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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Reverse Mutation Test, OECD 471): negative
Cytogenicity in mammalian cells (Chromosomal Aberration, OECD 473): negative
Gene mutation in mammalian cells (HGPRT, OECD 476): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions. Although 5 strains were tested no strain for detection of cross-linking or oxidising mutagens was included, but in accordance with guideline at time of testing.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Strain for detection of oxidising or cross-linking mutagens is missing, but in accordance with guideline at the time of testing.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His-operon
Species / strain / cell type:
other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Very low water solubility of test substance in common solvents. Appropriate solvents not suitable for bacterial culture; therefore, suspension of test substance in DMSO added to cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 without S-9 mix

Migrated to IUCLID6: 1 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 100 without S-9 mix

Migrated to IUCLID6: 0.5 µL/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4 NPD), 10 µg/plate
Remarks:
TA 98 and TA 1538 without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without S-9 mix

Migrated to IUCLID6: 60 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA), 0.5 µg/plate
Remarks:
All strains with S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background growth
Evaluation criteria:
Acceptability of Assay:
1) Negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range for each tester strain.
2) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance.
3) The selected dose range should include a clearly toxic concentration as demonstrated by a preliminary toxicity range-finding test with strain TA 100.
Statistics:
Mean and standard deviation of revertant numbers.
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA 98, 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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred up to limit concentrations.

RANGE-FINDING/SCREENING STUDIES:
Eleven serial dilutions of the test substance (0.1, 0.3, 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate) were plated with a diluted TA 100 culture on non-selective agar for viability counting. For viability counting equal numbers of bacterial cells were plated in the presence of test substance. The percentage of survival of an appropriately diluted TA 100 culture on non-selective agar was determined by comparing the number of colonies on the solvent control plate with those on the test substance plates. Even at the highest test substance concentration used (5000 µg/plate) there was no reduction in survival of TA 100. Therefore, the test substance was tested up to a concentration of 5000 µg/plate in the main test, which is the maximum test concentration to be used according to the OECD guideline.

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed numbers of revertants are well within the ranges of the historical controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Maximum mean numbers of revertants (± SD):

Strain

-S9

+S9

Control

Test substance (µg/plate)

Control

Test substance (µg/plate)

TA 1535

9 ± 2

9 ± 1 (100)

12 ± 4

14 ± 2 (333)

TA 1537

12 ± 4

14 ± 3 (1000)

9 ± 2

20 ± 19 (5000)

TA 1538

14 ± 2

19 ± 6 (100)

26 ± 6

28 ± 3 (100)

TA 98

36 ± 10

38 ± 9 (333)

30 ± 14

36 ± 8 (333)

TA 100

121 ± 6

119 ± 7 (5000)

98 ± 19

138 ± 9 (5000)

 

The test substance did not induce a statistically significant dose-related increase in the numbers of revertants in any of the tester strains.

Conclusion:

The test substance does not have to be considered as mutagenic to bacteria.

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions. Although 5 strains were tested no strain for detection of cross-linking or oxidising mutagens was included, but in accordance with guideline at time of testing.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Strain for detection of oxidising or cross-linking mutagens is missing, but in accordance with guideline at the time of testing.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His-operon
Species / strain / cell type:
other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: solubility of test compound in vehicle
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
hexane
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without S-9

Migrated to IUCLID6: 80 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
hexane
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA 100 and TA 1535 without S-9

Migrated to IUCLID6: 3 µg/plate (TA 100), 5 µg/plate (TA 1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
hexane
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 and TA 1538 without S-9

Migrated to IUCLID6: 1 µg/plate (TA 98) and 2 µg/plate (TA 1538)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
hexane
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: 0.5 µg/plate with TA 1538 and TA 98, 1 µg/plate with TA 100, and 2 µg/plate with TA 1535 and TA 1537.
Remarks:
all strains with S-9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: triplicates; independent repetition of complete test

