: S9 mix [Cofactors (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and Liver homogenate] was prepared prior to use in the study. The post-mitochondrial fraction (liver homogenate) was used at the concentration of 10 % (v/v) in the S9 mix.
- concentration or volume of S9 mix in the final culture medium : 0.5 ml
- quality controls of S9: Efficiency check of S9 was performed during the assay
Test concentrations with justification for top dose:
Test concentrations: 0.0 (NC), 0.0 (VC), 0.0048828, 0.0097656, 0.01953125, 0.0390625 and 0.078125 mg/plate.
Justification: Test concentrations were selected based on a preliminary cytotoxicity test. This pretest was performed with TA98 and TA100 strains, both in the presence and absence of metabolic activation using the plate incorporation method. The following concentrations were tested in triplicates along with the negative, vehicle and positive controls: 0.0 (NC), 0.0 (VC), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. In both strains, complete inhibition of background lawn with no presence of any revertant colonies were observed at 0.3125 mg/plate, both with and without S9 metabolic activation. A substantial reduction in the number of revertant colonies accompanied by a moderate inhibition of background lawn was observed at 0.15625 mg/plate compared to the vehicle control data. A slight reduction in the number of revertant colonies accompanied by a moderate inhibition of background lawn was observed at 0.078125 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. Hence, cconsidering the criteria that the highest test concentration should yield a decrease in the revertant colony count and/or diminution of the background lawn, 0.078125 mg/plate was selected as the highest concentration of the Test Item for the main assay
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulfoxide)

- Justification for choice of solvent/vehicle: The test Item was found to be insoluble in distilled water (50 mg/ml) and found to be soluble in dimethyl sulfoxide (50 mg/ml). Hence, dimethyl sulfoxide was selected as the vehicle for the study
Controls
Untreated negative controls:
yes
Remarks:
Distilled Water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 -2 ×109 bacteria/ml
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition.
Evaluation criteria:
A Test Item is considered as a mutagen if a biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) 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.
A dose-dependent increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent 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 experiment.
Statistics:
The mean number of revertant colonies and the standard deviation within the plates for each concentration were compared to the spontaneous reversion rates of the control. Microsoft Office Excel-based calculations were used for descriptive statistical analysis.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test Item was found to be insoluble in distilled water (50 mg/ml)
Remarks on result:
other: No mutagenic potential observed

Any other information on results incl. tables

Table1: 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)

23

(NI)

1.15

23

(NI)

1.53

102

(NI)

4.00

99

(NI)

3.51

VC

(Dimethyl sulfoxide)

22

(NI)

2.08

22

(NI)

1.53

99

(NI)

4.51

95

(NI)

4.73

T1

(0.0390625)

20

(NI)

1.53

20

(NI)

2.00

90

(NI)

4.04

91

(NI)

5.69

T2

(0.078125)

16

(MI)

0.58

15

(MI)

1.53

81

(MI)

2.65

79

(MI)

3.79

T3

(0.15625)

8

(MI)

1.73

8

(MI)

1.73

26

(MI)

6.00

22

(MI)

4.00

T4

(0.3125)

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

T5

(0.625)

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

T6

(1.25)

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

T7

(2.5)

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

T8

(5.0)

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

0

(CI)

0.00

PC

324

(NI)

12.50

314

(NI)

11.06

676

(NI)

13.01

676

(NI)

14.42

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, NI = No Inhibition, MI = Moderate Inhibition, CI = Complte Inhibition.

Positive Controls:

2-Nitrofluorene

:

TA98 (absence of metabolic activation)

Sodium azide

:

TA100 (absence of metabolic activation)

Benzo[a]pyrene

:

TA98 and TA100 (presence of metabolic activation)

Table2 Mean Revertant Colony Count in Trial I(Plate Incorporation Method)

Absence of metabolic activation

Presence of metabolic activation (+S9 10% v/v S9 Mix)

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

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (Distilled water)

21

2.65

102

5.00

14

1.15

7

1.53

227

3.51

22

2.00

101

2.08

12

2.08

6

1.15

232

8.19

VC (Dimethyl sulfoxide)

20

2.52

96

4.36

13

2.00

6

1.53

233

5.29

21

2.52

99

6.81

13

2.08

6

1.53

233

6.66

T1 (0.0048828)

