Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance did not cause a positive response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.
Based on the findings in a chromosome aberration study, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 4-hour and 20-hour exposure groups.
Under the conditions of a mouse lymphoma assay, the test article was concluded to be positive in the 24-hour exposure without S9 activation and negative in the 4-hour exposure both with and without S9 activation in the L5178Y TK+/- mouse lymphoma assay.

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:
April - May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2B
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate with and w/out S9 activation (Preliminary toxicity and initial mutagenicity assay) – Plate incorporation method

2.0, 6.0, 20, 60, 200 and 600 micrograms per plate with all Salmonella tester strains and
60, 200, 600, 1800 and 5000 micrograms per plate with tester strain WP2 uvrA. (Independent repeat/confirmatory mutagenicity assay) – Plate incorporation method. Due to toxicity profiles that differed from that observed in the initial assay, tester strain TA100 in the absence of S9 activation and tester strain TA1537 in the presence of S9 activation were retested with an adjustment in dose levels: 0.15, 0.50, 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate.
Vehicle / solvent:
DMSO (CAS No. 67-68-5); from EMD Chemicals Incorporated
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
Preliminary Toxicity/Initial Mutagenicity Assay: The preliminary toxicity/initial mutagenicity assay was used to establish the dose range over which the test article would be assayed and to provide a preliminary mutagenicity evaluation. A vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.

Confirmatory Mutagenicity Assay: The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six to 10 dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures: The test system was exposed to the test article via the plate incorporation method. On the day of its use, minimal top agar was melted and supplemented. Top agar, not used with S9 or Sham mix, was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was supplemented Vogel Bonner minimal medium E.

Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation.

Test article dilutions were prepared immediately before use. One half (0.5) milliliter of S9 or Sham mix, 100 microL of tester strain and 50 microL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45+/-2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 microL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37+/-2°C. Plates that were not counted immediately following the incubation period were stored at 2 8°C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation was scored relative to the vehicle control plate.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Generally observed beginning at 150, 500 or 5000 microg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Generally observed beginning at 150, 500 or 5000 microg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Generally observed beginning at 60, 200, 500, 600, or 1500 microg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Generally observed beginning at 60, 200, 500, 600, or 1500 microg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the initial mutagenicity assay no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1500 microg/plate. Toxicity was observed beginning at 150, 500 or at 5000 microg/plate with all test conditions except tester strain WP2 uvrA in the absence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 600 microg/plate with all Salmonella tester strains and 5000 microg/plate with tester strain WP2 uvrA.

For the Confirmatory Mutagenicity Assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 600 microg/plate. Toxicity was observed beginning at 60, 200 or at 600 microg/plate with all Salmonella tester strains except TA1537 in the presence of activation. Due to toxicity profiles that differed from that observed in the initial assay, tester strains TA100 and TA1537 in the absence or presence of S9 activation, respectively, were retested with adjustments to the dose levels. In experiment B3, no positive mutagenic responses were observed in either TA100 or TA1537 in the absence or presence of S9 activation, respectively. Precipitate was observed beginning at 500 microg/plate and toxicity was observed beginning at 500 or 1500 microg/plate.
Remarks on result:
other: Initial Mutagenicity Assay

Initial Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: None

Dose (microg/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

Vehicle (DSMO)

13 ± 4

98 ± 6

6 ± 1

5 ± 1

22 ± 2

1.5

10 ± 2

93 ± 5

11 ± 1

7 ± 1

19 ± 5

5.0

11 ± 4

91 ± 13

7 ± 1

5 ± 1

21± 0

15

8 ± 1

95 ± 8

7 ± 3

6 ± 1

17 ± 6

50

9 ± 2

74 ±4

8 ± 4

4 ± 1

17 ± 3

150

0 ± 0

0 ± 0

6 ± 1

4 ± 1

15 ± 3

500

0 ± 0 P

0 ± 0 P

0 ± 0 P

4 ± 1 P

21 ± 12

1500

0 ± 0 P

0 ± 0 P

0 ± 0 P

3 ± 0 P

12 ± 0 P

5000

0 ± 0 P

0 ± 0 P

0 ± 0 P

2 ± 1 P

19 ± 1 P

Positive Control

199 ± 33

511 ± 6

456 ± 28

365 ± 58

385 ± 8

P = precipitate observed

 

Initial Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: S9

 

Dose (microg/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

Vehicle (DSMO)

13 ± 1

104 ± 8

9 ± 2

10 ± 1

38 ± 8

1.5

14 ± 9

99 ± 12

10 ± 2

8 ± 3

40 ± 1

5.0

12 ± 7

86 ± 9

8 ± 3

5 ± 1

33 ± 8

15

13 ± 2

72 ± 5

11 ± 2

5 ± 1

30 ± 6

50

15 ± 5

77 ± 16

7 ± 1

4 ± 1

28 ± 0

150

12 ± 1

91 ± 8

9 ± 0

4 ± 1

28 ± 1

500

0 ± 0 P

0 ± 0 P

0 ± 0 P

5 ± 3 P

27 ± 0 P

1500

0 ± 0 P

0 ± 0 P

0 ± 0 P

5 ± 4 P

26 ± 8 P

5000

0 ± 0 P

0 ± 0 P

0 ± 0 P

3 ± 0 P

11 ± 0 P

Positive Control

237 ± 18

590 ± 55

83 ± 14

47 ± 5

189 ± 13

P = precipitate observed

Confirmatory Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: None

 

Dose (microg/plate)

TA98

TA100

TA1535

TA1537

Vehicle (DSMO)

23 ± 9

139 ± 11

11 ± 3

5 ± 5

2

20 ± 6

65 ± 8

13 ± 1

6 ± 3

6

24 ± 5

79 ± 7

10 ± 5

2 ± 2

20

24 ± 4

87 ± 15

11 ± 3

4 ± 1

60

18 ± 3

62 ± 13

11 ± 3

4 ± 3

200

10 ± 3

72 ± 9

11 ± 5

7 ± 3

600

0 ± 0 P

0 ± 0 P

0 ± 0 P

4 ± 1 P

Positive Control

305 ± 40

473 ± 34

629 ± 26

738 ± 53

P = precipitate observed

 

Dose (microg/plate)

WP2uvrA

Vehicle (DSMO)

14 ± 5

60

10 ± 3

200

11 ± 2

600

12 ± 1 P

1800

13 ± 3 P

5000

9 ± 2 P

Positive Control

295 ± 22

P = precipitate observed

 