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: reduction of background growth
Evaluation criteria:
Positive: increase in revertant colony numbers of at least twice of those of the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in presence or absence of S9-mix. No statistical analysis is performed.
Negative: no reproducible increases in revertant colony numbers of at least 1.5 times of those of the concurrent solvent controls, at any dose level with any bacterial strain. No statistical analysis is performed.
Ambiguous: If the results fail to satisfy the criteria for a clear positive or negative response as described the following approach is taken: 1) Repeat tests with modified experimental method may be performed. These modifications include (but are not restricted to) the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies the 'positive' criteria the material is considered to be mutagenic. No statistical analysis is performed. 2) If no clear 'positive' response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedure used is normally analysis of variance followed by Student's t-test.
Statistics:
In case of ambiguous results analysis of variance followed by Student's t-test is performed. In case of unequivocally positive or negative results according to the described criteria, no statistical analysis is performed.
Species / strain:
other: TA 1535, TA 1537, TA100, TA98, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation starting at 1500 µg/plate in test 1 and at 5000 µg/plate in test 2.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Starting at 1500 µg/plate with and without metabolic activation in all strains in test 1 and at 5000 µg/plate with and without activation in all strains in test 2.

RANGE-FINDING/SCREENING STUDIES:
Dose range finding tests were performed with 5, 50, 500 and 5000 µg/plate. No cytotoxicity was observed, therefore 5000 µg/plate was chosen as maximum concentration for main test.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Maximum mean number of revertants (Test with highest revertant numbers chosen):

 

Mean revertant numbers ±SD

 

without S-9

with S-9

Strain

Solvent control

Test substance (µg/plate)

Solvent control

Test substance (µg/plate)

TA 1535

10±2.6

11±2.1 (50)

13±1.5

13±4.0 (50)

TA 1537

12±2.5

11±5.1 (150)

9±1.4

10±0.6 (500)

TA 1538

11±1.5

10±4.0 (50)

10±1.7

13±1.7 (1500)

TA 98

23±1.2

26±3.2 (50)

23±3.6

25±4.2 (500)

TA 100

75±5.1

89±8.3 (500)

93±5.5

97±13.4 (50)

 

The concurrent positive controls demonstrated the validity of the test system.

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation.

Conclusion:

Under the conditions chosen the test substance was not mutagenic in bacteria.

Conclusions:
Interpretation of results (migrated information):
negative
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: GLP guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
ISBN 92-64-12221-4, Paris, 1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA 40 CFR Part 798, 27 September 1985
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: primary cells from peripheral rat blood
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 0.25 µg/mL Fungizone, 100 U/mL penicillin G, 0.1 mg/mL streptomycin sulfate, 2 mM L-glutamine and 20 µg/mL PHA. Treatment was done in medium without serum and PHA with or without 2% (v/v) S-9 mix. Thereafter, the old medium was applied to the cultures again.
- Properly maintained: yes, freshly prepared whole blood was cultured at 37 °C in T25 plastic tissue culture flasks
- Periodically checked for karyotype stability: not required, karyotype of cultured rat lymphocytes is stable in contrast to the relative karyotype instability of cell lines.
- Periodically "cleansed" against high spontaneous background: not applicable, freshly prepared cultures
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
40, 133, 400 µg/mL (additionally 4 and 13 µg/mL for initial determination of mitotic indices)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: isopropyl alcohol
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and various other solvents
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% (v/v) isopropyl alcohol (final concentration)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S-9 mix

Migrated to IUCLID6: 1000 µg/mL (8 mM)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% (v/v) isopropyl alcohol (final concentration)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S-9 mix

Migrated to IUCLID6: 4.2 µg/mL (15 µM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 and 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 28 and 52 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 200 metaphases (100 of each replicate culture) (positive control: 50 metaphases/replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Only those metaphases that contained 42 chromosomes were scored with the exception of severely damaged cells, in which case accurate counts of the chromosomes were not possible. Those cells having 10 or more aberrations/cell were classified as severely damaged cells. Gaps were not included in calculations of total cytogenetic aberrations.
Statistics:
The frequencies of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled and analysed by constructing two-dimensional contingency tables. The total Chi-square was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no difference in average scores (average number of aberrations per cell) among the dose groups and (2) no linear trend of increasing scores with increasing dose. An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at alpha=0.01 versus a two-sided alternative, pairwise tests (i.e. control vs treatment) were performed at each dose level and evaluated again at alpha=0.01 versus a two-sided alternative.

The statistical procedure is designed to accommodate scoring of individual cells where the score is equal to the number of aberrations observed in the cell. Scoring is then averaged within each dose group in a method similar to one used by Bhapkar, 1968. The mean scores are then analysed using a chi-square statistic that is partitioned to provide a test of trend (Landis et al., 1978; Mantel and Haenszel, 1959).
Species / strain:
lymphocytes: primary cells from peripheral rat blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: Precipitation occurred in the cultures treated with 133 and 400 µg/mL test substance in the culture medium; the choice of doses for the chromosomal aberration test was made due to solubility.

