Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation

Chromosome aberration test (OECD 473): positive in human peripheral blood lymphocytes with and without metabolic activation

Reconstructed skin micronucleus assay (RSMN, comparable to OECD 487): negative in human skin tissue (EpiDerm )

Mouse lymphoma assay (OECD 490): positive in L5178Y cells without metabolic activation; negative with metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Sep - 9 Oct 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
his operon
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone (80/100 mg/kg bw/day for 3 consecutive days)
Test concentrations with justification for top dose:
Pre-experiment:
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

First and second experiment: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate with and without metabolic activation
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was fully soluble at 50 mg/mL in DMSO.
Negative controls:
yes
Remarks:
untreated controls
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-dihydroxyanthraquinone (DAN), 2-aminoanthracene (2-AA), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG), 9-aminoacridine (9-AA), mitomycin C (MMC), 4-nitroquinoline-N-oxide (4-NQO), benzo(a)pyrene (BaP)
Remarks:
+S9: 2-AA (1 µg/plate, TA100; 2 µg/plate, TA1535, TA1537); BaP (5 µg/plate, TA98); DAN (10 µg/plate, TA102) -S9: ENNG (3 µg/plate, TA100; 5 µg/plate, TA1535); 9-AA (80 µg/plate, TA1537); MMC (0.5 µg/plate, TA102); 4-NQO (0.2 µg/plate, TA98)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony frequency or reduction of the bacterial background lawn
Evaluation criteria:
Several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation, were considered. However, biological relevance of the results was considered first. Additionally, statistical methods were used as an aid to evaluate a positive response.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(exp. 1: -S9: at 150 µg/plate in TA1535 and TA102; at 500 µg/plate in remaining strains; +S9: at 150 µg/plate in TA102; at 500 µg/plate in rem. strains; exp. 2: -S9: at 150 µg/plate; +S9: at 500 µg/plate in TA1537; at 150 µg/plate in rem. strains)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for the main study, a preliminary experiment was carried out to determine the toxicity of the test material. The test substance was toxic at and above 1500 µg/plate in TA100.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance caused a visible reduction in the growth of the bacterial background lawn of all tester strains in both the presence and absence of metabolic activation, initially from 150 µg/plate for TA100 and 500 µg/plate for the remaining tester strains. Several of the bacterial strains also exhibited substantial decreases in revertant colony frequency at 150 µg/plate in both the presence and absence of metabolic activation. The toxicity of the test substance to the tester strains varied slightly between experiment number and strain type and was of sufficient severity to prevent the test substance from being tested up to the maximum recommended dose level of 5000 µg/plate.

Table 2. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

135

25

316

14

9

0 (DMSO)

138 ± 21.7

28 ± 2.3

350 ± 4.9

14 ± 0.6

10 ± 0.6

1.5

145 ± 19.7

30 ± 5.0

329 ± 19.1

16 ± 5.7

9 ± 2.0

5

149 ± 8.7

31 ± 6.7

349 ± 50.1

18 ± 6.8

9 ± 0.6

15

134 ± 20.5

25 ± 4.6

360 ± 10.8

12 ± 3.8

10 ± 2.5

50

124 ± 8.5

27 ± 5.5

278 ± 34.9

17 ± 4.4

9 ± 1.2

150

151 ± 3.2

11 ± 1.0

91 ± 14.2

14 ± 0

6 ± 1.2

500

0T

0T

0T

0T

0T

1500

0T

0T

0T

0T

0T

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

[μg/plate]

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

495 ± 25.2

319 ± 19.1

2525 ± 5.5

455 ± 22.7

626 ± 48.2

+

0 (DMSO)

146 ± 25.1

17 ± 7.0

369 ± 16.2

24 ± 5.2

11 ± 3.6

+

1.5

143 ± 14.2

14 ± 8.4

385 ± 12.5

26 ± 1.5

12 ± 1.0

+

5

140 ± 20.0

11 ± 2.1

364 ± 28.9

25 ± 2.0

12 ± 2.1

+

15

129 ± 7.9

14 ± 7.0

311 ± 11.0

21 ± 1.2

13 ± 4.4

+

50

116 ± 13.4

14 ± 1.2

320 ± 12.1

19 ± 1.2

12 ± 6.2

+

150

96 ± 11.5

11 ± 2.3

188 ± 14.0

15 ± 5.2

7 ± 2.1

+

500

0T

0V

0T

0T

0T

+

1500

0T

0T

0T

0T

0T

Positive controls, +S9

Name

2-AA

2-AA

DAN

BaP

2-AA

Concentrations

[μg/plate]

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

660 ± 49.0

498 ± 53.6

1080 ± 95.2

435 ± 80.0

397 ± 33.3

DMSO: dimethylsulphoxide

BaP: benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

MMC: mytomycin C

T: toxic, no bacterial background lawn

V: very weak bacterial background lawn

 

Table 3. Test results of Experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

101

20

318

19

11

0 (DMSO)

94 ± 9.7

30 ± 1.5

275 ± 13.3

19 ± 3.0

14 ± 2.6

1.5

88 ± 4.6

30 ± 2.3

256 ± 15.8

16 ± 4.4

10 ± 1.5

5

95 ± 11.9

31 ± 7.8

261 ± 5.7

15 ± 6.0

11 ± 1.0

15

87 ± 5.5

26 ± 2.9

230 ± 20.1

16 ± 1.5

13 ± 2.1

50

96 ± 16.1

20 ± 2.1

229 ± 18.6

13 ± 8.5

13 ± 3.1

150

32 ± 3.2S

8 ± 1.2

45 ± 7.6

5 ± 1.0

4 ± 0.6

500

0T

0T

0T

0T

0T

1500

0T

0T

0T

0T

0T

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

[μg/plate]

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

505 ± 28.2

182 ± 27.2

1541 ± 68.8

131 ± 2.5

884 ± 243.8

+

0 (DMSO)

86 ± 4.0

12 ± 1.7

322 ± 9.9

21 ± 2.6

12 ± 3.8

+

1.5

85 ± 14.0

12 ± 4.0

290 ± 4.6

24 ± 6.0

8 ± 5.0

+

5

97 ± 9.2

14 ± 2.5

251 ± 20.0

24 ± 1.2

15 ± 3.5

+

15

86 ± 9.9

13 ± 4.2

247 ± 20.3

16 ± 2.6

10 ± 4.6

+

50

76 ± 5.3

11 ± 0.6

216 ± 20.0

20 ± 2.3

9 ± 3.5

+

150

34 ± 7.2S

5 ± 1.5

111 ± 5.3

5 ± 0.6

7 ± 3.6

+

500

0T

0T

0T

0T

0T

+

1500

0T

0T

0T

0T

0T

Positive controls, +S9

Name

2-AA

2-AA

DAN

BaP

2-AA

Concentrations

[μg/plate]

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

596 ± 45.5

275 ± 42.0

1407 ± 114.5

203 ± 16.8

183 ± 37.5

DMSO: dimethylsulphoxide

BaP: benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

MMC: mytomycin C

S: sparse bacterial background lawn

T: toxic, no bacterial background lawn

Conclusions:
Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and TA102) tested with and without metabolic activation.
Executive summary:

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 1500 µg/plate in the first experiment. The experiment was repeated using the same dose range as Experiment 1. Additional dose levels and an expanded dose range were selected to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn of all tester strains in both the presence and absence of S9, initially from 150 µg/plate for TA100 and 500 µg/plate for the remaining tester strains. Several of the bacterial strains also exhibited substantial decreases in revertant colony frequency at 150 µg/plate in both the presence and

absence of S9. The toxicity of the test material to the tester strains varied slightly between experiment number and strain type and was of sufficient severity to prevent the test material from being tested up to the maximum recommended dose level of 5000 lag/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Dec 2016 - 10 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)
Test material information:
Composition 1
Target gene:
not applicable
Species / strain:
lymphocytes:
Remarks:
human
Details on mammalian cell lines (if applicable):
CELLS USED
Blood samples were drawn from one healthy non-smoking male donor (33 years old) not receiving medication. The lymphocytes of the donor have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

MEDIA USED
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.
- Type and identity of media including CO2 concentration if applicable:
Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) supplemented with 200 mM GlutaMAX™, penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment/ Experiment 1:
- S9: 16.8, 29.3, 51.4, 89.9*, 157*, 275*, 482, 722, 939, 1221
+ S9: 16.8, 29.3, 51.4, 89.9, 157*, 275*, 482*, 722, 939, 1221

*concentrations evaluated

Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Since the test item was considered to be clastogenic after 4 hours treatment a second experiment was not performed.
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium (pulse treatment)

DURATION
- Exposure duration:
4 h
- Expression time (cells in growth medium): 18 h
- Fixation time (start of exposure up to fixation or harvest of cells):
22 h

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

STAIN (for cytogenetic assays):
Giemsa

NUMBER OF REPLICATIONS:
2 parallel cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip.

NUMBER OF CELLS EVALUATED:
At least 150 well-spread metaphases were evaluated per culture, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:
yes
- Determination of endoreplication:
yes


Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test substance concentrations should be within the range of the laboratory historical solvent control data
The test substance is then considered unable to induce chromosomal aberrations in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test substance is then considered able to induce chromosomal aberrations in this test system.
Statistics:
The statistical significance is confirmed by the Fisher’s exact test (modified) (p < 0.05) using a validated test script of “R”, a language and environment for statistical computing and graphics.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
-S9: at 157 and 275 µg/mL; +S9: 275 and 482 µg/mL
Cytotoxicity:
yes
Remarks:
at and above 482 µg/mL
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
The pH was adjusted to physiological values.
- Effects of osmolality:
No relevant influence on osmolarity was observed.
- Precipitation:
no precipitation of the test item was observed


RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. With regard to the molecular weight of the test item, 1221 μg/mL (approx. 10 mM) were applied as top concentration. Test item concentrations ranging from 16.8 to 1221 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic indices
In the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration.

Table 3. Results after 4 h exposure with and without metabolic activation

Experiment Preparation interval Test item concentration (µg/mL) Mitotic indices (% of control) Aberrant cells (%)
incl. gaps* excl. gaps* carrying exchanges
Exposure period 4 hrs without S9 mix            
1 22 hrs Solvent control 1 100 1 1 0
    Positive control 2 116.2 9 9.0s 2.7
    89.9 77.7 1.7 1.7 0
    157 68.6 6 6.0s 0.3
    275 62.2 4.3 4.0s 0.3
Exposure period 4 hrs with S9 mix
1 22 hrs Solvent control 1 100 2 2 0
    Positive control 3 56 8.3 8.3s 1.7
    157 74.6 2.7 2.7 0.3
    275 79.3 6.7 6.0s 0.7
    482 42.9 5 4.7s 0.3

*  Including cells carrying exchanges

s Aberration frequency statistically significant higher than corresponding control values

Deionized water: 10.0 % (v/v)

2 EMS: 825.0 µg/mL

CPA: 5.0 µg/mL

Statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed in the absence of S9 mix after treatment with 157 and 275 μg/mL (6.0 and 4.0 % aberrant cells, excluding gaps) and in the presence of S9 mix after treatment with 275 and 482 μg/mL (6.0 and 4.7 % aberrant cells, excluding gaps). The values clearly exceeded the range of the laboratory historical solvent control data (without S9 mix: 0.0 – 2.9 % aberrant cells, excluding gaps; with S9 mix: 0.0 – 3.3 % aberrant cells, excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Either EMS (825.0 μg/mL) or CPA (5.0 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
Under the experimental conditions of the in vitro chromosome aberration test, the test substance did induce structural chromosomal aberrations in human lymphocytes with and without metabolic activation. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test, when tested up to cytotoxic or to the highest evaluable concentration.
Executive summary:

The test item Tropolone, dissolved in deionized water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in one experiment. Exposure period was 4h. In each experimental group two parallel cultures were analyzed. Per culture 150 metaphases were evaluated for structural chromosomal aberrations. The highest applied concentration in this study (1221 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

