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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-10-10 until 2002-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: An up to date OECD guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 471: "Bacterial Reverse Mutation Test", adopted July 21,1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, Ll 362000, Annexe 4D", dated May 19, 2000
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:
444-860-9
EC Name:
-
Cas Number:
474510-57-1
Molecular formula:
C21 H24 O4
IUPAC Name:
2-hydroxy-1-(4-(4-(2-hydroxy-2-methylpropionyl) benzyl) phenyl)-2-methylpropan-1-one

Method

Target gene:
his, trp
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:
Mammalian microsomal fraction S9 mix
Test concentrations with justification for top dose:
33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation

Migrated to IUCLID6: for strains TA1535, TA100
Positive controls:
yes
Remarks:
for strains TA1537, TA98
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
Without metabolic activation
Positive controls:
yes
Remarks:
for WP2 uvrA
Positive control substance:
other: methyl methane sulfonate, MMS
Remarks:
Without metabolic activation
Positive controls:
yes
Remarks:
for strains TA 1535, TA 1537. TA 98, TA 100, WP2 uvrA
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
With metabolic activation
Details on test system and experimental conditions:
- Experiment 1: The test item was assessed for its potential to induce gene mutations according to the plate incorporation test
- Experiment 2: pre-incubation test using Salmonella typhimurium strains TA 1535, TA 1537, TA98, and
TA 100, and the Escherichia coli strain WP2 uvrA
- The test item was tested in both experiments at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate
- Each concentration and the controls were tested in triplicate
- The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar
- In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.
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 ofthe 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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxic effects, evident as a reduction in the number of revertants, were observed at 2500 µg/plate without S9 mix in strain TA 1537 in experiment I, and at 5000 µg/plate with S9 mix in tester strain TA 100 at 5000 µg/plate in experiment II.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Irregular background growth was observed at 2500 and 5000 µg/plate in strain TA 1537 in experiment I. An irregularity in the background growth was also observed in strain TA 1535 at 33 µg/plate without S9 mix in experiment I. This effect was judged to be biologically irrelevant since it was only observed in one out of three plates and the number of revertant colonies remains well within the historical control range
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance
- In experiment I, the data in the solvent control of strain TA 100 (with S9 mix) were slightly above the historical control range. In the presence of metabolic activation, the number of colonies did not quite reach the lower limit of the negative control of the historical control data in strains TA 1535 and TA 1537 (exp. II), and in strain WP2 uvrA (exp. I). Since these deviations were rather small, these effects were considered to be based upon biologically irrelevant fluctuations in the number of colonies
- The historical range of positive controls was exceeded in strains TA 1535 and TA 100 in experiment I without metabolic activation. This effect indicateed the sensitivity of the strains rather than compromising the assay
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative