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EC number: 201-815-6 | CAS number: 88-26-6
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15 - 24 Aug 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Aug 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
- Version / remarks:
- June 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 3,5-di-tert-butyl-4-hydroxybenzyl alcohol
- EC Number:
- 201-815-6
- EC Name:
- 3,5-di-tert-butyl-4-hydroxybenzyl alcohol
- Cas Number:
- 88-26-6
- Molecular formula:
- C15H24O2
- IUPAC Name:
- 2,6-Di-tert-butyl-4-(hydroxymethyl)phenol
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in a tightly closed container, protected from light and humidity.
FORM AS APPLIED IN THE TEST (if different from that of starting material)
liquid
Method
- Target gene:
- his operon, tryp operon
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats, t reated with phenobarbital and β-naphthoflavone
- Test concentrations with justification for top dose:
- Based on a range-finding study (performed in tester strains TA 98 and TA 100; doses applied: 5, 16, 50, 160, 500, 1600, 5000 μg/plate), the following concentrations were used in the main experiments:
10, 32, 100, 320, 800 and 1000 μg/plate with and without metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in DMSO, this solvent was selected as the vehicle.
Controls
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO (vehicle control for test substance and some positive control substances) and ultrapure water (vehicle control for some positive control substances)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first experiment); preincubation (second experiment)
DURATION
- Preincubation period: 20 min (second experiment)
- Exposure duration: 48 h (first and second experiment)
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and inspection of the bacterial background lawn - Evaluation criteria:
- Acceptance criteria
The study was considered valid if:
- the phenotypes of the tester strains could be confirmed
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains
- the tester strain culture titers are in the 10^9 cells/mL order
- the batch of S9 used in this study shows the appropriate biological activity
- the reference mutagens show an increase of at least: 3-fold in induced revertant colonies over the mean value of the respective vehicle control
- at least five analyzable concentrations were presented in all strains of the main tests (a minimum of three non-toxic dose levels is required to evaluate assay data)
Evaluation criteria
When the test substance shows a biologically relevant and dose-related increase in the number of revertant colonies of more than two times (TA 100) or three times (TA 98, TA 1535, TA 1537 and WP2 uvrA) compared to that of the solvent control, the response is judged to be positive. Additionally, the positive response should be reproducible for at least one of the dose groups and should occur in at least one strain with or without metabolic activation. The biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is therefore not regarded as necessary.
The test item is considered as non-mutagenic in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- Mean values and standard deviations were calculated.
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
- Remarks:
- Number of revertant colonies decreased to less than half compared to solvent control in exp. 1 starting at 800 μg/ plate (58%) without S9 mix and at 1000 μg/ plate (51%) with S9 mix.
- 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:
- cytotoxicity
- Remarks:
- Number of revertant colonies decreased to less than half compared to solvent control in exp. 1 starting at 800 μg/ plate (58%) without S9 mix and at 1000 μg/ plate (65%) with S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Number of revertant colonies decreased to less than half compared to solvent control in exp. 1 at 1000 μg/ plate (53%) without S9 mix and in exp. 2 starting at 800 μg/ plate (82%) with S9 mix.
- Vehicle controls validity:
- valid
- 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:
- cytotoxicity
- Remarks:
- Number of revertant colonies decreased to less than half compared to solvent control in exp. 1 starting at 800 μg/ plate (75%) without S9 mix and at 1000 µg/plate (55%) with S9 mix and in exp. 2 starting at 800 μg/ plate (71%) without S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Number of revertant colonies decreased to less than half compared to solvent control in exp. 1 at 1000 μg/ plate (57%) with S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes (for further details please refer to "Results and discussions"(Remarks on results))
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
yes (for further details please refer to "Any other information on results incl. tables", Table 3) - Remarks on result:
- other: Precipitation started in exp. 1 at 800 µg/plate without S9 mix and at 320 µg/plate with S9 mix and in exp. 2 at 800 µg/plate with and at 1000 µg/plate without S9 mix.
