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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 - 22 Oct 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted in 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
EC Number:
212-990-3
EC Name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
Cas Number:
903-19-5
Molecular formula:
C22H38O2
IUPAC Name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
Test material form:
solid: crystalline

Method

Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli)
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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats treated with Phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
First experiment (dose setting): 2.29, 6.86, 20.6, 61.7, 185, 556, 1667 and 5000 µg/plate with and without metabolic activation in all tested strains.
Second experiment (main): 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation in all strains except S. typhimurim TA 1537.

With strain TA 1537, growth inhibition occured in the dose setting study, so doses in the main study were adjusted accordingly, as follows:
TA 1537 (-S9): 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
TA 1537 (+S9): 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle/solvent.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-furyl)3-(5-nitro2-furyl)acrylamide [AF-2] in DMSO; 2-Aminoanthracene [2-AA] in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation was utilized as the application method in both the dose setting and the main studies.
- Cell density at seeding (if applicable): for use in studies viability required to be ≥1 x 10E9 cells/mL.

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: triplicate in each of the dose setting and main studies

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition (antibacterial activity) and examination of the bacterial background lawn.
Evaluation criteria:
Results were considered positive if the relevant colony count increased in tandem with dose concentration, and the increase was at least double that of the negative control, and the result was reproducible.

Results were considered negative if none of the positive results were observed.
Statistics:
No statistics were used for the data analysis.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
main study (experiment 2) without S9: ≥156 µg/plate; main study (experiment 2) with S9: ≥313 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was noted at ≥556 µg/plate of test substance with and without metabolic activation in both studies.


Test Period: From Nov 2007 to Jun 2008

Negative Control (Revertants per plate)
Bacterial Strain TA 100 TA 1535 WP2uvrA TA 1537 TA 98
Strain S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 102 107 12 12 69 97 9 12 17 22
SD 8 11 3 3 10 13 3 4 4 2
Min 78 81 4 2 44 61 2 0 5 16
Max 126 132 19 22 93 134 15 24 30 28


Positive Control (Revertants per plate)
Bacterial Strain TA 100 TA 1535 WP2uvrA TA 1537 TA 98
Strain S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 640 1523 360 289 1064 901 558 240 479 508
SD 42 115 39 21 167 65 106 31 39 48
Min 552 1224 268 225 646 760 338 163 364 395
Max 728 1823 451 354 1482 1042 778 317 593 620

Applicant's summary and conclusion

Conclusions:
Under the conditions of this OECD Guideline 471 study, the test substance AF-317, was found to be negative for mutagenicity with and without metabolic activation.