In the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. Statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed in the absence of S9 mix after treatment with 157 and 275 µg/mL (6.0 and 4.0 % aberrant cells, excluding gaps) and in the presence of S9 mix after treatment

with 275 and 482 µg/mL (6.0 and 4.7 % aberrant cells, excluding gaps). The values clearly exceeded the range of the laboratory historical solvent control data (without S9 mix: 0.0 – 2.9 % aberrant cells, excluding gaps; with S9 mix: 0.0 – 3.3 % aberrant cells, excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Tropolone is considered to be clastogenic in this chromosome aberration test, when tested up to cytotoxic or to the highest evaluable concentration.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May - 09 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
EpiDermTM skin model used with functional phase I and phase II enzyme system
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
(2014)
Deviations:
yes
Remarks:
EpiDermTM Skin Model used with functional phase I and phase II enzyme system
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Target gene:
not applicable
Species / strain:
other: reconstructed human 3D skin model (EpiDermTM)
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells:
MatTek Corporation, Ashland, MA, USA
- Suitability of cells:
The EpiDermTM model is a multilayered, differentiated tissue consisting of basal, spinous, granular and cornified layers resembling the normal human epidermis. The EpiDermTM tissues used in this assay have been demonstrated to have functional Phase I and Phase II enzyme system and capable of metabolizing the indirectly acting mutagens. This system has been demonstrated to be sensitive to the clastogenic and aneugenic activity of a variety of chemicals (Curren et al., 2006).
- Methods for maintenance in cell culture if applicable:
Tissue was maintained in New Maintenance Medium (NMM, containing keratinocyte growth factor) and Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline (CMF-DPBS) at 37±1 °C in a humidified atmosphere of 5±1% CO2 in air


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Tissue was cultivated in New Maintenance Medium (NMM, containing keratinocyte growth factor) and Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline (CMF-DPBS) at 37±1 °C in a humidified atmosphere of 5±1% CO2 in air.
- Properly maintained: yes
Metabolic activation:
not applicable
Metabolic activation system:
The EpiDermTM tissues used in this assay have been demonstrated to have functional Phase I and Phase II enzyme system and capable of metabolizing the indirectly acting mutagens (Cyclophosphamide, Benzo (a) pyrene and others).
Test concentrations with justification for top dose:
Preliminary toxicity test:
0.0061 - 100 mg/mL

Main Tests:
Initial assay (48 h-treatment): 1, 2, 4, 4.25, 4.5, 4.75, 5, 5.25, and 5.5 mg/mL
Repeat assay (48 h-treatment): 1*, 2*, 3.5*, 4, 4.5, 5, 5.5, 6.5, and 7 mg/mL
Confirmatory assay (72 h-treatment): 0.5*, 1, 2, 2.5*, 3, 3.25, 3.5, 4, and 4.5* mg/mL

*concentrations used for evaluation of micronuclei



Vehicle:
- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: Acetone was chosen based on the solubility of the test article, and compatibility with the target cells. The test article was soluble in acetone at a concentration of approximately 100 mg/mL, the maximum concentration tested for solubility.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and conditions:
METHOD OF APPLICATION: incorporation of the dose solution on the tissue surface
- Dose volume: 10 µL

DURATION
- Exposure duration:
48 (initial and repeat assay) and 72 h (confirmatory assay)


CYTOKINESIS BLOCK AGENT: Cytochalasin B (3 µg/mL in DMSO)

STAIN (for cytogenetic assays):
Acridine orange

NUMBER OF REPLICATIONS:
triplicate tissues

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cells were harvested by trypsinising and single cell solution was stained with Trypan blue for counting. After fixation of the cells with ice-cold methanol/acetic acid (3:1), a single drop (i.e. 15 to 20 μL) of the cell suspension was applied on to a clean, dry microscope slide. At least two slides were prepared from each EpiDerm™ model, whenever possible (one slide was kept as back up). Slides were air dried at room temperature and stained with acridine orange.

NUMBER OF CELLS EVALUATED:
1000 binucleated cells (500 per tissue)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
• Micronuclei must be stained the same color and intensity as the main nuclei.
• The diameter of the micronuclei must be approximately 1/3 or less the diameter of a main nucleus.
• The micronuclei must be non-refractile, located in the cytoplasm, no overlapping with the nucleus and not connected by cytoplasmic bridges.
• The micronuclei were not linked or connected to the main nuclei.
• The micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary.
• Other effects such as nucleoplasmic bridges, apoptosis, necrosis, micronuclei in mononucleated cells, etc. can also be analyzed in this assay, but the assessment of genotoxicity to date has been based on induction of micronuclei in binucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: relative viable cell count (RVCC) and cytokinesis-blocked proliferation index (CBPI)

Cytotoxicity based on RVCC:
The relative viable cell count was determined by the Trypan-blue exclusion method. Average live cell count from the control/solvent tissue was calculated as follows.
% Relative viable cells of treated tissue (% RVCC) = (Total viable cells of treated tissue / Average viable cell count from solvent control tissues) x 100
Cytotoxicity = 100 - % RVCC

Cytotoxicity based on CBPI:
CBPI = 1 x Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells
Total number of cells scored
% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test article treatment culture
c = vehicle control culture
Evaluation criteria:
Assessment of the test substance:
• Positive Response - The test article was considered positive overall if it had at least one experiment with two or more statistically significant concentrations, or one concentration that was statistically significant (one-sided Fisher’s Exact test) and biologically relevant, as defined below in one independent study.
• Negative Response - The test article was considered negative overall if, in two independent studies (one 48-hour exposure and one 72-hour exposure) the test article produced no statistically significant or biologically relevant increase in the frequency of micronuclei in a binucleated cell (MN-BN).
• Equivocal Response - Despite extensive testing, a test article may produce results that are neither clearly positive nor clearly negative. In those rare instances, the test article may be considered to have produced equivocal responses.
• Biological relevance of the findings were considered, and took into account the above mentioned points (dose-dependence, strength of the effect in relation to the 95%CI of the vehicle control) as well as reproducibility between tissues and experiments. To help judge dose-dependence of the effect, the Cochran-Armitage trend test (p<0.05) may be used in the overall judgment of the effect.
Statistics:
Statistical analysis employed a Fisher's exact test for pair-wise comparisons between each treated and vehicle control treatment group. The Cochran-Armitage trend test was also conducted. One-sided tail probabilities for an increase or positive trend were used to evaluate statistical significance (p< 0.05).
Key result
Species / strain:
other: reconstructed human 3D skin model (EpiDermTM)
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Preliminary cytotoxicity test: ≥ 6.25 mg/mL (CBPI); Initial toxicity: no cytotoxicity; Repeat assay: ≥ 3.5 mg/mL (CBPI); Confimatory assay: ≥ 2.5 mg/mL (RVCC)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test item concentrations ranging from 0.0061 - 100 mg/mL were chosen for the evaluation of cytotoxicity. Cytotoxicity was observed at concentrations ≥ 6.25 mg/mL (CBPI method). Based upon these results, the initial micronucleus assay was conducted using triplicate tissues at concentrations of 1, 2, 4, 4.25, 4.5, 4.75, 5, 5.25, and 5.5 mg/mL.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative viable cell count (RVCC) and cytokinesis-blocked proliferation index (CBPI)

