Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro. Gene mutation in bacteria (Ames)

The mutagenicity of the test material was evaluated in a bacterial reverse mutation assay (Ames test) conducted to a method equivalent to OECD 471 and EU Method B13/14. The study was conducted with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli WP2 in the presence and absence of S9 metabolic activation up to limit concentrations (5 mg/plate) or up to cytotoxic concentrations.

Under the conditions of the test, the test material was determined to be non-mutagenic in all strains tested. The assays were conducted in the presence and absence of metabolic activation up to cytotoxic concentrations (where appropriate), or up to limit concentrations.

 

Genetic toxicity in vitro. Chromosome Aberration

The clastogenicity of the test material was evaluated in an in vitro mammalian chromosome aberration test conducted to a method comparable to OECD 473. Chinese hamster ovary cells were exposed to the test material in the presence and absence of metabolic activation (S9) up to cytotoxic concentrations. Under the conditions of the test, the test material was concluded to be non-clastogenic in the presence and absence of metabolic activation.

 

Genetic toxicity in vitro. Gene mutation in mammalian cells

The mutagenicity of the test material was evaluated in a study conducted in accordance with OECD 476 and EU Method B.17. Two experiments were conducted up to precipitating cytotoxic concentrations in the presence and absence of metabolic activation for 3-hour and 24-hour exposure times. Under the conditions of the test, no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cell were observed and is therefore considered to be non-mutagenic under the conditions of the test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 1993 to 16 August 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro cytogenicity / chromosome aberration study in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The Merck Institute for Therapeutic Research, West Point, PA
- Cell cycle length, doubling time or proliferation index: Doubling time 14 hours
- Number of passages if applicable: 9 and 10
- Methods for maintenance in cell culture if applicable: Master vials were stored at -80 °C. Working cultures were maintained in a 37 °C, 5 % CO2 and 95 % realtive humidity incubator in culture medium.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 5 % CO2. Maintained in McCoy's 5A with NaHCO3 buffer, 10 % foetal bovine serum (FBS), 50 units/mL penicillin, 50 µg/mL streptomycin and 2 mL L-glutamine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 microsome fraction
Test concentrations with justification for top dose:
0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/mL - Nonactivated, 24 and 14 hour assays
0.015, 0.05, 0.15, 0.5, 1.5, 5, and 15 µg/mL - Activated, 24 hour assay
0.00051, 0.0015, 0.0051, 0.015, 0.051, 0.15, 0.51, 1.5, 5.1 and 15 µg/mL - Activated, 14 hour assay
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding : Seeded at a cell density of 5E+05 cells per flask

DURATION
- Exposure duration:14 or 24 hours in the nonactivated assay and 2 hours in the activated assay
- Expression time (cells in growth medium): 14 or 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were fixed at the end of the expression period

SPINDLE INHIBITOR (cytogenetic assays): Vinblastine sulfate (0.26 µg/mL added 2 hours prior to harvest)

STAIN : 5 % Giemsa

NUMBER OF REPLICATIONS: Performed in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the incubation period, metaphase cells were collected by treatment with trypsin (0.25 % trypsin and 0.02 % EDTA) followed by concentration of cells by centrifugation. Due to the presence of the test material and positive control in the flasks in the nonactivated assay, these cells were washed with phosphate buffered saline (PBS) solution (pH 7.3) or with medium. The cells for all assays were suspending in hypotonic solution (0.03 M KCl and 0.01 M sodium citrate) for 12 minutes at 37 °C and fixed 3 times 3:1 methanol:acetic acid by centrifugation and resuspension. Drops of the concentrated cell suspension were placed on glass slides, air dried and stained in 5 % Giemsa for approximately 5 minutes at room temperature.