Repeat Confirmatory Mutagenicity Assay for TA100

Mean Number of Revertants Per Plate

Activation: None

 

Dose (microg/plate)

TA100

Vehicle (DSMO)

83 ± 3

0.15

79 ± 1

0.50

86 ± 10

1.5

85 ± 2

5.0

86 ± 9

15

83 ± 1

50

82 ± 4

150

82 ± 5

500

55 ± 5 P

1500

51 ± 9 P

5000

55 ± 3 P

Positive Control

573 ± 9

P = precipitate observed

Confirmatory Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: S9

 

Dose (microg/plate)

TA98

TA100

TA1535

TA1537

Vehicle (DSMO)

32 ± 9

137 ± 14

10 ± 2

7 ± 2

2

26 ± 8

121 ± 14

9 ± 5

6 ± 3

6

28 ± 5

121 ± 18

11 ± 2

5 ± 1

20

30 ± 3

132 ± 12

11 ± 2

7 ± 4

60

25 ± 2

126 ± 4

10 ± 5

7 ± 3

200

25 ± 6

134 ± 16

12 ± 5

5 ± 3

600

0 ± 0 P

0 ± 0 P

12 ± 3 P

6 ± 2 P

Positive Control

577 ± 40

671 ± 159

94 ± 14

51 ± 14

P = precipitate observed

  

Dose (microg/plate)

WP2uvrA

Vehicle (DSMO)

20 ± 3

60

19 ± 2

200

19 ± 1

600

16 ± 4 P

1800

16 ± 5 P

5000

12 ± 1 P

Positive Control

188 ± 10

P = precipitate observed

 

Repeat Confirmatory Mutagenicity Assay for TA1537

Mean Number of Revertants Per Plate

Activation: S9

 

Dose (microg/plate)

TA1537

Vehicle (DSMO)

11 ± 1

0.15

9 ± 3

0.50

9 ± 3

1.5

8 ± 3

5.0

9 ± 4

15

9 ± 4

50

7 ± 2

150

8 ± 2

500

7 ± 4 P

1500

7 ± 3 P

5000

4 ± 0 P

Positive Control

76 ± 13

P = precipitate observed

Conclusions:
negative with metabolic activation
negative without metabolic activation

All criteria for a valid study were met. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, TMBPA did not cause a positive response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
obtained from American Type Culture Collection, Manassas, VA, USA
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
0.2804, 0.8412, 2.804, 8.412, 28.04, 84.12, 280.4, 841.2, and 2804 microg/mL (Preliminary toxicity assay)
10, 20, 25, 32, 40, and 50 microg/mL (Chromosome Aberration Assay - 4-hour treatment without S9 activation)
10, 20, 25, 32, 40, and 50 microg/mL (Chromosome Aberration Assay – 4 hour treatment with S9 activation
2.5, 5, 10, 20, 25, and 32 microg/mL (Chromosome Aberration Assay – 20-hour treatment without S9 activation)
Vehicle / solvent:
DMSO (CAS No. 67-68-5); from EMD Chemicals
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Description of test procedure: A preliminary toxicity assay was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of an evaluation of test article effect on cell growth. The osmolality in treatment medium of the vehicle, the highest test article dose level, the lowest precipitating test article dose level and the highest soluble test article dose level was measured. The pH of the highest dose level of dosing solution in the treatment medium was measured using test tape. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.2804 microg/mL to 2804 microg/mL in the absence and presence of an S9 reaction mixture. The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. For the cells treated for only 4 hours, at completion of the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. For all three treatment groups, the flasks with visible precipitation were washed twice with 3-5 mL CMF-PBS to avoid precipitation interference with cell counts. At 20 hours after the initiation of treatment, cells were harvested by trypsinization and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. Cell growth in the treatment group relative to vehicle control, was calculated based on the following formula:

[(mean viable cells in treatment group – mean viable cells baseline) / (mean viable cells in solvent control group – mean viable cells baseline)] x 100

For the chromosome aberration assay, CHO cells were seeded for each treatment condition at approximately 5 x 10^5 cells/25 cm^2 flask and were incubated at 37 +/- 1°C in a humidified atmosphere of 5 +/- 1% CO2 in air for 16 24 hours. At the time of treatment, the cell count was determined from a minimum of two flasks to determine the number of cells being treated (baseline). Treatment was carried out by refeeding duplicate flasks with 5 mL of appropriately supplemented complete medium for the non-activated study or 5 mL S9 reaction mixture for the S9-activated study, to which was added 50 microL of dosing solution of test article, positive control in solvent or vehicle alone. The pH (using test tape) of the highest concentration of test article dosing solution in treatment medium was measured.

The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9 at 37 +/- 1°C in a humidified atmosphere of 5 +/- 1% CO2 in air. At completion of exposure for the 4 hour exposure groups, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. Two hours prior to cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 microg/mL and the flasks were returned to the incubator until cell collection.

Two hours after the addition of Colcemid®, metaphase cells were harvested for both the nonactivated and S9-activated studies by trypsinization. The cells were collected approximately 20 hours after initiation of treatment by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 5 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL of fixative (methanol:glacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8°C.

To prepare slides, the fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant was aspirated (except for approximately 0.2 mL above the cell pellet), and 1 mL of cold fresh fixative was added. After additional centrifugation the supernatant fluid was decanted except for 0.1 to 0.3 mL fixative above the cell pellet. A sufficient amount of cell suspension was dropped onto the center of a glass slide and allowed to air dry. The dried slides were appropriately identified, stained with 5% Giemsa, air dried and permanently mounted.

A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. Test article precipitate was assessed using the unaided eye. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control.

Evaluation of metaphase cells:
The selection of dose levels for analysis of chromosome aberrations in CHO cells was based on toxicity. The highest dose evaluated for chromosome aberrations was the lowest dose with at least 50% reduction in cell growth and/or mitotic inhibition, relative to the vehicle control, with sufficient number of scorable metaphase cells. Two additional lower dose levels were included for analysis.

To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20 +/- 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate flask was reduced if the percentage of aberrant cells reached a statistically significant level before 100 cells were scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (greater than or equal to 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.

Plates/test: Samples were run in duplicate, with and without metabolic activation.