Preliminary toxicity test:
Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at concentrations ≥ 6.25 mg/mL. Cytotoxicity [≥ 50% reduction in viable cell count (RVCC) relative to the vehicle control] was observed at concentrations ≥ 25 mg/mL.

Initial assay: no cytotoxicity observed, therefore the assay was repeated with additional higher concentrations

Repeat assay:
Cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at concentrations ≥ 3.5 mg/mL. Cytotoxicity (≥ 50% RVCC) was observed at 7 mg/mL.

Confirmatory assay:
Cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at 4 mg/mL. Cytotoxicity (≥ 50% RVCC) was observed at concentrations 2.5, 3, and 3.25 mg/mL.

Table 2. Results of preliminary cyctotoxicity test

Treatment Tissue Cells Cytotoxicity Based on CBPI 1 Cytotoxicity Based On RVCC3
(mg/mL) No Counted Cells with Nuclei CBPI % Cyto2 Cell Counts Cells Per Tissue (x105) % Cyto2
      1 2 >2 Live Dead Live Dead Total
Acetone 1 500 179 314 7 1.656   40 0 2.4 0 2.4  
0.0061 2 500 199 294 7 1.616 6 40 1 2.4 0.06 2.46 0
0.0122 3 500 235 262 3 1.536 18 36 1 2.16 0.06 2.22 10
0.0244 4 500 212 286 2 1.58 12 42 0 2.52 0 2.52 -5
0.0488 5 500 233 264 3 1.54 18 43 0 2.58 0 2.58 -8
0.0977 6 500 243 251 6 1.526 20 37 1 2.22 0.06 2.28 8
0.195 7 500 202 294 4 1.604 8 42 1 2.52 0.06 2.58 -5
0.391 8 500 229 268 3 1.548 16 38 1 2.28 0.06 2.34 5
0.781 9 500 234 265 1 1.534 19 32 0 1.92 0 1.92 20
1.56 10 500 202 293 5 1.606 8 40 1 2.4 0.06 2.46 0
3.13 11 500 228 271 1 1.546 17 39 1 2.34 0.06 2.4 3
6.25 12 500 436 64 0 1.128 80 39 1 2.34 0.06 2.4 3
12.5 13 500 478 21 1 1.046 93 24 1 1.44 0.06 1.5 40
25 14 0 0 0 0 - - 5 0 0.3 0 0.3 88
50 15 0 0 0 0 - - 0 0 0 0 0 100
100 16 0 0 0 0 - - 0 0 0 0 0 100

1 CBPI = Cytokinesis-Block Proliferation Index relative to vehicle control

2 % Cyto = % Cytotoxicity

3 RVCC = Relative Viable Cell Count relative to vehicle control

Table 3. Results of initial micronucleus assay

      Cytotoxicity Based on CBPI1 Cytotoxicity Based on RVCC3
Treatment (mg/mL) Tissue No. Cells Counted Cells with Nuclei   %Cyto2 Cells Counted Cells per Tissue (x105) %Cyto2
      1N 2N >2N CBPI Live Dead Live Dead Total
Acetone 1 500 283 214 3 1.44   45 1 2.7 0.06 2.76  
2 500 268 231 1 1.466   42 1 2.52 0.06 2.58  
3 500 153 345 2 1.698   42 1 2.52 0.06 2.58  
Mean         1.535 0     2.58     0
1 4 500 238 257 5 1.534 0 43 2 2.58 0.12 2.7 0
5 500 221 276 3 1.564 -5 39 0 2.34 0 2.34 9
6 500 237 261 2 1.53 1 44 0 2.64 0 2.64 -2
Mean         1.543 -1     2.52     2
2 7 501 172 326 3 1.663 -24 39 0 2.34 0 2.34 9
8 500 224 272 4 1.56 -5 42 1 2.52 0.06 2.58 2
9 500 236 261 3 1.534 0 43 1 2.58 0.06 2.64 0
Mean         1.586 -10     2.48     4
4 10 500 256 239 5 1.498 7 36 2 2.16 0.12 2.28 16
11 500 241 256 3 1.524 2 40 1 2.4 0.06 2.46 7
12 500 244 255 1 1.514 4 37 1 2.22 0.06 2.28 14
Mean         1.512 4     2.26     12
4.25 13 500 225 275 0 1.55 -3 35 0 2.1 0 2.1 19
14 500 232 267 1 1.538 -1 36 1 2.16 0.06 2.22 16
15 500 210 289 1 1.582 -9 37 0 2.22 0 2.22 14
Mean         1.557 -4     2.16     16
4.5 16 500 264 234 2 1.476 11 36 1 2.16 0.06 2.22 16
17 500 251 248 1 1.5 6 33 0 1.98 0 1.98 23
18 500 249 250 1 1.504 6 39 1 2.34 0.06 2.4 9
Mean         1.493 8     2.16     16
4.75 19 500 253 247 0 1.494 8 31 1 1.86 0.06 1.92 28
20 500 267 232 1 1.468 12 32 2 1.92 0.12 2.04 26
21 500 259 241 0 1.482 10 35 0 2.1 0 2.1 19
Mean         1.481 10     1.96     24
5 22 500 273 224 3 1.46 14 38 1 2.28 0.06 2.34 12
23 500 239 260 1 1.524 2 36 2 2.16 0.12 2.28 16
24 500 297 202 1 1.408 24 38 1 2.28 0.06 2.34 12
Mean         1.464 13     2.24     13
5.25 25 500 233 266 1 1.536 0 24 0 1.44 0 1.44 44
26 500 259 241 0 1.482 10 26 0 1.56 0 1.56 40
27 500 245 255 0 1.51 5 29 0 1.74 0 1.74 33
Mean         1.509 5     1.58     39
5.5 28 500 263 237 0 1.474 11 34 1 2.04 0.06 2.1 21
29 500 334 166 0 1.332 38 34 3 2.04 0.18 2.22 21
30 500 299 200 1 1.404 24 38 2 2.28 0.12 2.4 12
Mean         1.403 25     2.12     18
MMC, 5 µg/mL 31 500 149 350 1 1.704 -32 31 1 1.86 0.06 1.92 28
32 500 238 262 0 1.524 2 35 0 2.1 0 2.1 19
33 500 295 205 0 1.41 23 34 2 2.04 0.12 2.16 21
Mean         1.546 -2     2     22
MMC, 6 µg/mL 34 500 245 252 3 1.516 3 34 1 2.04 0.06 2.1 21
35 500 259 240 1 1.484 9 28 0 1.68 0 1.68 35
36 500 286 214 0 1.428 20 32 1 1.92 0.06 1.98 26
Mean         1.476 11     1.88     27