NUMBER OF CELLS EVALUATED: 100

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (a minimum of 500 cells were counted)
Rationale for test conditions:
Two range finder assays were performed to determine toxic concentrations of the test material in the presence of metabolic activation in the test system. The first range finding assay was performed using concentrations of 523, 1577 and 5257 µg/mL. The second assay was performed with concentrations of 1.4 and 148 µg/mL. The test concentrations of the definitive test were selected based of the results of the two range finding tests.
Evaluation criteria:
Metaphase cells were analysed for chromosomal aberrations through a microscope using a 100 x objective. The mitotic index was determined by counting a minimum of 500 total cells. A minimum of 25 cells were analysed for each positive control flask. In the 14 hour activated assay, the highest test concentration, 15 µg/mL, was analysed uncoded due to the low number of metaphase cells (0.7 %) observed coupled with the low percentage of confluency noted (less than 10 %, toxic spots). One hundred metaphase cells per flask for at least three selected test sample concentrations and the negative control were analysed for the following types of chromosomal aberrations:
Chromatid gap
Chromatid break
Chromosome gap
Chromosome break
Quadriradial
Interstitial deletions
Double minute chromosomes
Triradial
Dicentric chromosome
Cell with at least one pulverised chromosome
Complex rearrangements
Rings
Cells with greater than 10 aberrations
A positive response was determined by a statistically significant increase in aberrations.
Statistics:
Coordinates of cells with aberrations were recorded.
% Cells with aberrations = (Number of cells with aberrations/total number of cells analysed) * 100
Number of Aberrations per Cell = Total number aberrations/Total cells analysed
% Mitotic Index (MI) = (Number of metaphase cells/Total number of cells) * 100
The number of aberrations per cell and the percent of cells with aberrations were calculated by two methods. In one calculation the number of chromatid and chromosome gaps were counted as aberrations. IN the other calculation, the more traditional method was followed in which gaps were not counted as aberrations. A cell with a large number of aberrations was counted as having 10 aberrations. No cells with pulverised chromosomes were observed.
The data were analysed statistically using the method described in Margolin et al (1986) Statistical Analyses for In Vitro Cytogenetic Assays Using Assays Chinese Hamster Ovary Cells, Environ. Mutagen. 8: 183-204.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test material was found to be cytotoxic at concentrations greater than 49.3 µg/mL in the presence of metabolic activation.

Table 1: Results of the range finding studies

Concentration
(µg/mL)

1st Range Finding Study
(Mitotic Index)

2nd Range Finding Study
(Mitotic Index)

5257

Toxic

1577

Toxic

523

Toxic

148

Toxic

49.3

Toxic

14.8

11/543 (2.0 %)

5/506 (0.79 %)

4.93

18/506 (3.6 %)

16/545 (2.9 %)

1.48

13/534 (2.4 %)

17/514 (3.3 %)

1 % DMSO

35/498 (7.0 %)

14/509 (2.8 %)

28/514 (5.4 %)

10/502 (2.0 %)

 

Table 2: 14-hour assay without metabolic activation (individual results)

Treatment

No. Cells

Analysed

%

Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Mitomycin C

0.5 µg/mL

25

2.6

12

3

0

1

4

3

0

0

0

0

0

8

3

25

1.8

9

5

0

1

7

4

0

0

0

0

1

6

1

DMSO

1.00%

100

10.4

1

0

0

0

0

0

0

0

0

0

0

4

1

100

6.4

0

0

0

0

0

0

1

0

0

0

0

1

0

Test material

1.5 µg/mL

100

9.4

1

2

0

0

0

0

1

0

0

0

0

2

1

100

7.2

2

0

0

0

0

0

0

0

0

0

0

4

0

5.0 µg/mL

100

11.8

0

0

0

0

0

0

0

0

0

0

0

3

0

100

4.8

0

1

0

0

0

0

0

0

0

0

0

4

0

15 µg/mL

100

3.7

0

1

0

0

0

0

0

1

0

0

0

7

0

100

8.7

0

3

0

0

0

0

0

0

0

0

0

3

0

50 µg/mL

Toxic

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+

 

Table 3: 14-hour assay without metabolic activation (summary)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Mitomycin C

