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Genetic toxicity in vitro

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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
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
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 / 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
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)
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'.

- 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.

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).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study performed under GLP without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
liver metabolizing system (S9 mix)
Test concentrations with justification for top dose:
Experiment I, without S9: 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500, 5000 µg/mL
Experiment I, with S9: 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500, 5000 µg/mL
Experiment II, without S9: 156.3, 312.5, 625, 1250, 2500, 5000 µg/mL
Experiment II, with S9: 156.3, 312.5, 625, 1250, 2500, 5000 µg/mL
Experiment III, without S9: 1000, 2000, 2500, 3750, 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Without S9 mix: mitomycin C (MMC). With S9 mix: cyclophosphamide (CPA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I, without S9: 3 h
Experiment I, with S9: 3 h
Experiment II, without S9: 20 h and 44 h
Experiment II, with S9: 3 h and 3 h
Experiment III, without S9: 20 h
- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment I, without S9: 20 h
Experiment I, with S9: 20 h
Experiment II, without S9: 20 h and 44 h
Experiment II, with S9: 20 h and 44 h
Experiment III, without S9: 20 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analyzed when at least 10% cells with structural chromosome aberration were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the Chi square test, in which p = 0.05 was used as the lowest level of significance.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
None.

RANGE-FINDING/SCREENING STUDIES: The highest dose-level for treatment was selected on the basis of pH, osmolality and solubility. This did not take into account the molecular weight of the test item (214.3 g/mol) due to the unavailability of this information on due time. In the culture medium, the dose-level of 5000 μg/mL (corresponding to 23.33 mM) showed no precipitate. At this dose-level, the pH was approximately 7.1 (7.1 for the vehicle control) and the osmolality equal to 330 mOsm/kg H2O (306 for the vehicle control). 5000 µg/mL was therefore chosen as the highest dose.

COMPARISON WITH HISTORICAL CONTROL DATA: The frequency of cells with structural chromosome aberration in the controls was consistent with the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Following the 3-hour treatment without S9 mix, no noteworthy decrease in mitotic index was noted at all dose-levels tested. Following the 20-hour treatment in the second experiment (without S9), a moderate to marked decrease in mitotic index was observed at dose-levels ≥ 2500 μg/mL (49-65% decrease). This decrease in mitotic index was not reproduced in the third experiment performed under the same experimental conditions. Following the 44-hour treatment (experiment II, without S9 mix), a marked to severe decrease in mitotic index was observed at dose-levels ≥ 2500 μg/mL (76-89% decrease). No noteworthy decrease in the mitotic index was observed at the 20-hour as well as at the 44-hour harvest times in the experiments with S9 mix but the substance was tested up to the limit concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Metaphase analysis

No significant increase in the frequency of cells with structural chromosomal aberration was noted after 3- and 44-hour treatments without S9 mix. Following the 20-hour treatment without S9, neither significant increase in the frequency of cells with structural chromosome aberrations nor any clear reproducible tendency to a dose-relationship were noted at dose-levels as high as 4167 μg/mL (corresponding to 19.44 mM) and including a dose-level near the highest recommended dose-level by the international guidelines (2500 μg/mL corresponding to 11.67 mM). At the highest dose-level used for treatment (5000 μg/mL corresponding to 23.33 mM), a slight significant increase in the frequency of aberrant cells was noted (6.5% versus 0.5% for the vehicle control and 7% versus 2% for the vehicle control, in the second and third experiments, respectively).

Taking into account the following considerations:

- absence of significant chromosomal aberrations at dose-levels equivalent to the highest recommended dose-level by the international guidelines (10 mM if lower than 5000 μg/mL) and even higher (up to 19.44 mM),

- observation of a slight increase in the frequency of aberrant cells (not exceeding 7%) only at the dose-level of 23.33 mM which is 2.33-fold higher than the highest recommended dose-level,

- well-known effects of concentrations higher than 10 mM (or 5000 μg/mL whichever is lower) can induce artefactual increases in aberration,

the increase in the frequency of aberrant cells observed at this highest dose of 23.33 mM (5000 μg/mL) was not considered as biologically relevant.

No significant increase in the frequency of cells with structural chromosomal aberration was noted in both experiments with S9 mix and at both harvest times.

Controls

The frequency of cells with structural chromosome aberration of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

Conclusions:
Interpretation of results (migrated information):
negative

Under our experimental conditions, the test item did not induce any increase in the frequency of chromosome aberrations which could be considered as biologically relevant, in cultured human lymphocytes.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce chromosome aberrations in cultured human lymphocytes. The study was performed according to the international guidelines (OECD 473, Commission Directive No. B10) and in compliance with the Principles of Good Laboratory Practice Regulations.