1 CBPI = Cytokinesis-Block Proliferation Index relative to vehicle control

2 % Cyto = % Cytotoxicity

3 RVCC = Relative Viable Cell Count relative to vehicle control

Table 4. Results of repeat micronucleus assay

      Cytotoxicity Based on CBPI1 Cytotoxicity Based on RVCC3
Treatment (mg/mL) Tissue No. Cells Counted Cells with Nuclei   %Cyto2 Cells Counted Cells per Tissue (x105) %Cyto2
      1N 2N >2N CBPI Live Dead Live Dead Total
Acetone 1 500 165 331 4 1.678   44 2 2.64 0.12 2.76  
2 500 174 322 4 1.66   42 0 2.52 0 2.52  
3 500 185 306 9 1.648   43 3 2.58 0.18 2.76  
Mean         1.662 0     2.58     0
1 4 500 196 298 6 1.62 6 43 2 2.58 0.12 2.7 0
5 500 172 321 7 1.67 -1 43 2 2.58 0.12 2.7 0
6 500 183 312 5 1.644 3 39 1 2.34 0.06 2.4 9
Mean         1.645 3     2.5     3
2 7 500 248 250 2 1.508 23 36 1 2.16 0.06 2.22 16
8 500 225 271 4 1.558 16 38 0 2.28 0 2.28 12
9 500 210 286 4 1.588 11 40 0 2.4 0 2.4 7
Mean         1.551 17     2.28     12
3.5 10 500 356 144 0 1.288 56 35 0 2.1 0 2.1 19
11 500 344 156 0 1.312 53 38 1 2.28 0.06 2.34 12
12 500 361 139 0 1.278 58 37 1 2.22 0.06 2.28 14
Mean         1.293 56     2.2     15
4 13 500 386 114 0 1.228 66 27 0 1.62 0 1.62 37
14 500 418 81 1 1.166 75 29 0 1.74 0 1.74 33
15 500 397 102 1 1.208 69 30 1 1.8 0.06 1.86 30
Mean         1.201 70     1.72     33
4.5 16 500 452 48 0 1.096 85 31 1 1.86 0.06 1.92 28
17 500 436 63 1 1.13 80 28 0 1.68 0 1.68 35
18 500 449 51 0 1.102 85 30 1 1.8 0.06 1.86 30
Mean         1.109 83     1.78     31
5 19 500 451 48 1 1.1 85 30 1 1.8 0.06 1.86 30
20 500 446 54 0 1.108 84 29 1 1.74 0.06 1.8 33
21 500 442 58 0 1.116 82 31 0 1.86 0 1.86 28
Mean         1.108 84     1.8     30
5.5 22 500 450 48 2 1.104 84 29 0 1.74 0 1.74 33
23 500 463 36 1 1.076 89 32 0 1.92 0 1.92 26
24 500 466 34 0 1.068 90 25 1 1.5 0.06 1.56 42
Mean         1.083 88     1.72     33
6.5 25 500 407 92 1 1.188 72 23 0 1.38 0 1.38 47
26 500 436 64 0 1.128 81 23 1 1.38 0.06 1.44 47
27 500 480 20 0 1.04 94 25 0 1.5 0 1.5 42
Mean         1.119 82     1.42     45
7 28 500 471 29 0 1.058 91 20 0 1.2 0 1.2 53
29 500 475 25 0 1.05 92 21 0 1.26 0 1.26 51
30 500 488 12 0 1.024 96 20 0 1.2 0 1.2 53
Mean         1.044 93     1.22     53
MMC, 5 µg/mL 31 500 205 290 5 1.6 9 20 0 1.2 0 1.2 53
32 500 265 234 1 1.472 29 19 0 1.14 0 1.14 56
33 500 212 288 0 1.576 13 18 0 1.08 0 1.08 58
Mean         1.549 17     1.14     56
MMC, 6 µg/mL 34 500 269 229 2 1.466 30 11 0 0.66 0 0.66 74
35 500 241 259 0 1.518 22 12 0 0.72 0 0.72 72
36 500 256 244 0 1.488 26 11 0 0.66 0 0.66 74
Mean         1.491 26     0.68     74