RANGE-FINDING/SCREENING STUDIES:
The determination of the mitotic indices after exposure to test substance concentrations of 4 to 400 µg/mL and 24 and 48 hours expression time served as kind of a range finder; precipitation was observed at test substance concentrations of 133 and 400 µg/mL in the absence and presence of metabolic activation. The average mitotic index of cultures treated with 400 µg/mL in the absence of S-9 was 6.1% (24 hours) and 4.1% (48 hours) compared to negative control values of 6.0 and 6.5%, respectively. In the presence of S-9, the average mitotic index for cultures treated with 400 µg/mL was 3.8 (24 hours) and 4.0% (48 hours) compared to negative control values of 4.8 and 3.9%, respectively.

Since no evidence of toxicity was observed (with and without S-9) in cultures treated with concentrations up to and exceeding the limit of solubility, the three top concentrations (40, 133, 400 µg/mL) were selected for determining chromosomal aberration frequencies at the 24-hour harvest with and without S-9.
At the 48-hour harvest, cultures treated with the three top doses (40, 133, 400 µg/mL) were scored in the absence of S-9; in the presence of S-9 only the 400 µg/mL dose level was scored. No positive controls were included at the 48-hour harvest.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the chromosomal aberration assay after exposure up to 400 µg/mL and expression times of 24 and 48 hours.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of the chromosomal aberration assay in cultured rat lymphocytes:

Concentration (µg/mL)

Mitotic index (%)

Chromatid Gaps

Total Aberrations (excluding gaps)

No. of cells with Aberrations (excluding gaps)

absolute

(%)

absolute

(%)

24 hours after treatment, without S-9

Solvent

6.0

7

4

2.0

4

2.0

40

6.2

15

8

4.0

6

3.0

133

5.5

7

0

0

0

0

400

6.1

14

5

2.5

5

2.5

EMS(1000)

4.3

25

32

32.0

25*

25.0

24 hours after treatment, with S-9

Solvent

4.8

7

1

0.5

1

0.5

40

5.7

11

1

0.5

1

0.5

133

4.8

5

1

0.5

1

0.5

400

3.8

10

2

1.0

2

1.0

CP (4.2)

2.5

18

44

44.0

19*

19.0

48 hours after treatment, without S-9

Solvent

6.5

16

3

1.5

3

1.5

40

3.9

11

1

0.5

1

0.5

133

6.0

8

3

1.5

3

1.5

400

4.1

16

0

0

0

0

48 hours after treatment, with S-9

Solvent

3.9

9

0

0

0

0

400

4.0

7

1

0.5

1

0.5

* alpha≤0.01

In the assays conducted without S-9 (both 24- and 48-hour harvest), the frequencies of cells with aberrations in the test chemical treated cultures ranged from 0 to 3.0%. In the assays with S-9, the highest percentage of cells with aberrations was 1.0%. These values were neither significantly different from the concurrent negative (solvent) controls nor outside the reasonable range of the laboratory historical background aberration frequencies. Significant increases in aberration rates were observed in cultures treated with the positive control chemicals.

Conclusion:

The test substance did not exhibit clastogenic activity in cultured rat lymphocytes when tested up to or exceeding the limits of solubility under the conditions of this study.

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA 40 CFR Part 798
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT-locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 nutrient mix supplemented with 5% (v/v) heat-inactivated (56 °C, 30 min) dialysed fetal bovine serum, 25 mM HEPES, 0.25 µg/mL Fungizone, 100 U/mL penicillin and 0.1 mg/mL streptomycin sulfate. The selection medium used for the detection of HGPRT-deficient mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6-thioguanine, 5% serum, 25 mM HEPES, 2 mM L-glutamine and the above-mentioned antibiotics-antimycotics. Treatment was conducted in medium without serum with or without 2% (v/v) S-9 mix.
- Properly maintained: yes, stocks stored at approx. -100 °C or below; cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of 5% CO2 in air at 37 °C in a humidified incubator.
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
Survival test: 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, 400.0 µg/mL
Assay 1+2: 50, 100, 200, 400 µg/mL
Assay 3: 10, 20, 30, 40, 50, 60 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: isopropyl alcohol
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and various other solvents
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% (v/v) isopropyl alcohol (final concentration)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S-9 mix