The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix). An additional experiment was performed without S9 mix. The highest dose-level for treatment was selected on the basis of pH, osmolality and solubility. This did not take into account the molecular weight of the test item (214.3 g/mol) due to the unavailability of this information on due time. For selection of the dose-levels, toxicity indicated by the reduction of mitotic index if any was also taken into account.

In the culture medium, the dose-level of 5000 μg/mL (corresponding to 23.33 mM) showed no precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. The dose-levels used for treatment were as follows:

- 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL, for the first experiment, both with and without S9 mix,

- 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL, for the second experiment, both with and without S9 mix,

- 1000, 2000, 2500, 3750, 4167 and 5000 μg/mL, for the third experiment without S9 mix.

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment. The second experiment without S9 mix, cells were exposed continuously to the test or control items until harvest, with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment. In a third confirmatory experiment without S9 mix, cells were exposed continuously to the test or control items until harvest which was performed 20 hours after the beginning of treatment. One and a half hours before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

Following the 3-hour treatment without S9 mix, no noteworthy decrease in mitotic index was noted at all dose-levels tested. Following the 20-hour treatment without S9 mix in the second experiment, a moderate to marked decrease in mitotic index was observed at dose-levels ≥ 2500 μg/mL (49-65% decrease). This decrease in mitotic index was not reproduced in the third experiment performed under the same experimental conditions. Following the 44-hour treatment without S9 mix, a marked to severe decrease in mitotic index was observed at dose-levels ≥ 2500 μg/mL (76 -89% decrease). No noteworthy decrease in the mitotic index was observed at the 20-hour as well as at the 44-hour harvest times in the experiments with S9 mix.

No significant increase in the frequency of cells with structural chromosomal aberration was noted after 3- and 44-hour treatments without S9 mix. Following the 20-hour treatment, neither significant increase in the frequency of cells with structural chromosome aberrations nor any clear reproducible tendency to a dose-relationship were noted at dose-levels as high as 4167 μg/mL (corresponding to 19.44 mM) and including a dose-level near the highest recommended dose-level by the international guidelines (2500 μg/mL corresponding to 11.67 mM). At the highest dose-level used for treatment (5000 μg/mL corresponding to 23.33 mM), a slight significant increase in the frequency of aberrant cells was noted (6.5% versus 0.5% for the vehicle control and 7% versus 2% for the vehicle control, in the second and third experiments, respectively).

Taking into account the following considerations:

- absence of significant chromosomal aberrations at dose-levels equivalent to the highest recommended dose-level by the international guidelines (10 mM if lower than 5000 μg/mL) and even higher (up to 19.44 mM),

- observation of a slight increase in the frequency of aberrant cells (not exceeding 7%) only at the dose-level of 23.33 mM which is 2.33-fold higher than the highest recommended dose-level,

- well-known effects of concentrations higher than 10 mM (or 5000 μg/mL whichever is lower) which can induce artefactual increases in aberrations,

the increase in the frequency of aberrant cells observed at this highest dose of 23.33 mM (5000 μg/mL) was not considered as biologically relevant. No significant increase in the frequency of cells with structural chromosomal aberration was noted in both experiments with S9 mix at both harvest time.

The frequency of cells with structural chromosome aberration of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

Under the experimental conditions, the test item did not induce any increase in the frequency of chromosome aberrations which could be considered as biologically relevant, in cultured human lymphocytes.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2008-12-01 to 2009-05-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD TG 487 compliant with some deviation
Qualifier:
according to guideline
Guideline:
other: OECD guideline 487
Deviations:
yes
Remarks:
: Due to technical error, the final concentrations of MMC in treated cultures were 10-fold less than those stated in the Definitive Protocol. This deviation had no impact on the validity of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
- Type and identity of media: human peripheral blood lymohocytes from two healthy, non-smoking female volunteers
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no (short-term culture and maintain a stable karyotype)
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
400.0, 550.0 and 750.0 µg/mL for treatment of 24+0 hour, -S-9
1370, 1712 and 2140 µg/mL (approximately equivalent to 0.01 M) for treatment of 3+21 hour, +/-S-9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: vinblastine and mitomycin c (in the absence of rat liver S-9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h treatment + 21 h recovery (with and without S-9), 24 h treatment + 0 h recovery (without S-9)
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): 4% (v/v) Giemsa in pH 6.8 buffer

NUMBER OF CELLS EVALUATED: a minimum of 200 cells per concentraiton (range-finder experiment), a minimum of 500 cells per culture (main experiment only)