1 CBPI = Cytokinesis-Block Proliferation Index relative to vehicle control

2 % Cyto = % Cytotoxicity

3 RVCC = Relative Viable Cell Count relative to vehicle control

Table 5. Summary of results of repeat micronucleus assay

Treatment (mg/mL) Tissue No. % Cytotoxicity Based on BN counted % MNBN % MNBN per Dose
    CBPI RVCC     Mean ±SD
  1     1000 0.1    
Acetone 2     1000 0 0.1 0.1
  3     1000 0.2    
  4 6 0 1000 0    
1 5 -1 0 1000 0.1 0.1 0.1
  6 3 9 1000 0.2    
  7 23 16 1000 0.1    
2 8 16 12 1000 0.1 0.07 0.06
  9 11 7 1000 0    
  10 56 19 1000 0    
3.5 11 53 12 1000 0.1 0.07 0.06
  12 58 14 1000 0.1    
  31 9 53 1000 1.1    
MMC, 5 µg/mL 32 29 56 1000 1 1.07 0.06**
  33 13 58 1000 1.1    

BN = Binucleated cells

MNBN =Micronucleated binucleated cells

SD=Standard Deviation

** p ≤ 0.01, Fisher's Exact Test, relative to the vehicle control

Table 6. Results of confirmatory micronucleus assay

      Cytotoxicity Based on CBPI1 Cytotoxicity Based on RVCC3
Treatment (mg/mL) Tissue No. Cells Counted Cells with Nuclei   %Cyto2 Cells Counted Cells per Tissue (x105) %Cyto2
      1N 2N >2N CBPI Live Dead Live Dead Total
Acetone 1 500 109 361 30 1.842   40 0 2.4 0 2.4  
2 500 110 374 16 1.812   43 1 2.58 0.06 2.64  
3 500 128 360 12 1.768   40 1 2.4 0.06 2.46  
Mean         1.807 0     2.46     0
1 4 500 105 365 30 1.85 -5 43 3 2.58 0.18 2.76 -5
5 500 115 368 17 1.804 0 44 1 2.64 0.06 2.7 -7
6 500 119 338 43 1.848 -5 39 0 2.34 0 2.34 5
Mean         1.834 -3     2.52     -2
2 7 500 74 396 30 1.912 -13 32 0 1.92 0 1.92 22
8 500 135 342 23 1.776 4 34 1 2.04 0.06 2.1 17
9 500 119 360 21 1.804 0 35 1 2.1 0.06 2.16 15
Mean         1.831 -3     2.02     18
3.5 10 500 132 360 8 1.752 7 22 0 1.32 0 1.32 46
11 500 121 370 9 1.776 4 26 0 1.56 0 1.56 37
12 500 110 381 9 1.798 1 23 2 1.38 0.12 1.5 44
Mean         1.775 4     1.42     42
4 13 500 137 357 6 1.738 9 20 1 1.2 0.06 1.26 51
14 500 123 373 4 1.762 6 20 0 1.2 0 1.2 51
15 500 131 363 6 1.75 7 19 0 1.14 0 1.14 54
Mean         1.75 7     1.18     52
4.5 16 500 120 375 5 1.77 5 20 0 1.2 0 1.2 51
17 500 127 367 6 1.758 6 20 1 1.2 0.06 1.26 51
18 500 157 336 7 1.7 13 21 1 1.26 0.06 1.32 49
Mean         1.743 8     1.22     50
5 19 500 167 332 1 1.668 17 21 0 1.26 0 1.26 49
20 500 180 320 0 1.64 21 19 1 1.14 0.06 1.2 54
21 500 202 298 0 1.596 26 20 1 1.2 0.06 1.26 51
Mean         1.635 21     1.2     51
5.5 22 500 290 210 0 1.42 48 21 0 1.26 0 1.26 49
23 500 214 283 3 1.578 28 20 0 1.2 0 1.2 51
24 500 241 259 0 1.518 36 22 0 1.32 0 1.32 46
Mean         1.505 37     1.26     49
6.5 25 500 316 184 0 1.368 54 20 0 1.2 0 1.2 51
26 500 312 187 1 1.378 53 22 0 1.32 0 1.32 46
27 500 301 199 0 1.398 51 25 0 1.5 0 1.5 39
Mean         1.381 53     1.34     46
7 28 500 291 208 1 1.42 48 25 1 1.5 0.06 1.56 39
29 500 303 196 1 1.396 51 19 2 1.14 0.12 1.26 54
30 500 296 203 1 1.41 49 22 1 1.32 0.06 1.38 46
Mean         1.409 49     1.32     46
MMC, 5 µg/mL 31 500 278 222 0 1.444 45 20 0 1.2 0 1.2 51
32 500 237 261 2 1.53 34 19 0 1.14 0 1.14 54
33 500 226 273 1 1.55 32 19 0 1.14 0 1.14 54
Mean         1.508 37     1.16     53
MMC, 6 µg/mL 34 500 193 303 4 1.622 23 21 0 1.26 0 1.26 49
35 500 211 287 2 1.582 28 14 1 0.84 0.06 0.9 66
36 500 254 242 4 1.5 38 19 2 1.14 0.12 1.26 54
Mean         1.568 30     1.08     56

1 CBPI = Cytokinesis-Block Proliferation Index relative to vehicle control

2 % Cyto = % Cytotoxicity

3 RVCC = Relative Viable Cell Count relative to vehicle control

Table 7. Summary of results of confirmatory micronucleus assay

Treatment (mg/mL) Tissue No. % Cytotoxicity Based on BN counted % MNBN % MNBN per Dose
    CBPI RVCC     Mean ±SD
  1     1000 0.1    
Acetone 2     1000 0 0.1 0.1
  3     1000 0.2    
  4 -5 -5 1000 0.3    
0.5 5 0 -7 1000 0.1 0.13 0.15
  6 -5 5 1000 0    
  13 9 51 1000 0    
2.5 14 6 51 1000 0.1 0.03 0.06
  15 7 54 1000 0    
  28 48 39 1000 0.2    
4.5 29 51 54 1000 0.1 0.1 0.1
  30 49 46 1000 0    
MMC, 31 45 51 1000 1.8    
5 µg/mL 32 34 54 1000 3.2 2.1 0.98**
  33 32 54 1000 1.3    

BN = Binucleated cells

MNBN =Micronucleated binucleated cells

SD=Standard Deviation

** p ≤ 0.01, Fisher's Exact Test, relative to the vehicle control

Conclusions:
Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in a reconstructed human skin model (EpiDermTM).
Executive summary:

The test article was evaluated for its potential to induce micronuclei in the reconstructed skin micronucleus assay (RSMN) in EpiDermTM . Acetone was used as the vehicle.