Activation system: Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 (Lot No. 2877) was obtained from Molecular Toxicology Inc. (Boone, NC) , stored frozen and thawed immediately prior to use.
Evaluation criteria:
The toxic effects of treatment were based upon cell growth and mitotic inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically aberrant cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test article dose level, the Cochran Armitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations was increased in a dose responsive manner with one or more dose levels being statistically significant (p less than or equal to 0.05). However, values that are statistically significant and fall within or just outside the range of historical solvent control values may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations are concluded to be negative.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
63% at 32 microg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
81% at 40 microg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
60% at 20 microg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Upon sonication for approximately 5 minutes at 26.3°C, the test article formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity test, TMBPA was soluble in at all dose levels. Visible precipitate was observed in treatment medium at dose levels greater than or equal to 841.2 microg/mL, while dose levels less than or equal to 280.4 microg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels greater than or equal to 280.4 microg/mL, while dose levels less than or equal to 84.12 microg/mL were soluble in treatment medium.

The osmolality in treatment medium of the highest dose level tested, 2804 microg/mL, was 408 mmol/kg. The osmolality in treatment medium of the lowest precipitating dose level tested, 841.2 microg/mL, was 435 mmol/kg. The osmolality in treatment medium of the highest soluble dose level, 280.4 microg/mL, was 441 mmol/kg. The osmolality of the solvent (DMSO) in treatment medium was 400 mmol/kg. The osmolality of the TMBPA concentrations in treatment medium were acceptable because they did not exceed the osmolality of the solvent by more than 20%. The pH of the highest dose level of test article in treatment medium was 7.0.

Substantial toxicity (i.e. at least 50% cell growth inhibition relative to the solvent control) was observed at dose levels greater than or equal to 28.04 microg/mL for the non-activated 4- and 20 hour exposure groups, and at dose levels greater than or equal to 84.12 microg/mL in the S9 activated 4-hour exposure group. The dose levels selected for testing in the chromosome aberration assay were based on the results of this preliminary assay.

In the chromosome aberration assay, TMBPA was soluble in treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The pH of the highest concentration of test article in treatment medium was approximately 7.0.

Toxicity of TMBPA in CHO cells treated for 4-hours without metabolic (S9) activation was 63% at 32 microg/mL, the highest dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations (32 microg/mL) was 51% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 10, 20 and 32 microg/mL. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p greater than 0.05, Fisher’s Exact test). The percentage of structurally damaged cells in the MMC (positive control) treatment group (26.0%) was found to be statistically significant.

Toxicity of TMBPA in CHO cells when treated for 4-hours with S9 activation was 81% at 40 microg/mL, the highest dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations (40 microg/mL) was 60% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 10, 25 and 40 microg/mL. The percentage of cells with structural aberrations in the test article-treated group was statistically increased above that of the solvent control at 40 microg/mL. The Cochran-Armitage test was also positive for a dose response. However, the percentage of cells with structural aberrations (5.0%) in the TMBPA-treated group at 40 microg/mL was within the historic solvent control range of 0.0% to 5.5%. Therefore, this would not be considered biologically significant. The percentage of cells with numerical aberrations in the test article-treated groups was also increased statistically above that of the solvent control at 25 microg/mL. However, the percentage of cells with numerical aberrations in the 25 microg/mL group (6.5%) was within the historic solvent control range of 0.0% to 7.5%. Therefore, this would not be considered biologically significant. The percentage of structurally damaged cells in the positive control (CP) group (25.0%) was statistically significant.

Toxicity of TMBPA in CHO cells treated for 20 hours in the absence of S9 activation was 60% at 20 microg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (50 microg/mL) was 50% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 5, 10 and 20 microg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (Fisher’s exact test). The percentage of structurally aberrant cells in the positive control (MMC) group (20.0%) was statistically significant.

The chromosome aberration assay indicated no biologically significant increase in structural and numerical chromosome aberrations in the test article-treated groups relative to the respective vehicle controls in both presence and absence of the S9 metabolic activation system. The percentages aberrant cells in the test article-treated groups were within the historical vehicle control range. The positive and solvent controls fulfilled the requirements for a valid test. Based on these criteria, the negative result is justified and does not require a repeat of any portions of the study.
Remarks on result:
other: 4-hour exposure

Summary of Test Results:

Treatment

(microg/mL)

S9

Activation

Treatment

Time

(hours)

Flask

Mean

Mitotic

Index

Cells

Scored

Cells with Numerical

Aberrations

(%)

Cells with Structural Aberrations

(%)

Mean Aberrations

Per Cell

Vehicle (DMSO)

-

4

A

B

13.0

13.4

100

100

0

2

0

0

0.000

0.000

TMBPA

 

 

 

 

 

 

 

 

10

-

4

A

B

12.8

12.0

100

100

0

3

0

1

0.000

0.010

20

-

4

A

B

12.0

12.2

100

100

0

3

3

1

0.030

0.010

32

-

4

A

B

6.4

6.6

100

100

0

3

1

3

0.020

0.030

Positive control (MMC)

0.2

-

4

A

B

10.4

9.6

100

100

0

1

32

20

0.420

0.260

 

Vehicle (DMSO)

+

4

A

B

13.2

12.8

100

100

0

3

0

0

0.000

0.000

TMBPA

 

 

 

 

 

 

 

 

10

+

4

A

B

12.2

12.4

100

100

1

6

2

1

0.020

0.010

25

+

4

A

B

12.0

12.4

100

100

6

7

3

1

0.030

0.010

40

+

4

A

B

4.8

5.6

100

100

1

4

4

6

0.040

0.060

Positive control (CP)

7.5

+

4

A

B

5.8

6.0

100

100

0

1

26

24

0.420

0.3000

 

Vehicle (DMSO)

-

20

A

B

12.2

12.4

100

100

0

2

0

1

0.000

0.010

TMBPA

 

 

 

 

 

 

 

 

5

-

20

A

B

11.2

10.2

100

100

0

2

0

1

0.000

0.010

10

-

20

A

B

10.0

9.8

100

100

0

3

1

0

0.010

0.000

20

-

20

A

B

6.0

6.2

100

100

0

3

0

1

0.000

0.010

Positive control (MMC)