Any other information on results incl. tables
Table 1: Summary of test results (exp. 1; Initial Mutation Test, Plate Incorporation Method)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA1537 |
TA98 |
TA100 |
TA1535 |
WP2 uvrA |
||
– |
Solvent control (ultrapure water) |
- |
- |
102.7 ± 2.31 |
7.3 ± 1.15 |
17.7 ± 3.79 |
Solvent control (DMSO) |
5.3 ± 0.58 |
19.0 ± 1.73 |
85.7 ± 6.03 |
11.3 ± 4.04 |
17.0 ± 3.00 |
|
Untreated control |
6.7 ± 2.08 |
18.0 ± 1.00 |
104.0 ± 9.17 |
8.7 ± 5.13 |
20.0 ± 0.00 |
|
10 |
6.3 ± 3.51 |
17.0 ± 2.65 |
89.0 ± 5.29 |
14.7 ± 5.51 |
16.7 ± 2.08 |
|
32 |
6.3 ± 2.08 |
15.0 ± 3.61 |
81.7 ± 8.74 |
10.7 ± 2.08 |
20.3 ± 5.77 |
|
100 |
3.7 ± 0.58 |
14.3 ± 2.31 |
82.3 ± 6.11 |
8.7 ± 1.53 |
20.7 ± 4.16 |
|
320 |
5.3 ± 2.08 |
16.3 ± 5.69 |
81.7 ± 6.81 |
11.3 ± 4.51 |
17.7 ± 2.89 |
|
800 |
1.3 ± 0.58 |
8.0 ± 4.00 |
35.7 ± 4.93 |
7.3 ± 3.51 |
19.3 ± 3.79 |
|
1000 |
0.0 ± 0.00 |
1.7 ± 2.08 |
33.3 ± 10.02 SB, P |
5.3 ± 3.79 |
12.0 ± 4.36 SB, P |
|
Positive controls (unit/plate) |
9AA |
NPD |
SA |
SA |
MMS |
|
Mean No. of colonies/plate (average of 3 plates) |
750.0 ± 84.59 |
342.7 ± 26.10 |
1245.3 ± 185.44 |
672.0 ± 66.81 |
818.7 ± 118.68 |
|
+ |
Solvent control (DMSO) |
7.3 ± 3.06 |
21.7 ± 7.02 |
128.0 ± 18.33 |
14.0 ± 2.56 |
28.0 ± 2.00 |
Untreated control |
6.3 ± 1.53 |
21.7 ± 6.11 |
132.0 ± 11.14 |
12.3 ± 4.93 |
29.3 ± 4.16 |
|
10 |
6.0 ± 1.00 |
25.3 ± 5.51 |
105.7 ± 16.62 |
11.0 ± 4.58 |
34.7 ± 2.08 |
|
32 |
10.7 ± 2.08 |
20.7 ± 2.52 |
86.0 ± 8.19 |
12.7 ± 4.51 |
26.0 ± 7.55 |
|
100 |
10.3 ± 2.52 |
24.3 ± 2.08 |
84.7 ± 2.89 |
9.0 ± 1.73 |
27.3 ± 5.51 |
|
320 |
6.0 ± 3.00 SP |
25.0 ± 3.00 SP |
78.3 ± 18.15 SP |
13.0 ± 1.00 SP |
29.7 ± 6.35 SP |
|
800 |
5.0 ± 2.00 P |
17.3 ± 1.15 P |
66.7 ± 4.04 P |
7.3 ± 0.58 P |
24.3 ± 2.31 P |
|
1000 |
3.3 ± 0.58 |
10.7 ± 1.15 |
44.7 ± 3.21 |
7.3 ± 1.53 |
12.0 ± 3.46 |
|
Positive controls (µg/plate) |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Mean No. of colonies/plate (average of 3 plates) |
110.0 ± 17.06 |
1512.0 ± 246.45 |
2200.0 ± 381.58 |
199.3 ± 37.65 |
234.0 ± 15.62 |
|
SD = standard deviation
9AA = 9-aminoacridine
NPD = 4-nitro-1,2-phenylene-diamine
SA = sodium azide
MMS = methylmethanesulfonate
2AA = 2-aminoanthracene
P = precipitate
SP = slight precipitate
SB = slightly reduced background lawn development
Table 2: Summary of test results (exp. 2; Confirmatory Mutation Test, Preincubation Method)
With or without S9-Mix |
Test substance (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA1537 |
TA98 |
TA100 |
TA1535 |
WP2 uvrA |
||
– |
Solvent control (ultrapure water) |
- |
- |
93.3 ± 6.66 |
11.7 ± 3.79 |
28.3 ± 1.15 |
Solvent control (DMSO) |
8.0 ± 4.00 |
16.7 ± 0.58 |
78.0 ± 9.64 |
11.0 ± 3.00 |
24.3 ± 6.43 |
|
Untreated control |
8.0 ± 2.65 |
17.7 ± 9.07 |
90.3 ± 13.20 |
9.3 ± 2.52 |
19.3 ± 2.52 |
|
10 |
7.3 ± 4.16 |
17.0 ± 0.00 |
88.0 ± 7.21 |
11.7 ± 4.62 |
22.3 ± 7.09 |
|
32 |
7.3 ± 3.21 |
18.7 ± 5.77 |
78.0 ± 5.29 |
11.0 ± 4.36 |
23.3 ± 6.66 |
|
100 |
7.7 ± 0.58 |
20.3 ± 7.09 |
79.3 ± 9.02 |
11.7 ± 2.52 |
17.3 ± 4.73 |
|
320 |
9.3 ± 7.57 |
15.0 ± 4.36 |
82.0 ± 11.14 |
9.3 ± 4.93 |
14.0 ± 3.61 |
|
800 |
2.3 ± 4.04 B |