The preliminary toxicity test was conducted using a 2-day dosing regimen (48-hour treatment) by exposing a single tissue per concentration to vehicle alone and fifteen concentrations (0.0061 to 100 mg/mL) of the test article. Based upon the results, the micronucleus assay was conducted using triplicate tissues at concentrations ranging from 1 to 5.5 mg/mL.

In the initial micronucleus assay, due to shift in cytotoxicity, the assay was repeated using triplicate tissues at concentrations ranging from 1 to 7 mg/mL. In the repeat assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at concentrations ≥ 3.5 mg/mL. Cytotoxicity (≥ 50% RVCC) was observed at 7 mg/mL. The concentrations selected for evaluation of micronuclei were 1, 2, and 3.5 mg/mL. No significant or dose-dependent increases in micronuclei induction were observed at any concentration (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). Since the result of the micronucleus assay using a 2-day dosing regimen (48-hour treatment) was negative, a confirmatory assay was conducted with a 3-day dosing regimen (72-hour treatment) at concentrations ranging from 0.5 to 4.5 mg/mL using triplicate tissues. In the confirmatory micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle

control) was observed at 4 mg/mL. Cytotoxicity (≥ 50% RVCC) was observed at concentrations 2.5, 3, and 3.25 mg/mL. The concentrations selected for evaluation of micronuclei were 0.5, 2.5, and 4.5 mg/mL.

No significant or dose-dependent increases in micronuclei induction were observed at any concentration (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). These results indicate the test item was negative for the induction of micronuclei in the reconstructed skin micronucleus assay (RSMN) in EpiDermTM .

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
summarised data
Reference:
Composition 0
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted: 29 Jul 2016
Deviations:
yes
Remarks:
invalid solvent control
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted: 21 Jul 1997
Deviations:
no
GLP compliance:
not specified
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Test material information:
Composition 1
Target gene:
TK locus
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells:
Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC, USA)
- Methods for maintenance in cell culture if applicable: cells were maintained in growth medium at 37 ( ± 1) °C


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD, USA) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI, USA)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arochlor 1254
Test concentrations with justification for top dose:
Toxicity assay: 0.0005 - 10000 µg/mL
Mutagenicity assay: -S9: 3.9 - 250 µg/mL; +S9: 7.8 - 125 µg/mL
Vehicle:
- Vehicle/solvent used: DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
7,12-dimethylbenzanthracene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and conditions:
METHOD OF APPLICATION: soft agar method
- Cell density at seeding: toxicity assay: 6 x 10E5/mL (6 x 10E6 cells total); mutagenicity assay: 1.2 x 10E7 cells

DURATION
- Exposure duration:
4 h
- Expression time (cells in growth medium):
48 h
- Selection time (if incubation with a selection agent):
10 - 12 days


SELECTION AGENT (mutation assays):
trifluorothymidine (TFT; 3 µg/mL)

NUMBER OF REPLICATIONS:
duplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
The doses selected for testing were within the range yielding approximately 0-90% cytotoxicity.


OTHER:
Only colonies larger than ~0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10E6 surviving cells.
Evaluation criteria:
The substance is considered positive if a concentration-related increase in mutant frequency is observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of ≥ 100 mutants per 10E6 clonable cells over the background level.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
lowest effective dose: 7.8 µg/mL
Cytotoxicity:
yes
Remarks:
≥ 16 µg/mL
Vehicle controls valid:
no
Remarks:
< 35-140 mutants/106 cells (according to OECD guideline 490)
Negative controls valid:
not applicable
Positive controls valid:
not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
≥ 125 µg/mL
Vehicle controls valid:
no
Remarks:
< 35-140 mutants/106 cells (according to OECD guideline 490)
Negative controls valid:
not applicable
Positive controls valid:
not specified
Additional information on results:
OTHER:
All induced colonies were small in size (~0.2 - 1.1 mm) when compared to the untreated control.

Table 1. Results of mouse lymphoma assay

Non-Activated Cultures
Dose (µg/mL) Average TFT Average VC Mut Freq RTG
3.9 to 250        
3.9 81 136 1.19 76
  78 132 1.18 70
7.8 119 90 2.64 18
16 131 35 7.49 4
  119 54 4.41 7
63 43 42 2.05 3
  72 34 4.24 3
250 37 52 1.42 4
  33 58 1.14 4
Solvent Control (DMSO) 23 153 0.3  
Positive Control 385 67 11.49 25
S9-Activated Cultures
Dose (µg/mL) Average TFT Average VC  Mut Freq RTG
         
7.8 39 143 0.55 84
  45 158 0.57 91
16 32 131 0.49 37
  27 128 0.42 39
31 29 122 0.48 32
  42 117 0.72 30
63 39 80 0.98 14
  33 99 0.67 18
125 39 53 1.47 7
  40 78 1.03 8
Solvent Control (DMSO) 22 153 0.29  
Positive Control 196 90 4.36 36

TFT: Resistance to trifluorothymidine

Mut Freq: mutant frequency (no unit given)

RTG: relative total gowth

Conclusions:
Under the experimental conditions of the gene mutation assay in mouse lymphoma cells L5178Y the test item induced gene mutations at and above 7.8 µg/mL in the absence of metabolic activation, while it did not induce gene mutations in the presence of metabolic activation. Mutant frequencies of solvent control were below acceptable range of 35-140 mutants/10E6 cells according to the current criteria of OECD TG 490 and therefore invalid. Since an increase of mutant frequency was reported for 1 of 2 replicates only and according to OECD guideline 490 positive results found between 10 and 20% relative total growth are questionable in their biological relevance, this study was judged to be "inconclusive" with respect to mutagenicity.
Executive summary:

An in vitro mammalian cell gene mutation assay was performed in mouse lymphoma L5178Y cells according to the protocol described by Clive & Spector (1975) and similar to OECD Test Guidelines 476 (1997) and to OECD guideline 490 and under GLP conditions (Seifried, 2006). L5178Y cells were treated for 4 hours in the absence and presence of Aroclor-induced rat liver S9 mix, grown for 48 hrs to allow any mutations to be expressed, and then mutant colonies selected in trifluorothymidine-containing selective medium using the agar method. The test substance concentrations were tested in duplicate and ranged from 3.9-250 µg/mL in the absence of S9 and from 7.8-125 µg/mL in the presence of S9. The top concentrations were apparently selected on induction of about 90% cytotoxicity, however, this limit was exceeded at 3 concentrations tested in the absence of S9, and at 1 concentration tested in the presence of S9. Therefore valid data were only available for 2 or 4 concentrations of the test substance; data from only 2 concentrations is not sufficient according to the guideline. Two different sets of criteria for a positive result were given, the initial evaluation being a doubling in mutant frequency relative to solvent controls, and the new evaluation being an increase over solvent control of at least 100 mutants/106 clonable cells in cultures showing >10% relative total growth. The latter criterion is in line with the latest OECD guideline, although for the agar method this would actually be an increase of 90 mutants/106 cells.