0.1

-

20

A

B

6.6

7.2

100

100

0

2

20

20

0.220

0.280

Conclusions:
negative

Based on the findings of this study, TMBPA was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 4-hour and 20-hour exposure groups.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2A and S2B
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y cells, clone 3.7.2C, were obtained from Glaxo Wellcome Inc., Research Triangle Park, NC - Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Prior to use on study the L5178Y cells were cleansed of spontaneous TK -/- cells by culturing in a restrictive medium.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 purchased from Moltox (Boone, NC), stored frozen and thawed immediately prior to use
Test concentrations with justification for top dose:
0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 2800 microg/mL with and without S9 activation (Preliminary assay)
5, 20, 25, 35, 40 and 50 microg/mL (Mutagenicity assay: 4-hour exposure without S9 activation)
0.1, 2.5, 7.5, 15 and 25 microg/mL (Mutagenicity assay: 4-hour exposure with S9 activation)
2.5, 7, 8, 9 10 and 12.6 microg/mL (Mutagenicity assay: 24-hour exposure without S9 activation)
Vehicle / solvent:
DMSO (CAS No. 67-68-5); from Sigma-Aldrich Chemical Company
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
Description of test procedure:
The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay. L5178Y cells were exposed to the solvent alone (in duplicate) and nine concentrations of test article ranging from 0.5 to 2800 microg/mL in both the absence and presence of S9-activation with a 4 hour exposure and in the absence of S9 with a 24-hour exposure. The osmolality of the solvent control and the highest soluble concentration in treatment medium at the beginning of treatment was determined.

For the 4-hour exposures, cell population density was determined 1 and 2 days after exposure to the test article. The cultures were adjusted to 3x10^5 cells/mL after the first day only. For the 24-hour exposure, cell population was determined 1, 2 and 3 days after the exposure. The cell population was adjusted to 3x10^5 cells/mL immediately after test article removal and 1 day after test article removal. Cultures with less than 3x10^5 cells/mL were not adjusted. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 2 or 3 days.

The mutagenesis assay was used to evaluate the mutagenic potential of the test article (with and without activation with a 4 hour exposure) and extended treatment assay (without activation with a 24-hour exposure). L5178Y mouse lymphoma cells were exposed to the solvent alone and ten concentrations of the test article in duplicate in both the presence and absence of S9. The positive controls with and without metabolic activation were tested concurrently in single cultures.

Treatment of target cells: Treatment was carried out in conical tubes by combining 6 x 10^6 L5178Y/TK+/- cells, F0P medium or S9 activation mixture, and 100 microL dosing solution of test or control article in solvent or solvent alone in a total volume of 10 mL. The positive controls were treated with MMS (at final concentrations in treatment medium of 15 and 20 microg/mL with a 4-hour exposure or 5.0 and 7.5 microg/mL with a 24-hour exposure without activation) or 7,12 DMBA (at final concentrations in treatment medium of 1.25 and 1.5 microg/mL in the presence of S9 activation). Treatment tubes were gassed with 5+/-1% CO2 in air, capped tightly, and incubated with mechanical mixing for 4 or 24 hours at 37+/-1°C. The preparation and addition of the test article dosing solutions were carried out under yellow lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice with F0P or supplemented F0P(F10P) and resuspended in F10P medium, gassed with 5+/-1% CO2 in air and placed on the roller drum apparatus at 37+/-1°C.

Expression of the mutant phenotype: For expression of the mutant phenotype in the 4-hour exposures, the cultures were counted using the electronic cell counter and adjusted to 3 x 10^5 cells/mL 1 and 2 days after treatment in 20 and 10 mL total volume, respectively. For the 24-hour exposures, cultures were adjusted to 3 x 10^5 cells/mL in 20 mL immediately after test article removal, then 2 and 3 days after treatment in 20 and 10 mL total volume, respectively. Cultures with less than 3 x 10^5 cells/mL were not adjusted. For expression of the TK-/- cells, cells were placed in cloning medium (CM). Two flasks per culture to be cloned were labeled with the test article concentration, activation condition, and either TFT (trifluorothymidine, the selective agent) or VC (viable count). Each flask filled with 100 mL CM and placed in an incubator shaker at 37+/-1°C until used. The cells were centrifuged at 1000 rpm for 10 minutes and the supernatant was decanted. The cells were then diluted in CM to concentrations of 3 x 10^6 cells/100 mL CM for the TFT flask and 600 cells/100 mL CM for the VC flask. After the dilution, 1.0 mL of stock solution of TFT was added to the TFT flask (final concentration of 3 microg/mL) and both this flask and the VC flask were placed on the shaker at 125 rpm and 37±1°C for 15 minutes. After 15 minutes, the flasks were removed and the cell suspension was divided equally into each of three appropriately labeled Petri dishes. To accelerate the gelling process, the plates were placed in cold storage (2-8°C) for approximately 30 minutes. The plates were then incubated at 37+/-1°C in a humidified 5+/-1% CO2 atmosphere for 10 - 14 days.

Scoring procedures: After incubation, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were counted for each culture with greater than or equal to 10% total relative growth (including at least one concentration with greater than or equal to 10% but less than or equal to 20% total growth, if possible). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test article-treated cultures were determined over a range of approximately 0.2 to 1.1 mm.
Evaluation criteria:
The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10^6 surviving cells) for each treatment condition was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2x10^-4) then multiplying by 10^6. In evaluation of the data, increases in mutant frequencies that occurred only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. The following criteria are presented as a guide to interpretation of the data: (1) A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited induced mutant frequencies of greater than or equal to 90 mutants per 10^6 clonable cells (based on the average mutant frequency of duplicate cultures). (2) A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants per 10^6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test article was soluble (formed a clear solution) in DMSO at approximately 500 mg/mL, the maximum concentration evaluated in the solubility test.

The maximum concentration tested in the preliminary toxicity assay was 2800 microg/mL (10mM). Visible precipitate was present in treatment medium at concentrations greater than or equal to 150 microg/mL at the beginning of treatment and at concentrations greater than or equal to 50 microg/mL at the end of treatment. The osmolality of the solvent control was 452 mmol/kg and the osmolality of the highest soluble dose at the beginning of treatment, 50 microg/mL, was 452 mmol/kg. Suspension growth relative to the solvent controls was 0% at concentrations greater than or equal to 15 microg/mL without activation with a 24-hour exposure and a concentration of greater than or equal to 50 microg/mL with and without S9 activation with a 4-hour exposure with the exception of the 500 microg/mL concentration with S9 activation for which the suspension growth relative to the solvent control was 5%. Based on the results of the toxicity test, the concentrations chosen for the mutagenesis assay ranged from 1 to 50 microg/mL for the non-activated cultures with 4-hour exposure; from 0.1 to 25 microg/mL for the S9-activated cultures with a 4hour exposure; and from 0.5 to 15 microg/mL for the non-activated cultures with a 24-hour exposure.

