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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:
2019-01-08 - 2019-02-01 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997
“Bacterial Reverse Mutation Test“
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
472-110-0
EC Name:
-
Cas Number:
71868-15-0
Molecular formula:
C20H22O5
IUPAC Name:
2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl]-2-methylpropan-1-one
Test material form:
solid: particulate/powder
Remarks:
off white to pale yellow powder
Details on test material:
Storage: Fridge (2 – 8 °C); keep away from light; keep away from humidity
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage: Fridge (2 – 8 °C); keep away from light; keep away from humidity.
- Stability in solvents: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Method

Target gene:
his
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S9 produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
Test concentrations with justification for top dose:
- 5000 / 1500 / 500 / 150 / 50 µg/plate (plate incorporation method; experiment 1a)
- 5000 / 1500 / 500 / 150 / 50 / 15 µg/plate (plate incorporation method; experiment 1b)
- 5000 / 2500 / 1250 / 625 / 313/ 156 / 78 µg/plate (pre-incubation method; experiment 2)
- top dose was selected according to the guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone. The solid test item was sufficiently soluble in DMSO, only. Based on this non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Remarks:
H2O
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min (Pre-incubation method)
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
Minimal histidine agar

NUMBER OF REPLICATIONS: Per strain and dose, three plates with and three plates without S9 mix were used. Two experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
Two independent experiments with variations in methodology should be performed.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Evaporation from medium: based on the substance' vapour pressure, evaporation is highly unlikely
- Water solubility: substance is sufficiently soluble
- Precipitation: none stated

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
COMPARISON WITH HISTORICAL DATA
In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 04. Dec. 2018 is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.
For the historical data, the plate incorporation method and the pre- incubation method were used.

Table Historical Data of Spontaneous Revertants

Strain TA97a TA98 TA100 TA102 TA1535
Induction - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
Demin.water Mean 88 95 26 27 94 98 286 305 17 17
Min 60 63 6 8 51 64 85 67 6 7
Max 144 138 52 53 147 141 445 587 36 40
SD 16 16 13 12 15 15 61 73 6 6
Exp 1a 79 107 49 42 100 91 421 324 12 13
Exp 1b 97 91 41 42 90 103 293 293 9 11
Exp. 2 77 86 40 47 88 102 297 304 11 9

DMSO Mean 88 98 25 26 90 94 287 298 17 17
Min 58 67 7 8 44 62 79 80 8 6
Max 139 144 50 51 138 199 465 499 35 37
SD 17 16 13 13 15 16 63 65 6 6
Exp 1a 97 94 50 43 107 97 339 324 12 11
Exp 1b 95 97 43 42 81 88 229 293 10 10
Exp. 2 76 80 47 43 102 117 289 291 12 12

Pos.Control* Mean 539 536 388 149 523 809 1119 1214 269 149
Min 264 228 77 39 220 273 491 408 55 45
Max 1165 1181 s.g. 488 1256 1912 2331 6083 515 s.g.
SD 163 173 172 114 205 287 387 517 85 109
Exp 1a 564 456 300 287 1021 s.g. 1208 1184 252 188
Exp 1b 480 539 90 86 716 1003 1267 1320 377 176
Exp. 2 264 431 309 312 1192 1261 1344 1365 176 96

* Different positive controls were used
s.g.= strong growth, too strong for counting of revertants

The incubation of the bacteria strains TA98, TA100 and TA1535 with the respective positive control induced such a high increase in revertant colonies, which overlapped and made counting impossible. So, it could not be determined whether colony count was in the range of historical control, but an >2-fold increase over control was obvious.

Applicant's summary and conclusion

Conclusions:
not mutagenic in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535, in the absence and presence of metabolic activation
Executive summary:

The study was performed according to the most recent OECD and EC guidelines, i.e. OECD 471 and EU method B.13/14.

The test item 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

All negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. 

In the experiment 1a and 1b, precipitations of the test item 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one was observed at the two highest tested concentrations (5000 μg/plate and 1500 μg/plate). In experiment 2, precipitations were observed in the two highest concentrations, too (5000 and 2500 μg/plate). In all three experiments, the test item caused cytotoxicity towards all bacteria strains

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.

All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Since all criteria for acceptability have been met, the study is considered valid.

Based on the results of this study it is concluded that 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.