There was no evidence of a mutagenic response in the presence of S9 at acceptable levels of cytotoxicity. However, the data in the absence of S9 are difficult to interpret for the following reasons:

  • For the one treatment that gave an increase in mutant frequency that would be considered biologically relevant (i.e. at 7.8 µg/mL in the absence of S9) data were reported for only 1 of the 2 replicate cultures.

  • The extent of cytotoxicity at 7.8 µg/mL was >80%, and the OECD guideline states that “care should be taken when interpreting positive results only found between 20 and 10% RTG”. Since the next lowest concentration (3.9 µg/mL) induced only 27% reduction in relative total growth. Therefore, it is unclear whether the test substance induced mutations at more acceptable levels of cytotoxicity close to, but below, 80%.

 

According to the materials and methods, mutant colonies were categorised according to size (large and small). It is stated that all chemicals giving positive results (so presumably this also applies to the test substance) induced predominantly small colonies (possibly indicative of a chromosome breakage mode of action), but the supplementary data only show total mutant colony counts and mutant frequencies. Therefore the possible mode of action cannot be confirmed from the data.

 

Thus, although the data from Seifried et al (2006) indicate a positive mutagenic response with the test substance in the absence of S9, there are many reasons to question the biological relevance of the single positive response. Therefore, this study is judged “inconclusive” with respect to mutagenicity.

 

References

Clive, D., and Spector, J. F. S. (1975) Laboratory procedure for assessing specific locus mutations at the TK locus incultured L5178Y TK+/-mouse lymphoma cells. Mutat.Res. 31, 17-29.

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2008). In this study the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation up to cytotoxic concentrations (150 µg/plate).

Beyond data requirements for Annex VII, data on the genetic toxicity in mammalian cells are available for the test substance:

Cytogenicity in mammalian cells

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured human lymphocytes performed according to OECD Guideline 473 and GLP (2017). In this test significant increases of structural chromosomal aberrations were observed after 4 h exposure (additional 18 h recovery) with the test substance at 157 and 275 µg/mL without metabolic activation and at 275 and 482 µg/mL with metabolic activation.

The potential of the test substance to induce mirconuclei was investigated in the reconstructed skin micronucleus assay (RSMN) in EpiDerm™ tissues performed according to a protocol similar to OECD Guideline 487 and GLP (2017). In this test no significant or dose dependent increases in micronuclei induction were observed after 48 and 72 h exposure at test substance concentrations up to 4.5 mg/mL (limited due to toxicity).

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed in mouse lymphoma L5178Y cells according to a protocol similar to OECD guideline 490 and under GLP conditions (Seifried, 2006). Under conditions of this test the test substance was mutagenic after 4 h exposure at and above 7.8 µg/mL in the absence of metabolic activation, while in the presence of metabolic activation it was not mutagenic. Mutant frequencies of solvent control were below the acceptable range of 35-140 mutants/10E6 cells according to the criteria of the current OECD TG 490 and therefore invalid. Since an increase of mutant frequency was reported for 1 of 2 replicates only and according to OECD guideline 490 positive results found between 10 and 20% relative total growth are questionable in their biological relevance, this study was judged to be "inconclusive" with respect to mutagenicity.

Overall conclusion

Since data on the genetic toxicity of the test substance in mammalian cells are heterogeneous, no final conclusion on the genotoxic potential can be made. In principle, further testing for genetic toxicity (in vivo) shall be considered. As the substance is used as a cosmetic ingredient, the animal testing ban as set out in the Cosmetic Regulation (EC) No 1223/2009 applies. Therefore, no further animal testing is proposed for the test substance.

However, there are in vivo data on carcinogenicity available for a structurally related natural derivate of the test substance to be registered which also showed positive results in an in vitro chromosome aberration test.

In a 2 -year carcinogenicity study in F344 rats equivalent to OECD Guideline 451 conducted with the monoterpenoid hinokitiol (beta-thujaplicin; CAS 499-44-5), groups of 50 rats of each sex were given a diet containing the test substance at doses of 0, 0.005, 0.015, and 0.05% (excluding 0.005% in females; corresponding intakes were calculated to be 7.8 and 25.9 mg/kg bw/day for females and 2.1, 6.4 and 20.9 mg/kg bw/day for males, respectively) for 104 weeks (Inai et al., 2006). No treatment-related changes in survival rate, general condition, body weight, food consumption, hematology and organ weights were noted. Detailed histopathological examination revealed no treatment-related increase in the incidences of any neoplastic lesions. The results demonstrate that hinokitiol was not carcinogenic in F344 rats of either sex under the conditions of this study.

Thus, a carcinogenic potential related to the positive results observed in genetic toxicity tests in mammalian cells for the substance for registration is not assumed.

References

Derelanko, M. J. (2008) The toxicologist´s handbook - 2nd edition. CRC Press Taylor & Francis Group, Boca Raton, FL, USA

Inai et al. (2006) Lack of hinokitiol (beta-thujaplicin) carcinogenicity in F344/DuCrj rats. The Journal of Toxikological Sciences 31 (4): 357-370

Justification for classification or non-classification

The available data on genetic toxicity of the test substance are heterogeneous and do not allow classification according to Regulation (EC) 1272/2008, and they are therefore inconclusive.