In the first mutation assay, visible precipitate was present in the treatment medium in the absence of activation at a concentration of 50 microg/mL, at the end of treatment only. In the non-activated system after 4 hours of exposure, cultures treated with concentrations of 5, 20, 25, 35, 40 and 50 microg/mL were cloned and produced a range in suspension growth of 17% to 109%. One culture at 40 microg/mL was too toxic to clone with a suspension growth of only 8%. In the S9-activated system after 4 hours of exposure, cultures treated with concentrations of 0.1, 2.5, 7.5, 15 and 25 microg/mL were cloned and produced a range in suspension growth from 13% to 110%. No cloned cultures exhibited induced mutant frequencies greater than or equal to 90 mutants per 10^6 clonable cells over that of the solvent control. There was no concentration-related increase in mutant frequency. The % relative total growth ranged from 13% to 112% for the non-activated cultures at concentrations from 5 to 50 microg/mL and 11% to 108% for the S9-activated cultures at concentrations from 0.1 to 25 microg/mL. The results of the initial assay were negative in the absence and presence of S9 activation.

Because no unique metabolic requirements were known about the test article, an extended treatment assay was performed only in the absence of S9 for a 24-hour exposure period.

The concentrations treated in the extended mutagenesis assay ranged from 0.5 to 15 microg/mL. No visible precipitate was present in the treatment medium at the beginning or end of treatment. Cultures treated with concentrations of 2.5, 7, 8, 9 and 10 microg/mL were cloned and produced a range in suspension growth from 12% to 98%. Two cloned cultures exhibited induced mutant frequencies greater than or equal to 90 mutants per 10^6 clonable cells. The mean induced mutant frequency at 9 microg/mL was 118 mutants per 10^6 cells. A concentration-related increase in mutant frequency was observed. The total growth ranged from 6% to 89%. No mutant frequency data were gathered from the cultures with less than 10% total growth.

The trifluorothymidine-resistant colonies for the cloned test article-treated cultures in the extended mutagenesis assay and the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

All criteria for a valid test were met.
Remarks on result:
other: 4-hour exposure

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with TMBPA in the Absence of Exogenous Metabolic Activation Initial Assay (4-hour exposure)

Dose Level (microg/mL)

Rep

% Susp. Growth

VC Colonies

(Mean)

TFT

Colonies

(Mean)

Total Mutant Freq.a

Induced Mutant Freq.b

% Relative Total Growthc

0 (solvent)

A

100

174

50

58

N/A

100

0 (solvent)

B

205

51

50

Mean Solvent Mutant Frequency = 54

5

A

109

175

36

41

-13

101

5

B

106

201

48

48

-6

112

20

A

63

211

45

43

-11

70

20

B

65

215

53

50

-4

74

25

A

30

182

42

47

-7

28

25

B

28

165

53

64

10

24

35

A

17

144

54

74

21

13

35

B

37

180

45

50

-4

35

40

A

18

152

54

71

18

15

40

B

8

+

+

 

 

 

50

A

21

177

51

57

3

19

50

B

28

178

46

52

-2

27

Positive Control - MMS (microg/mL)

20

--

53

64

260

813

759

18

15

--

58

115

254

440

387

35

Rep = Replicate; Solvent = DMSO

+ = Too toxic to clone

* = Precipitating concentration

a Total mutant frequency (per 10^6 surviving cells) = (average # TFT colonies / average # VC colonies) x 200

b Induced mutant frequency (per 10^6 surviving cells) = total mutant frequency - average mutant frequency of solvent controls

c % Total growth = (% suspension growth x % cloning growth) / 100

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with TMBPA in the Presence of Exogenous Metabolic Activation Initial Assay (4-hour exposure)

Dose Level (microg/mL)

Rep

% Susp. Growth

VC Colonies

(Mean)

TFT

Colonies

(Mean)

Total Mutant Freq.a

Induced Mutant Freq.b

% Relative Total Growthc

0 (solvent)

A

100

178

63

71

N/A

100

0 (solvent)

B

201

79

79

Mean Solvent Mutant Frequency = 75

0.1

A

100

194

66

68

-7

102

0.1

B

110

185

57

61

-13

108

2.5

A

56

179

66

73

-2

53

2.5

B

51

177

87

98

23

48

7.5

A

29

176

74

84

10

27

7.5

B

28

139

67

97

22

20

15

A

21

172

116

135

60

19

15

B

20

156

126

162

87

16

25

A

13

162

122

151

76

11

25

B

15

158

114

144

69

12

Positive Control - DMBA (microg/mL)

 

1.5

--

23

127

296

468

393

15

 

1.25

--

30

147

289

393

318

24

 

Rep = Replicate; Solvent = DMSO

+ = Too toxic to clone

* = Precipitating concentration

a Total mutant frequency (per 10^6 surviving cells) = (average # TFT colonies / average # VC colonies) x 200

b Induced mutant frequency (per 10^6 surviving cells) = total mutant frequency - average mutant frequency of solvent controls

c % Total growth = (% suspension growth x % cloning growth) / 100

 

 

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with TMBPA in the Absence of Exogenous Metabolic Activation Assay (24-hour exposure)

Dose Level (microg/mL)

Rep

% Susp. Growth

VC Colonies

(Mean)

TFT

Colonies

(Mean)

Total Mutant Freq.a

Induced Mutant Freq.b

% Relative Total Growthc

0 (solvent)

A

100

194

35

36

N/A

100

0 (solvent)

B

181

39

43

Mean Solvent Mutant Frequency = 40

2.5

A

96

160

43

53

14

82

2.5

B

98

172

39

45

6

89

7

A

56

141

69

98

58

42

7

B

59

146

67

92

52

45

8

A

39

151

77

101

62

32

8

B

36

130

80

122

83

25

9

A

23

118

82

138

99

14

9

B

25

112

99

177

137

15

10

A

12

103

++

 

 

7

10

B

12

91

++

 

 

6

12.5

A

4

+

+

 

 

 

12.5

B

4

+

+

 

 

 

Positive Control - MMS (microg/mL)

