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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-01-15 to 2009-04-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline compliant GLP compliant

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:
as at 1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Mexoryl SBO
IUPAC Name:
Mexoryl SBO
Details on test material:
- Name of test material (as cited in study report): Mexoryl SBO

Method

Target gene:
Organism Strain Type of mutation in the histidine gene
S. typhimurium TA98 frame-shift
S. typhimurium TA100 base-pair substitution
S. typhimurium TA1535 base-pair substitution
S. typhimurium TA1537 frame-shift
S. typhimurium TA102 base-pair substitution
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9), prepared from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
- Experiment 1 (plate incorpotration assay): 1.6, 8, 40, 200, 1000, 5000 µg/plate
- Experiment 2 (pre-incubation assay): 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not reported, but DMSO is a standard vehicle for use in the Ames Test
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (2NF), Sodium azide (NaN3), 9-aminoacridine (AAC), Mitomycin C (MMC), Benzo[a]pyrene (B[a]P), 2-aminoanthracene (AAN); see Table 2 for details
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, experiment 1); preincubation (experiment 2);

DURATION
- Preincubation period: 1 h (only in experiment 2)
- Exposure duration: 3 d
- Expression time (cells in growth medium): 3 d
- Selection time (if incubation with a selection agent): 3 d
- Fixation time (start of exposure up to fixation or harvest of cells): 3 d

SELECTION AGENT (mutation assays): histidine free medium ( traces of histidine available to allow first cell division)

NUMBER OF REPLICATIONS:
- treatment groups: triplicate
- negative (vehicle) and positive controls: quintuplicate

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;

OTHER EXAMINATIONS:
- not applicable
Evaluation criteria:
Evaluation criteria
For valid data, the test article was considered to be mutagenic if:
1. significant response (p ≤ 0.01) as determined by Dunnett's test + concentration relation of effects
2. reproducibility of positive trend/effects described above

The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
Dunnet's Test ( see above)

Results and discussion

Test resultsopen allclose all
Key result
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:
no cytotoxicity
Vehicle 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:
cytotoxicity
Remarks:
slight thinning of background bacterial lawn and/or a reduction in revertants at 5000 μg/plate in the absence of S-9at 1000 μg/plate and above in the presence of S-9
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none noted
- Effects of osmolality: none noted
- Evaporation from medium: not noted
- Water solubility: test article completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed
- Precipitation: no, see above
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES:
presented as Experiment 1

COMPARISON WITH HISTORICAL CONTROL DATA:
see Table 3
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

- Table 3: Historical negative (vehicle) control values for S. typhimurium strains

Strain

S-9

No. of studies

No. of plates

Mean

99% reference range(1)

99% confidence interval for group mean of:

4 values (2)

5 values (2)

6 values (2)

TA98

-

50

503

25

10.0-43.0

16.4-33.3

17.1-32.3

17.7-31.5

TA98

+

50

525

35

15.0-56.0

24.7-46.2

25.6-44.9

26.4-44.0

TA100

-

50

572

111

72.0-160.0

88.8-134.1

90.9-131.5

92.6-129.6

TA100

+

50

588

119

77.0-178.0

92.9-145.4

95.4-142.4

97.2-140.1

TA1535

-

50

505

17

5.0-33.0

9.8-24.8

10.4-23.9

10.9-23.2

TA1535

+

50

524

19

6.0-35.0

11.6-26.8

12.2-25.9

12.7-25.2

TA1537

-

50

512

11

2.0-27.0

5.4-17.7

5.9-16.9

6.2-16.3

TA1537

+

50

534

15

4.0-32.0

8.3-22.9

8.9-21.9

9.4-21.2

TA102

-

48

475

281

178.0-435.0

222.8-342.7

228.5-335.7

232.7-330.6

TA102

+

48

499

238

152.0-341.0

194.4-283.4

198.7-278.3

201.9-274.6

Reference ranges are calculated from percentiles of the observed distributions

Calculated from square-root transformed data

Ranges calculated in August 2008 by CLEH Statistics, using data selected without bias from studies# started during the periods given below:

S.typhimurium strains (except TA102): Mar 07 to Oct 07

S.typhimurium strain TA102: Feb 07 to Oct 07

# All studies had been audited prior to data collection.

- For detailed test data, please see the attached document.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Mexoryl SBO was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments according to OECD 471 and GLP.
It was concluded that the test item did not induce mutation in the five tester strains when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

Mexoryl SBO was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments according to OECD 471 and GLP.

An initial toxicity Range-Finder Experiment was carried out in the absence and in the presence of S-9 in strain TA100 only, using final concentrations of Mexoryl SBO at 1.6, 8, 40, 200, 1000 and 5000 μg/plate, plus negative (vehicle) and positive controls. Following these treatments, no evidence of toxicity was observed These data were considered acceptable for mutation assessment and were used as the TA100 mutagenicity data for Experiment 1.

Treatments of the remaining test strains were performed in the absence and in the presence of S-9 in Experiment 1, and retained the same test concentrations employed for the Range-Finder Experiment treatments. Following these treatments, evidence of toxicity was observed solely in strain TA102 at the highest one (treatments without S-9) or two (treatments with S-9) concentrations.

Experiment 2 treatments of all the test strains were performed in the absence and in the presence of S-9 at concentrations up to 5000 μg/plate. In each strain narrowed concentration intervals were used (156.25 to 5000 μg/plate), in order to more closely investigate those concentrations of the test item approaching the limit concentration, and therefore considered most likely to provide evidence of any mutagenic activity. In addition, treatments in the presence of metabolic activation were further modified by the inclusion of a pre-incubation step, and in this way the range of mutagenic chemicals that can be detected in this assay system was increased. Following these treatments, evidence of toxicity was observed solely in strain TA102 at concentrations of 1250 μg/plate and above in the absence of S-9 and at 5000 μg/plate in the presence of S-9. Negative (vehicle) and positive control treatments were included for all strains in both experiments.

The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

Following Mexoryl SBO treatments of all the strains tested, both in the absence and in the presence of S-9, no statistically significant increases in revertant numbers were observed when the data were analysed at the 1% level using Dunnett’s test.

This study was therefore considered to have provided no evidence of any Mexoryl SBO mutagenic activity in this assay system.

It was concluded that Mexoryl SBO did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).