Migrated to IUCLID6: 621 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% (v/v) isopropyl alcohol (final concentration)
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S-9 mix

Migrated to IUCLID6: 4 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 8 days (replating on day 1, 3, 6)
- Selection time (if incubation with a selection agent): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-17 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: crystal violet after methanol fixation

NUMBER OF REPLICATIONS: 5 replicates/treatment

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
Determination of HGPRT-deficient mutants per 10e6 clonable cells:

Mutants per 10e6 clonable cells = (No. of mutant colonies per 10e6 plated cells/Cloning efficiency)

where Cloning efficiency = (Number of colonies/Number of cells plated)
Evaluation criteria:
Acceptability: The mutation frequency in positive controls should be significantly higher than the background frequency, and the mutation frequency in the negative controls should be within reasonable limits of the historical control values of this laboratory and the literature values.

Positive: Statistically significant and reproducible increase in mutation frequency at more than one of the dose levels tested. The final interpretation also considers such factors as mutation frequency and cloning efficiencies in the negative controls and dose-response relationships.
Statistics:
The frequencies of mutants per 10e6 clonable cells observed in the treated cultures were compared to the negative control with an appropriate chi-square statistic at an alpha level of 0.01, one-sided (Steel and Torrie, 1980). Examination of trends was conducted with least squares regression using an alpha level of 0.025 (Winer, 1971).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed in 1/3 assays in the absence of metabolic activation only without dose-response. Not reproducible in an identical assay.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no appreciable change in the pH of the culture medium following addition of the test material as determined by means of an Accumet pH meter in a solution containing 420 µg/mL of the test substance.
- Effects of osmolality: There was no appreciable change in the osmolality of the culture medium following addition of the test material as determined by means of an Accumet pH meter in a solution containing 420 µg/mL of the test substance.
- Water solubility: The test substance has only a very limited water solubility; therefore isopropyl alcohol was chosen as solvent.
- Precipitation: Starting at 40 µg/mL in the absence and presence of metabolic activation in assay #3. A remarkable variability in precipitation was observed from assay to assay; therefore, in assay #3 the maximum concentration was lowered to 60 µg/mL. In the preliminary toxicity assay, no precipitate was observed at 100 µg/mL or lower concentrations. In assay #1 precipitate was observed at 100 µg/mL, in assay #2 there was precipitation at the 50 µg/mL dose level, as well. This variability was considered likely to the poor solubility properties of the test material in aqueous and organic solvents.

RANGE-FINDING/SCREENING STUDIES:
In order to establish an appropriate dose range, survival of CHO cells treated with the test substance was determined in a preliminary toxicity test. The cells were exposed to 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, 400.0 µg/mL in triplicate cultures in the absence and presence of metabolic activation, and the relative cell survival was calculated as percentage of the ratio of mean number of colonies/dish in the treated cultures and the mean number of colonies/dish in the negative control. The highest concentration tested was based upon limitations imposed by solubility of the test material in the solvent. Precipitation was observed at 400 and 200 µg/mL in this assay. No cytotoxicity was observed at any of the concentrations tested. Based upon these results a dose level of 400 µg/mL was selected as highest test concentration for assays 1 and 2.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies observed from the control cultures are well comparable to the respective values of the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In assay #1 the relative cell survival (RCS) was not affected in cultures treated with the test material in the presence of S-9. However, in the assay conducted without S-9, the RCS was affected, albeit without a dose response, in the treated cultures. In assay #2 the test substance was not toxic at any dose level either in the presence or in the absence of S-9.

Results of the HGPRT Assays #1 and 2 in CHO cells:

Treatment (µg/mL)

Toxicity assay: RCS (%)

Mutation assay: TG-resistant colonies

Cloning Efficiency CE (%)