7.5

--

48

102

298

586

547

26

5

--

66

137

302

441

402

48

Rep = Replicate; Solvent = DMSO

+ = Too toxic to clone

++ = Too toxic to count

* = Precipitating concentration

a Total mutant frequency (per 10^6 surviving cells) = (average # TFT colonies / average # VC colonies) x 200

b Induced mutant frequency (per 10^6 surviving cells) = total mutant frequency - average mutant frequency of solvent controls

c % Total growth = (% suspension growth x % cloning growth) / 100

Conclusions:
positive in the 24-hour exposure without metabolic activation

All criteria for a valid study were met. Under the conditions of this study, test article Tetramethyl Bisphenol A was concluded to be positive in the 24-hour exposure without S9 activation and negative in the 4-hour exposure both with and without S9 activation in the L5178Y TK+/- mouse lymphoma assay.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
September, 2014
GLP compliance:
yes
Type of assay:
other: mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Stoneridge, New York)
- Age at study initiation: 7 weeks
- Weight at study initiation: 161.6 - 214.1 g
- Assigned to test groups randomly: yes
- Housing: Micro-Barrier cages, up to 5 animals per cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. Heat treated hardwood chips (P.J. Murphy Forest Products) were used for bedding to absorb liquids.
- Diet: certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet); ad libitum
- Water: tap water (met U.S. EPA drinking wtaer standards); ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Amount of vehicle: 10 mL/kg/day
- Supplier: MP Biomedical, LCC
- Lot/Batch No.: M6581
- Expiration date: 31 January 2017
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared daily. A suitable sized vial with a PTFE stir bar was calibrated to the final batch size and an appropriate amount of test article was transferred to the vial. An appropriate amount of vehicle was added until approximately 70% of the target volume was reached. The suspension was stirred magnetically for 12 to 57 minutes. Then, the appropriate amount of vehicle was added until the target volume was achieved.
Duration of treatment / exposure:
3 days
Frequency of treatment:
Daily
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (males)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide monohydrate for micronucleus assay; Ethyl methanesulfonate (EMS) and vinblastine sulfate (VB) for potential CREST analysis
- Doses / concentrations: EMS: 200 mg/kg/day; VB: 4 mg/kg/day
- Administration: EMS/VB: once daily on Study Day 2 and approximately 3 to 4 hours prior to euthanasia on Study Day 3.
- Other: Scoring positive control slides (fixed and unstained), generated from a different study, were included to verify scoring in the Micronucleus assay. These slides were generated from male rats treated once with cyclophosphamide monohydrate (CP) at 40 mg/kg, and the bone marrow harvested 24 hours after treatment
Tissues and cell types examined:
Polychromatic erythrocytes derived from femoral bone marrow were evaluated on micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the selected doses were based on a range-finding study. The dose levels tested in the dose range finding assay (DRF) were 500, 1000, and 2000 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day in males only, which is the highest guideline recommended dose for this assay.

TREATMENT AND SAMPLING TIMES: Daily, euthanisia by carbon dioxide inhalation took place 3 - 4 hours after the last treatment. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum.

DETAILS OF SLIDE PREPARATION:
- The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least 6 positive control (CP) slides) was stained with acridine orange for microscopic evaluation. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.
- After preparation of the two slides for micronucleus evaluation, the remaining bone marrow suspension, including that from the VB-treated animals, was processed through a cellulose column containing a 50:50 mixture of cellulose type 50:alpha-cellulose (one gram per column, one column per animal). The bone marrow suspension was eluted through the cellulose column using 4 mL of fetal bovine serum. The eluted cells were pelleted by centrifugation, and the supernatant drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least two slides for CREST were prepared from each animal, air dried and fixed by dipping in methanol. Each slide was identified by the harvest date, study number and animal number. Slides were placed in a slide box within a sealed plastic bag purged with nitrogen. The CREST slides were stored at -10 to -30ºC until disposal prior to finalization.

METHOD OF ANALYSIS: Bone marrow was evaluated by fluorescent microscopy. The staining procedure permitted the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus were counted as one micronucleated PCE (MnPCE), not two (or more) micronuclei. At least 4000 PCEs/animal were scored for the presence of micronuclei (MnPCEs), whenever possible. In addition, at least 500 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE/EC proportions <20% of vehicle control value were considered excessively cytotoxic and the animal data was excluded from evaluation.
Evaluation criteria:
The test article was considered to have induced a positive response if:
a) at least one of the test article doses exhibited a statistically significant increase when compared with the concurrent vehicle control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.
A test article was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the bone marrow was exposed to the test article (unless intravenous administration was used).
Statistics:
The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test article groups at the respective sampling time were compared using Levene’s test (significant level of p ≤ 0.05). Since the variation between groups was found not to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc analysis to compare each dose group to the concurrent vehicle control. A linear regression analysis was conducted to assess dose responsiveness in the test article treated groups (p ≤ 0.01). A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group to the concurrent vehicle control group.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Ratio of PCE/NCE: A statistically significant reduction in the PCEs/EC ratio was observed in male rats in the test article group treated with 2000 mg/kg/day compared to the vehicle control group. However, this reduction was determined to be biologically insignificant, and all dose levels could be scored.
- No statistically significant increase in the incidence of MnPCEs was observed in the test article treated groups relative to the vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
- The positive control, CP, induced a statistically significant increase in the incidence of MnPCEs (24,000 PCEs scored, 4000 PCEs/animal; Student’s t-test, p ≤ 0.05).
- The number of MnPCEs in the vehicle control group did not exceed the historical control range.

CLINICAL SIGNS

No mortality occurred. The only clinical effect observed was piloerection in all animals from the highest dose group, 2 hours after the latest dose on day 3. Animals gained weight during the study.

VALIDITY

All criteria for a valid test were met as mentioned under 'Any other information on materials and methods incl tables'. There were some deviations from the protocol, but these had no adverse impact on the integrity of the data or the validity of the study conclusion as determined by the study director.

Conclusions:
The test substance was not genotoxic in the in vivo Micronucleus assay.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
September, 2014
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Stoneridge, New York)
- Age at study initiation: 7 weeks
- Weight at study initiation: 161.6 - 214.1 g
- Assigned to test groups randomly: yes
- Housing: Micro-Barrier cages, up to 5 animals per cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. Heat treated hardwood chips (P.J. Murphy Forest Products) were used for bedding to absorb liquids.
- Diet: certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet); ad libitum
- Water: tap water (met U.S. EPA drinking wtaer standards); ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Amount of vehicle: 10 mL/kg/day
- Supplier: MP Biomedical, LCC
- Lot/Batch No.: M6581
- Expiration date: 31 January 2017
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared daily. A suitable sized vial with a PTFE stir bar was calibrated to the final batch size and an appropriate amount of test article was transferred to the vial. An appropriate amount of vehicle was added until approximately 70% of the target volume was reached. The suspension was stirred magnetically for 12 to 57 minutes. Then, the appropriate amount of vehicle was added until the target volume was achieved.
Duration of treatment / exposure:
3 days
Frequency of treatment:
Daily
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (males)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Ethyl methanesulfonate (EMS)
- Doses / concentrations: 200 mg/kg/day
- Administration: once daily on Study Day 2 and approximately 3 to 4 hours prior to euthanasia on Study Day 3.
Tissues and cell types examined:
Liver and duodenum cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the selected doses were based on a range-finding study. The dose levels tested in the dose range finding assay (DRF) were 500, 1000, and 2000 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day in males only, which is the highest guideline recommended dose for this assay.