20

3.21

98

4.58

12

1.73

6

1.73

237

5.00

20

2.52

95

2.52

11

1.15

6

1.73

236

10.60

T2 (0.0097656)

21

2.08

98

6.11

12

2.08

5

0.58

230

5.03

20

3.06

101

3.21

13

0.58

7

1.73

234

10.21

T3 (0.01953125)

21

2.52

99

4.16

12

1.53

4

0.58

228

11.00

19

1.15

95

4.04

12

1.15

5

1.73

229

4.51

T4 (0.0390625)

18

1.73

99

3.61

11

1.15

6

1.53

226

6.11

20

3.00

97

5.13

12

2.08

7

1.53

228

7.64

T5 (0.078125)

15

0.58

85

3.51

9

1.15

3

0.58

198

8.54

17

1.73

81

5.51

8

0.58

3

1.15

196

11.50

PC

323

14.01

684

6.43

316

14.50

191

6.51

1590

17.01

306

4.58

700

13.58

307

6.00

193

12.34

1599

17.44

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.

 

Positive Controls:

2-Nitrofluorene

:

TA98 (absence of metabolic activation)

Sodium azide

:

TA100 and TA1535 (absence of metabolic activation)

9-Aminoacridine

:

TA1537 (absence of metabolic activation)

Mitomycin-C

:

TA102(absence of metabolic activation)

Benzo[a]pyrene

:

TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table3  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 98

TA 100

TA 1535

TA 1537

TA 102

TA 98

TA 100

TA 1535

TA 1537

TA 102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (Distilled water)

21

2.5

102

5.51

12

2.08

7

1.15

235

7.55

21

2.65

102

5.51

13

2.00

6

1.15

236

5.57

VC (Dimethyl sulfoxide)

19

2.5

97

2.52

13

1.53

6

1.15

238

7.94

20

2.52

96

2.65

13

1.15

5

1.15

229

6.43

T1 (0.00488828)

19

2.3

99

7.55

12

1.15

6

1.53

235

6.66

23

1.15

100

3.79

12

1.15

6

1.73

232

6.24

T2 (0.0097656)

21

3.0

101

6.11

12

2.08

6

1.15

234

8.96

22

1.15

94

4.04

11

1.00

6

0.58

240

6.03

T3 (0.01953125)

19

1.2

95

3.21

14

1.73

7

1.73

234

6.66

22

1.53

94

6.03

13

0.58

6

1.73

226

6.03

T4 (0.0390625)

20

2.6

102

4.00

11

1.53

5

1.15

227

9.17

19

1.15

102

2.65

13

2.08

6

1.53

223

4.04

T5 (0.078125)

16

0.6

84

4.36

9

2.08

4

1.61

200

8.33

17

2.31

82

3.79

9

1.73

3

0.58

196

10.60

PC

335

8.0

684

10.60

333

9.07

203

16.01

1700

29.10

335

12.29

687

3.06

318

8.08

204

14.29

1686

11.79

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.

 

Positive Controls:

2-Nitrofluorene

:

TA98 (absence of metabolic activation)

Sodium azide

:

TA100 and TA1535 (absence of metabolic activation)

9-Aminoacridine

:

TA1537 (absence of metabolic activation)

Mitomycin-C

:

TA102 (absence of metabolic activation)

Benzo[a]pyrene  

:

TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table4 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)

0.94

0.94

0.94

0.98

0.95

1.08

0.95

1.12

1.03

1.01

0.94

0.97

0.95

0.93

1.08

1.03

0.95

0.94

1.01

0.97

T1 (0.0048828)

1.03

0.98

1.02

0.96

0.92

0.85

0.95

0.95

1.02

1.01

0.97

1.11

1.02

1.05

0.88

0.93

0.89

1.13

0.99

1.01

T2 (0.0097656)

1.05

0.98

1.02

1.01

0.95

0.95

0.84

1.11

0.99

1.00

1.09

1.07

1.04

0.99

0.93

0.83

0.89

1.19

0.98

1.05

T3 (0.01953125)

1.05

0.94

1.03

0.95

0.90

0.88

0.68

0.79

0.98

0.98

1.00

1.08

0.98

0.98

1.05

1.00

1.11

1.13

0.98

0.99

T4 (0.0390625)

0.92

0.97

1.03

0.98

0.87

0.93

1.00

1.05

0.97

0.98

1.03

0.95

1.06

1.06

0.85

0.95

0.84

1.06

0.95

0.98

T5 (0.078125)

0.78

0.82

0.88

0.82

0.72

0.63

0.42

0.42

0.85

0.84

0.84

0.85

0.87

0.86

0.70

0.68

0.58

0.63

0.84

0.86

PC

16.41

14.81

7.13

7.04

24.33

23.03

30.11

30.53

6.82

6.85

17.34

16.48

7.08

7.18

24.95

23.88

32.11

38.19

7.14

7.37

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.