TG-resistant colonies/10e6 clonable cells

Assay #1 without S-9

neg. control

100.0

0

44.0

0

50

37.7

1

62.2

1.6

100 P

80.8

2

53.2

3.8

200 P

7.2

1

63.0

1.6

400 P

12.5

1

55.9

1.8

pos. control

72.4

120

44.4

270.3*

Assay #1 with S-9

neg. control

100.0

0

53.0

0

50

95.2

3

56.6

5.3

100 P

99.3

4

63.5

6.3

200 P

106.1

7

62.0

11.3

400 P

100.0

0

73.1

0

pos. control

107.5

81

58.3

138.9*

Assay #2 without S-9

neg. control

100.0

3

71.6

4.2

50 P

115.6

11

68.8

16.0*

100 P

113.6

5

65.9

7.6

200 P

115.6

3

67.1

4.5

400 P

118.3

10

73.6

13.6

pos. control

70.9

110

51.4

214.0*

Assay #2 with S-9

neg. control

100.0

5

68.7

7.3

50 P

90.7

24

53.6

44.8*

100 P

104.1

9

76.9

11.7

200 P

96.3

8

62.8

12.7

400 P

114.5

10

77.6

12.9

pos. control

101.3

123

71.5

172.0*

* alpha≤0.01

P: precipitation

 

Results of HGPRT Assay #3 in CHO cells:

Treatment (µg/mL)

Toxicity assay: RCS (%)

Mutation assay: TG-resistant colonies

Cloning Efficiency CE (%)

TG-resistant colonies/10e6 clonable cells

Assay #3 without S-9

neg. control

100.0

7

83.2

8.4

10

105.8

7

81.8

8.6

20

108.4

1

76.8

1.3

30

96.1

4

81.3

4.9

40 P

99.0

7

77.3

9.1

50 P

102.2

4

83.2

4.8

60 P

103.1

4

77.8

5.1

pos. control

80.0

99

45.9

215.7*

Assay #3 with S-9

neg. control

100.0

14

70.9

19.7

10

103.9

16

66.6

24.0

20

101.4

15

59.4

25.3

30

93.1

3

60.8

4.9

40 P

103.9

4

62.8

6.4

50 P

101.6

6

63.6

9.4

60 P

97.2

3

62.8

4.8

pos. control

92.0

93

66.9

139.0*

* alpha≤0.01

P: precipitation

In Assay #1 the relative cell survival was affected in the absence of S-9; however, this effect could not be reproduced in any of the other assays, neither in absence nor in presence of metabolic activation. The test material did not significantly increase the mutation frequency either in the presence or absence of S-9 in this assay.

In Assay #2 the test material was not toxic at any dose level tested either in the presence or absence of S-9. Statistical analysis of the mutation frequency data from this assay indicated a significant increase at the lowest test concentration of 50 µg/mL, which was also outside the historical background range.

In order to ascertain the biological significance of the increased mutation frequency observed at the 50 µg/mL dose level in Assay #2, a third assay with concentrations from 10 to 60 µg/mL was conducted. In this assay precipitation was observed in treatment medium at 40 µg/mL or higher. There were no significant increases in mutation frequency in cultures treated with the test material. Hence, the isolated increase in mutation frequency in Assay #2 at 50 µg/mL has to be interpreted as chance occurrence unrelated to the treatment.

The positive control chemicals induced significant increases in mutation frequency in all assays and therefore demonstrated the adequacy of the experimental conditions for detecting induced mutations.

Conclusion:

The test substance did not induce gene mutations in mammalian cells in the CHO/HGPRT gene mutation assay under the experimental conditions chosen.

Conclusions:
Interpretation of results (migrated information):
negative

Genetic toxicity in vivo

Description of key information

Waiving - No in vivo testing required as none of the in vitro genetic toxicity studies were positive

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro

- Gene mutation in bacteria

The mutagenic potential of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) was assessed in a GLP-compliant gene mutation assay in bacteria (Ames test) according to OECD 471 (Jones, 1990). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to the test material diluted in hexane for 48 h at concentrations of 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). Consistent with the preliminary range finding test, no cytotoxic effects were observed in any tester strains up to the maximum concentration of 5000 µg/plate in the both experiments of the main study. In the first experiment, precipitation of the test substance was observed starting at 1500 µg/plate (with and without S9 mix) in all strains, whereas in the second experiment, precipitation was noted at 5000 µg/plate with and without activation. No increase in the mean number of revertants compared to controls was noted after treatment in either the presence or absence of S9 mix in both independent experiments. The concurrent positive and solvent controls demonstrated the expected results, and thus verified the sensitivity of the assay. Based on these results, the test substance was not considered mutagenic in the presence and absence of metabolic activation in the selected strains of S. typhimurium.