TREATMENT AND SAMPLING TIME: Daily, euthanisia by CO2 asphyxiation took place 3 - 4 hours after the last treatment.

TISSUE COLLECTION AND PROCESSING
- Animals were dissected and the liver and duodenum were extracted (removed) and collected.
- A section of the liver and duodenum was cut and placed in formalin for possible histopathology analysis.
- Another section of the liver and duodenum was placed in chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO) and was used in preparation of cell suspensions and Comet slides.

PREPARATION OF CELL SUSPENSIONS AND COMET SLIDES
A portion of each dissected liver was placed in cold mincing buffer, then the liver was finely cut (minced) with a pair of fine scissors to release the cells. A portion of each dissected duodenum was placed in cold mincing buffer; then, the duodenum was scraped using a plastic spatula to release the cells. Each cell suspension was strained through a Cell Strainer into a pre-labeled 50 mL polypropylene conical tube. An aliquot of the suspension was used to prepare the Comet slides.
- Preparation of the slides: from each liver and duodenum suspension, an aliquot of 2.5 or 7.5 μL, respectively, was mixed with 50 μL (0.5%) of low melting agarose (0.5%). The cell/agarose suspension was applied to glass microscope slides/wells, previously coated with 1% normal melting agarose. The slides were kept at 2 - 8°C for 16 minutes to allow the gel to solidify. At least two Trevigen, Inc. 3-well slides were prepared per animal per tissue. Three slides/wells were used in scoring and the other wells were designated as a backup. Following solidification of the agarose, the slides were placed in jars containing lysis solution.
- Lysis: Following solidification of agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution overnight at 2-8oC.
- Unwinding: After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-10°C and protected from light, allowing DNA to unwind.
- Electrophoresis: Using the same buffer, electrophoresis was conducted for 32 to 35 minutes at 0.7 V/cm, at 2-10°C and protected from light. The electrophoresis time was similar for all slides.
- Neutralization: After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for approximately 23 to 25 hours and stored at room temperature with desiccant.
- Staining: slides were stained with a DNA stain (i.e., Sybr-gold™) prior to scoring. The stain solution was prepared by diluting 1 μL of Sybr-gold™ stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

METHOD OF ANALYSIS: Three slides/wells per organ/animal were used. Fifty randomly selected cells per slide were scored resulting in a total of 150 cells evaluated per animal. If any of the slides did not have 50 scorable cells, additional cells were scored using the backup slides. If 150 cells were not available, then the calculations were performed using the number of scorable cells. The following endpoints of DNA damage were assessed and measured:
- Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
- % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
- Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100;]

OTHER: Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal (percentage of “clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). Every effort was made to score at least 150 cells, otherwise, the total number of scorable cells was used for calculations. The “clouds,” also known as “hedgehogs,” are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the Comet and the head is reduced in size, almost nonexistent. “Clouds” with visible gaps between the nuclei and the Comet tail were excluded from Comet image analysis.
Evaluation criteria:
The test article was considered to have induced a positive response if:
a) at least one of the test article doses exhibited a statistically significant increase when compared with the concurrent vehicle control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01) and
a) results of the group mean or of the individual animals of at least one group were outside the distribution of the historical negative control database for that tissue.
The test article was considered to have induced a clear negative response if none of the criteria for a positive response were met and direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue was demonstrated.
Statistics:
The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test article groups were compared using Levene’s test (significant level of p ≤ 0.05). Since the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). A linear regression analysis was conducted to assess dose responsiveness in the test article treated groups (p ≤ 0.01). A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group to the concurrent vehicle control group.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- The presence of ‘clouds’ in the test article groups for liver was ≤ 0.8%, which was lower than the % of clouds in the vehicle control group (1.2%).
- The presence of ‘clouds’ in the test article groups for duodenum was ≤23.5%, which was lower than the % of clouds in the vehicle control group (57.6%).
- Group variances for mean of medians of the % Tail DNA in the vehicle and test article groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
- No statistically significant response in the % Tail DNA (DNA damage) was observed in the test article groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
- No dose-dependent increase in the % Tail DNA was observed across three test article doses (regression analysis, p > 0.01).
- The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver and duodenum cells as compared to the vehicle control groups (Student’s t-test, p ≤ 0.05).
- In the vehicle control group, % Tail DNA was within the historical vehicle control range for the liver and duodenum.

HISTOPATHOLOGY EVALUATION

Per the study protocol, histopathology evaluation was not performed since biologically significant increases in DNA damage were not observed.

CLINICAL SIGNS

No mortality occurred. The only clinical effect observed was piloerection in all animals from the highest dose group, 2 hours after the latest dose on day 3. Animals gained weight during the study.

VALIDITY: all validity criteria were met.

Conclusions:
The test substance was not genotoxic in the in vivo Comet assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

A GLP compliant study was performed, according to OECD guideline 471, to evaluate the mutagenic potential of the test substance. The test substance was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on information provided by the Sponsor, solubility of the test article and compatibility with the target cells. After sonication at 26.3ºC for five minutes, the test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test. In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1500 μg per plate. Toxicity was observed beginning at 150, 500 or at 5000 μg per plate with all test conditions except tester strain WP2 uvrA in the absence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 600 μg per plate with all Salmonella tester strains and 5000 μg per plate with tester strain WP2 uvrA. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 2.0, 6.0, 20, 60, 200 and 600 μg per plate with all Salmonella tester strains and 60, 200, 600, 1800 and 5000 μg per plate with tester strain WP2 uvrA. Precipitate was observed beginning at 600 μg per plate. Toxicity was observed beginning at 60, 200 or at 600 μg per plate with all Salmonella tester strains except tester strain TA1537 in the presence of S9 activation. Due to toxicity profiles that differed from that observed in the initial assay, tester strain TA100 in the absence of S9 activation and tester strain TA1537 in the presence of S9 activation were retested with an adjustment in dose levels. In the retest of the confirmatory mutagenicity assay, no positive mutagenic responses were observed with tester strain TA100 in the absence of S9 activation and tester strain TA1537 in the presence of S9 activation. The dose levels tested were 0.15, 0.50, 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 500 μg per plate. Toxicity was observed beginning at 500 or 1500 μg per plate. Under the conditions of this study, the test substance was concluded to be negative in the Bacterial Reverse Mutation Assay.