Table5 S9 Efficiency Check- Summary

Summary of S9 efficiency check

 

TA100

TA1535

Mean

SD

Mean

SD

VC Dimethyl Sulfoxide (-S9)

98.67

4.51

13.00

2.00

VC Dimethyl Sulfoxide (+S9)

95.33

4.73

13.33

2.08

PC Benzo[a]pyrene (-S9)

97.67

3.51

13.33

2.08

PC Benzo[a]pyrene (+S9)

676.00

14.42

307.00

6.00

Key:   VC = Vehicle control, PC = Positive control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, it is concluded that p,p',p"-tris(dimethylamino)trityl alcohol [CAS No. 467-63-0] is non-mutagenic as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 or TA102) neither in the presence nor in the absence of metabolic activation system.
Executive summary:

The ability of  the registered substance, p,p’,p”-tris(dimethylamino)trityl alcohol [CAS No. 467-63-0] to induce point mutation and/or frameshift in the histidine operon was tested in five Salmonella typhimurium tester strains (TA1537, TA1535, TA98, TA100 or TA102) in the presence and absence of an exogenous metabolic activation. The S9 fraction was obtained from rats injected with the combination of phenobarbitone and β-naphthoflavone. Test concentrations were selected based on solubility, precipitation and a preliminary cytotoxicity test. The cytotoxicity test was performed with TA98 and TA100 strains, both in the presence and absence of metabolic activation using the plate incorporation method. The following concentrations were tested in triplicates along with the negative, vehicle and positive controls: 0.0 (NC), 0.0 (VC), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. In both strains, complete inhibition of background lawn with no presence of any revertant colonies were observed at 0.3125 mg/plate, both with and without S9 metabolic activation. A substantial reduction in the number of revertant colonies accompanied by a moderate inhibition of background lawn was observed at 0.15625 mg/plate compared to the vehicle control data. A slight reduction in the number of revertant colonies accompanied by a moderate inhibition of background lawn was observed at 0.078125 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. Hence, cconsidering the criteria that the highest test concentration should yield a decrease in the revertant colony count and/or diminution of the background lawn, 0.078125 mg/plate was selected as the highest concentration of the Test Item for the main assay, either in the presence or the absence of metabolic activation when compared to the vehicle control data. Hence the following doses were selected for the main test: 0.0 (NC), 0.0 (VC), 0.0048828, 0.0097656, 0.01953125, 0.0390625 and 0.078125 mg/plate. Trial I of the main test was conducted according to the plate incorporation method, both with (10 % v/v S9 mix) and without S9 metabolic activation system using the tester strains TA98, TA100, TA1535, TA1537 and TA102. Trial II  was performed to confirm the negative result of Trial I. Trial II was performed according to the preincubation method, both in the presence (10 % v/v S9 mix) and absence of S9 metabolic activation system using the tester strains TA1535, TA1537, TA98, TA100 and TA102. Results: In Trial I, a slight reduction in the revertant count and moderate inhibition in the background lawn was noted at the concentration of 0.078125 mg/plate both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. There was no increase in the number of revertant colonies or diminution of the background lawn was observed at the concentrations from0.0390625 to 0.00488mg/plate either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. Furthermore, no trend of an increased number of revertant colonies with increased Test Item concentration was observed. In Trial II, a slight reduction in the revertant count and moderate inhibition in the background lawn was noted at the concentration of 0.078125 mg/plate both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. There was no increase in the number of revertant colonies or diminution of the background lawn was observed at the concentrations from0.0390625 to 0.0048828mg/plate either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the Test Item was observed. Conclusion: Based on the results of the present study, it is concluded that p,p',p"-tris(dimethylamino)trityl alcohol [CAS No. 467-63-0] is non-mutagenic as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 or TA102) neither in the presence nor in the absence of metabolic activation system. The study was performed according to GLP.