DETERMINATION OF CYTOTOXICITY
- Method:
RI (replication index) = (number binucleate cells + 2(number multinucleate cells))/total number of cells in treated cultures
Relative RI (%) = RI of treated cultures/RI of vehicle controls
cytotoxicity (%) = (100 - Relative replication index)
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed
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. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result.
Statistics:
The proportions of Micronucleated Binucleate (MNBN) cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: human peripheral blood
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: no significant changes in pH were observed at the highest concentration tested (2140 µg/mL) as compared to the concurent vehicle controls.
- Effects of osmolality: significant (greater than 50 mOsm/kg) shifts in osmolarity were observed from post treatment medium taken from all treatments (range-finder and micronucleus experiments). As test article treated cultures analysed for micronuclei demonstrated no evidence of an increase in MNBN cells above vehicle or historical control data, the osmolality shifts noted had no impact on the data interpretation, or, validity of the study data.

RANGE-FINDING/SCREENING STUDIES: a maximum concentration of 2140 µg/mL, approximately equivalent to 10 mM (molecular weight of Mexoryl SBO = 214 g/mol) was selected for the cytotoxicity Range-Finder experiment, in order that treatments were performed up to a maximum recommended concentration according to current regulatory guidelines. Concentrations for the Main Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.
Remarks on result:
other: other: lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Mexoryl SBO did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were tested up to 2140 μg/mL (approximately equivalent to 10 mM), a recommended regulatory maximum concentration for in vitro cytogenetic assays.
Executive summary:

Mexoryl SBO was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by

narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest

concentration used in the Main Experiment, 2140 μg/mL (equivalent to 10 mM) was determined following a preliminary cytotoxicity Range-Finder Experiment.Treatments were conducted 48 hoursfollowing mitogen stimulation by Phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Mexoryl SBO on the replication index (RI). In the Main Experiment, micronuclei were analysed at three concentrations. Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate cells (MNBN)

in these cultures fell within current historical vehicle control (normal) ranges for the majority of cultures. A single exception to this was noted following 24+0 hour –S-9 treatment but was isolated with three other concurrent controls and all test article

treated cultures demonstrating normal MNBN cell values. As such, the vehicle control data was accepted as valid. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Main Experiment at 24 hours after the start of treatment; all compounds induced statistically

significant increases in the proportion of cells with micronuclei. The assay system was therefore considered as sensitive and valid. Treatment of cultures with Mexoryl SBO in the absence and presence of metabolic activation (S-9) resulted in frequencies of MNBN cells which were similar to and not significantly (p ≤ 0.05) different from those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all Mexoryl SBO treated cultures fell within historical vehicle control (normal) ranges. It is concluded that Mexoryl SBO did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were tested up to 2140 μg/mL

(equivalent to 10 mM), a recommended regulatory maximum concentration for in vitro cytogenetic assays.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three in vitro tests on genetic toxicity are available.

In an Ames test performed under GLP and according to OECD 471, five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium were treated with the registration item at concentrations up to 5000 µg/plate, both in the absence and in the presence of metabolic activation. Two independent experiments were conducted. Following the treatments, no statistically significant increases in revertant numbers were observed in any of the strains tested, both in the absence and in the presence of S-9. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system.

A GLP study was performed in accordance with OECD 473 to evaluate the potential of the test item to induce chromosome aberrations in cultured human lymphocytes. Two independant experiments with concentrations up to 5000 µg/mL (23.33 mM) were performed with and without metabolic activation. No significant increase in the frequency of cells with structural chromosomal aberration was noted except at the highest dose-level (5000 μg/mL corresponding to 23.33 mM) after 20 h continuous treatment, where a slight significant increase in the frequency of aberrant cells was noted, but was not considered as biologically relevant. The frequency of cells with structural chromosome aberration of the vehicle and positive controls was as specified in acceptance criteria. In conclusion, the test item did not induce any increase in the frequency of chromosome aberrations in cultured human lymphocytes which could be considered as biologically relevant.

An in vitro micronucleus assay was performed according to OECD 487 and under GLP. Duplicate human lymphocyte cultures prepared from the pooled blood of two female donors were used. Concentrations up to 2140 µg/mL (10 mM) were tested. Treatments were conducted 48 hours following mitogen stimulation by Phytohaemagglutinin (PHA). Treatment of cultures with the test item in the absence and presence of metabolic activation resulted in frequencies of MNBN cells which were not significantly different from those observed in concurrent vehicle controls for all concentrations analysed. It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system.

In conclusion, no genotoxic effects were observed in any of the available in vitro tests.

Short description of key information:

no adverse effects observed, OECD 471, OECD 473 and OECD 487

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No adverse effects were observed in the available in vitro tests on genotoxicity. It is therefore concluded that the substance does not have to be classified for genetic toxicity.