10.3 ± 6.11 SB |
64.3 ± 31.37 SB |
2.0 ± 3.46 B |
21.0±2.65 |
|
1000 |
0.0 ± 0.00 |
12.3 ± 1.53 |
92.0 ± 26.91 |
4.3 ± 0.58 |
20.7 ± 4.93 |
|
Positive controls (unit/plate) |
9AA |
NPD |
SA |
SA |
MMS |
|
Mean No. of colonies/plate (average of 3 plates) |
356.0 ± 107.63 |
254.0 ± 40.60 |
1210.7 ± 122.46 |
965.3 ± 53.27 |
962.7 ± 158.86 |
|
+ |
Solvent control (DMSO) |
6.3 ± 3.06 |
27.7 ± 2.89 |
108.7 ± 17.62 |
13.3 ± 3.21 |
27.7 ± 4.04 |
Untreated control |
7.7 ± 4.93 |
28.7 ± 3.51 |
129.7 ± 8.96 |
11.7 ± 0.58 |
32.3 ± 4.51 |
|
10 |
8.0 ± 3.00 |
25.3 ± 5.13 |
118.3 ± 10.21 |
13.3 ± 1.53 |
31.7 ± 5.03 |
|
32 |
10.7 ± 11.72 |
25.0 ± 9.85 |
105.3 ± 12.90 |
10.7 ± 2.08 |
35.0 ± 7.94 |
|
100 |
7.0 ± 1.73 |
28.3 ± 8.50 |
82.3 ± 6.03 |
10.7 ± 0.58 |
28.3 ± 5.69 |
|
320 |
9.3 ± 1.53 |
25.3 ± 10.50 |
94.7 ± 8.50 |
12.3 ± 5.13 |
36.0 ± 5.20 |
|
800 |
7.0 ± 2.65 |
27.0 ± 5.29 |
93.0 ± 19.00 SB, SP |
12.0 ± 4.58 |
31.3 ± 3.21 SP |
|
1000 |
8.3 ± 4.04 |
21.0 ± 6.08 |
72.0 ± 27.73 |
6.7 ± 3.21 |
38.3 ± 11.93 SB, P |
|
Positive controls (µg/plate) |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Mean No. of colonies/plate (average of 3 plates) |
164.7 ± 9.02 |
1117.3 ± 120.09 |
1114.7 ± 360.65 |
138.0 ± 7.81 |
170.3 ± 15.57 |
|
SD = standard deviation
9AA = 9-aminoacridine
NPD = 4-nitro-1,2-phenylene-diamine
SA = sodium azide
MMS = methylmethanesulfonate
2AA = 2-aminoanthracene
P = precipitate
SP = slight precipitate
SB = slightly reduced background lawn development
Table 3: Historical Control Values for Revertants/Plate (for the Period of 2008-2015)
|
S. typhimurium |
E. coli |
|||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||
Untreated |
-S9 |
Average |
21.4 |
106 |
10.4 |
8.1 |
25.6 |
SD |
3.7 |
27.3 |
1.5 |
2.5 |
5.5 |
||
Minimum |
9 |
65 |
3 |
2 |
11 |
||
Maximum |
39 |
157 |
23 |
19 |
45 |
||
+S9 |
Average |
28 |
117.1 |
11.9 |
9 |
34.3 |
|
SD |
4.2 |
19.4 |
1.5 |
2 |
5.4 |
||
Minimum |
12 |
75 |
4 |
3 |
18 |
||
Maximum |
48 |
166 |
24 |
20 |
56 |
||
DMSO |
-S9 |
Average |
20.9 |
101.4 |
10.3 |
7.9 |
24.9 |
SD |
3.5 |
26.2 |
1.4 |
2.5 |
4.9 |
||
Minimum |
10 |
65 |
3 |
2 |
11 |
||
Maximum |
39 |
150 |
23 |
20 |
44 |
||
+S9 |
Average |
27.1 |
114.7 |
12 |
8.8 |
34.2 |
|
SD |
4 |
19.3 |
1.5 |
2.1 |
5.2 |
||
Minimum |
15 |
71 |
4 |
3 |
16 |
||
Maximum |
48 |
161 |
24 |
20 |
56 |
||
Water |
-S9 |
Average |
22.4 |
105.5 |
10.4 |
7.5 |
26.3 |
SD |
3.6 |
27.6 |
1.6 |
2.3 |
5.9 |
||
Minimum |
12 |
67 |
3 |
2 |
13 |
||
Maximum |
36 |
156 |
24 |
15 |
47 |
||
+S9 |
Average |
28 |
117.4 |
11.5 |
8.7 |
35.2 |
|
SD |
4 |
19.8 |
1.4 |
2.3 |
5.2 |
||
Minimum |
15 |
83 |
4 |
4 |
18 |
||
Maximum |
43 |
166 |
22 |
16 |
56 |
||
Positive controls |
-S9 |
Average |
255.6 |
958.9 |
842.1 |
467.4 |
712.3 |
SD |
30.7 |
149.9 |
134 |
105.7 |
57.5 |
||
Minimum |
123 |
522 |
354 |
109 |
320 |
||
Maximum |
647 |
1927 |
1871 |
1498 |
1283 |
||
+S9 |
Average |
1224.8 |
1431.9 |
165.4 |
148 |
264.7 |
|
SD |
293.8 |
339.9 |
35.1 |
21.3 |
74.2 |
||
Minimum |
409 |
581 |
85 |
68 |
141 |
||
Maximum |
2587 |
2923 |
507 |
407 |
487 |
||
SD = standard deviation
Applicant's summary and conclusion
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