A GLP compliant study was performed, according to OECD guideline 473, to evaluate the clastogenic potential of the test substance based upon its ability to induce chromosome aberrations in CHO cells. The test article, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article. Dimethyl sulfoxide (DMSO) was used as the vehicle. Upon sonication for approximately 5 minutes at 26.3°C, the test article formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested for solubility. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the dose levels tested ranged from 0.2804 to 2804 μg/mL (10 mM). CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the vehicle control) was observed at dose levels ≥ 28.04 μg/mL for the non-activated 4 and 20-hour exposure groups, and at dose levels ≥ 84.12 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 50 μg/mL for the non-activated and the S9-activated 4-hour exposure groups, and ranged from 2.5 to 32 μg/mL for the non-activated 20-hour continuous exposure group. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. The highest dose analyzed under each treatment condition produced ≥ 50% reduction in cell growth index which met the dose limit as recommended by testing guidelines for this assay. No biologically significant increases in aberrant metaphases, or polyploid or endoreduplicated cells, were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met. These results indicate the test substance was negative in the in vitro chromosome aberration assay in CHO cells under the conditions, and according to the criteria of the study protocol.

A GLP compliant study was performed, according to OECD guideline 476, to evaluate the genotoxic potential of the test article based on quantitation of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells and sizing of the resulting colonies. The test article was tested in the L5178Y/TK+/- Mouse Lymphoma Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay established the concentration range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test article. Dimethyl sulfoxide (DMSO) was selected as the solvent. After sonication at 26.3ºC for five minutes, the test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test. In the preliminary toxicity assay, the maximum concentration of the test substance in treatment medium was 2800 μg/mL (10 mM). Visible precipitate was present in the treatment medium at concentrations ≥150 μg/mL at the beginning of treatment and at concentrations ≥50 μg/mL at the end of treatment. Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control. Suspension growth relative to the solvent control was 0% at concentrations ≥15 μg/mL without S9 activation with a 24-hour exposure and at concentrations ≥50 μg/mL with and without S9 activation with a 4-hour exposure with the exception of the 500 μg/mL concentration with S9 activation with a 4-hour exposure, for which the suspension growth relative to the solvent control was 5%. Based on the results of the preliminary toxicity assay, the concentrations treated in the initial mutagenesis assay ranged from 1 to 50 μg/mL for the non-activated cultures with a 4-hour exposure and from 0.1 to 25 μg/mL for the S9-activated cultures with a 4-hour exposure. Visible precipitate was present in the treatment medium at a concentration of 50 μg/mL at the end of treatment only. The concentrations chosen for cloning were 5, 20, 25, 35, 40 and 50 μg/mL for the non-activated cultures with a 4-hour exposure and 0.1, 2.5, 7.5, 15 and 25 μg/mL for the S9-activated cultures with a 4-hour exposure. No cloned cultures exhibited induced mutant frequencies ≥90 mutants per 10^6 clonable cells. There was no concentration-related increase in mutant frequency. Based on the results of the preliminary toxicity assay, the concentrations treated in the extended mutagenesis assay ranged from 0.5 to 15 μg/mL for the non-activated cultures with a 24-hour exposure. No visible precipitate was present in the treatment medium at the beginning or end of treatment. The concentrations chosen for cloning were 2.5, 7, 8, 9 and 10 μg/mL. Two cloned cultures exhibited induced mutant frequencies ≥90 mutants per 10^6 clonable cells. The mean induced mutant frequency at 9 μg/mL was 118 mutants per 10^6 cells. A concentration-related increase in mutant frequency was observed.

The trifluorothymidine-resistant colonies for the cloned test article-treated cultures in the extended mutagenesis assay and the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The data on colony size distributions showed an increase in the frequency of small and large colonies when the test article-treated cultures were compared to the solvent control cultures. An increase in the frequency of small colonies is consistent with damage to multiple loci on chromosome 11 in addition to functional loss of the TK locus. An increase in large colony mutants is indicative of localized damage in the form of a point mutation or small deletion within the TK locus. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies. Under the conditions of this study, the test article was concluded to be positive in the 24-hour exposure without S9 activation and negative in the 4-hour exposure both with and without S9 activation in the L5178Y TK+/-Mouse Lymphoma Assay.

The test article was evaluated for its genotoxic potential in bone marrow of male rats in a GLP-compliant study, performed according to OECD guideline 474. Corn oil was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg/day by oral gavage. In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000, and 2000 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day in males only, which is the highest guideline recommended dose for this assay. The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day.In the Micronucleus assay, no statistically significant increase in the incidence of MnPCEs was observed in the test article treated groups relative to the vehicle control group. Thus, the test article was negative (non-clastogenic). The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control group did not exceed the historical control range. No mortality occurred during the study and all animals gained weight.Under the conditions of this study, the administration of test substance at doses up to and including a dose of 2000 mg/kg/day, did not induce a significant increase in the incidence of MnPCEs relative to the concurrent vehicle control. Therefore, the test substance was concluded to be negative in the in vivoMicronucleus assay.

The test article was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver and duodenum cells in a GLP-compliant study, performed according to OECD guideline 489. Corn oil was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg/day by oral gavage. In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000, and 2000 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day in males only, which is the highest guideline recommended dose for this assay. The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day. In the Comet assay, the test article gave a negative (non-DNA damaging) response in the liver and duodenum for males in % Tail DNA. None of the test article treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver and duodenum tissues.Under the conditions of this study, the administration of the test substance at doses up to and including a dose of 2000 mg/kg/day, did not induce a significant increase in DNA damage in liver or duodenum tissues. Therefore, the test substance was concluded to be negative in the Comet assay.

Justification for classification or non-classification

The substance was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA and was negative for the induction of structural and numerical chromosome aberrations in CHO cells. In the mouse lymphoma assay with L5178Y cells, the substance tested positive after 24-hour exposure in the absence of metabolic activation, and negative after 4-hour exposure with and without metabolic activation. Based on these results, a conclusion on classification and labeling can only be made when in vivo data is available.