In a further GLP-compliant Ames test according to OECD 471, the same strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) in DMSO for 48 h (Debets and Enninga, 1986). Based on a preliminary range-finder test in TA 100 showing no cytotoxicity up to 5000 µg/plate, tester strains were treated with concentrations of 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of metabolic activation. No cytotoxicity and no precipitation were observed in any tester strain at any concentration with and without S9 mix. The mean numbers of revertants in treated cells were not increased compared to the vehicle control. The concurrent positive and vehicle controls demonstrated the expected results, and thus verified the sensitivity of the assay. Therefore, the test substance was not mutagenic in the presence and absence of metabolic activation in the selected strains of S. typhimurium.

In conclusion, the results of both gene mutation assays in bacteria consistently showed that the test substance is not mutagenic in S. typhimurium in the presence and absence of metabolic activation.

- Chromosome aberrations

An in vitro mammalian chromosome aberration test was performed with (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) in cultured rat lymphocytes according to OECD guideline 473 and in compliance with GLP (Linscombe, 1991). An initial cytotoxicity test was performed with concentrations of 4, 13, 40, 133 and 400 µg/mL in the presence or absence of metabolic activation (S9 mix) of 4 h followed by an expression time of 24 and 48 h to select suitable concentrations for the analysis of chromosomal aberrations. Precipitation occurred in cultures treated with 133 and 400 µg/mL of the test substance in the culture medium. Since no evidence of toxicity was observed (with and without S9 mix) in cultures treated with concentrations up to and exceeding the limit of solubility, the three top concentrations (40, 133, 400 µg/mL) were selected for determining chromosomal aberration frequencies at the 24-h harvest with and without S9 mix. At the 48-h harvest, cultures treated with the three top doses (40, 133, 400 µg/mL) were scored in the absence of S9 mix, whereas in the presence of S9 mix only the highest concentration of 400 µg/mL was scored. No statistically significant increase in treated cells compared to the concurrent negative (solvent) controls was observed. The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values in all experiments and the positive controls (only included in the 24-h harvest experiment) produced statistically significant increases in the frequency of metaphases with chromosomal aberrations. Therefore, under the conditions of this assay, the test substance did not exhibit clastogenic activity in cultured rat lymphocytes in the presence and absence of metabolic activation.

- Gene mutation in mammalian cells

(Z)-N-octadec-9-enylhexadecan-1-amide was investigated in a HGPRT gene mutation assay in cultured mammalian cells (Chinese hamster ovary (CHO) cells) according to OECD guideline 476 (Linscombe, 1991). In a preliminary cytotoxicity test, cells were exposed to concentrations ranging from 6.3 to 400 µg/mL in the presence and absence of metabolic activation (S9-mix) for a period of 4 h to establish an appropriate concentration range for the analysis of gene mutations. Precipitation of the test substance was observed at 400 and 200 µg/mL in this assay. Since no cytotoxic effects were observed at any concentration tested, gene mutations at the HGPRT locus in the main assay were investigated at concentrations of 100, 200, 400, 600, 800 and 1000 µg/mL in the presence and absence of metabolic activation (S9 mix) in two subsequent experiments. In the first experiment, the relative cell survival was affected in the absence of S9 mix, which, however, could not be reproduced in any of the other assays, neither in absence nor in presence of metabolic activation. Furthermore, the test material did not significantly increase the mutation frequency compared to controls in the presence and absence of S9 mix. In the second experiment, the test material was not toxic at any concentration tested, neither in the presence nor in the absence of S9 mix. In contrast to the first experiment, a statistically significant increase in the mutation frequency was observed at the lowest test concentration of 50 µg/mL, which was also outside the historical background range of the vehicle control. To ascertain the biological significance of the increased mutation frequency observed at the 50 µg/mL in the second experiment, a third independent experiment with a narrowed concentration range of 10 to 60 µg/mL was conducted. In this assay, precipitation was observed in treatment medium at 40 µg/mL or higher and no significant increases in mutation frequency in cultures treated with the test material were noted. Therefore, regarding the criteria for a positive test result in this assay, the isolated increase in mutation frequency in the second experiment at 50 µg/mL was considered to be incidental, and thus not toxicologically relevant. Under the conditions of the study, it was thus concluded that the test material was not mutagenic at the HGPRT locus of Chinese hamster ovary cells in the absence and presence of metabolic activation.

 

In vivo

According to Regulation (EC) No 1907/2006, Annex IX, column 2, testing for genetic toxicity in vivo is not indicated as the test substance did not demonstrate any genotoxic activity in bacteria or mammalian cells in vitro.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies addressed different endpoints and were negative.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC and are therefore conclusive but not sufficient for classification.