0.5 µg/mL

25

23

0.92

56

34

1.36

68

25

36*

1.44

68

43*

1.72

72

DMSO

1.00%

100

1

0.01

1

6

0.06

5

100

1

0.01

1

2

0.02

2

Test material

1.5 µg/mL

100

4

0.04

3

7

0.07

6

100

2

0.02

2

6

0.06

5

5.0 µg/mL

100

0

0

0

3

0.03

3

100

1

0.01

1

5

0.05

5

15 µg/mL

100

4

0.04

4

11

0.11

10

100

3

0.03

2

6

0.06

4

50 µg/mL

Toxic

* Data indicated pulverised cells and/or damaged cells with more than 10 chromosome aberrations per cell

 

Table 4: 24-hour assay without metabolic activation (individual results)

Treatment

No. Cells Analysed

% Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Mitomycin C

0.3 µg/mL

25

1.6

10

2

0

2

3

5

0

3

9

0

1

3

1

25

1.2

20

4

0

2

9

6

0

1

5

0

0

7

1

DMSO

1.00%

100

6.0

1

2

0

0

0

0

1

0

0

0

0

3

1

100

7.0

0

3

0

0

0

0

0

1

0

0

0

0

0

Test material

1.5 µg/mL

100

6.0

1

0

0

0

0

0

0

0

0

0

0

1

0

100

5.8

0

2

0

0

0

0

0

0

0

0

0

0

0

5.0 µg/mL

100

5.9

0

2

0

0

0

0

2

0

0

0

0

1

0

100

3.9

0

2

0

0

0

0

0

0

0

0

0

1

0

15 µg/mL

100

6.0

0

1

0

0

0

0

0

0

0

0

0

1

0

100

6.3

1

2

0

0

0

0

0

0

0

0

0

1

0

50 µg/mL

Toxic

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+

 

Table 5: 24-hour assay without metabolic activation (summary)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Mitomycin C

0.5 µg/mL

25

44*

1.76

84

48*

1.92

84

25

47

1.88

72

55

2.20

76

DMSO

1.00%

100

4

0.04

3

8

0.08

6

100

4

0.04

3

4

0.04

3

Test material

1.5 µg/mL

100

1

0.01

1

2

0.02

2

100

2

0.02

2

2

0.02

2

5.0 µg/mL

100

4

0.04

4

5

0.05

5

100

2

0.02

2

3

0.03

3

15 µg/mL

100

1

0.01

1

2

0.02

2

100

3

0.03

2

4

0.04

3

50 µg/mL

Toxic

* Data indicated pulverised cells and/or damaged cells with more than 10 chromosome aberrations per cell

 

Table 6: 14-hour assay with metabolic activation (individual results)

Treatment

No. Cells Analysed

% Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Benzo(a)pyrene

15 µg/mL

50

0.6

8

1

0

0

3

1

0

0

1

0

0

4

0

50

2.9

4

7

0

5

7

0

0

2

0

0

0

6

2

DMSO

1.00%

100

4.4

0

6

0

0

0

0

0

1

0

0

0

1

0

100

2.4

1

8

0

0

1

0

0

1

0

0

0

5

0

Test material

0.51 µg/mL

100

2.8

4

9

0

0

0

0

0

1

0

0

1

3

1

100

2.2

0

6

0

0

0

0

0

2

0

0

0

3

0

1.5 µg/mL

100

4.3

0

1

0

0

0

0

0

0

0

0

0

4

0

100

2.4

3

4

0

0

1

0

0

0

0

0

0

4

0

5.1 µg/mL

100

3.8

0

2

0

0

0

0

0

1

0

0

1

5

1

100

2.0

2

3

1*

0

0

0

0

0

0

0

0

6

0

15 µg/mL

100

2.2

1

4

0

0

0

0

0

2

0

0

0

0

1

100

0.7

2

0

0

0

0

0

0

1

0

0

0

2

1

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+
* 4 pairs of double minutes in one cell

 

Table 7: 14-hour assay with metabolic activation (summary of results)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Benzo(a)pyrene

15 µg/mL

50

14

0.28

24

18

0.36

30

50

25

0.50

36

33

0.66

44

DMSO

1.00%

100

7

0.07

5

8

0.08

6

100

11

0.11

7

16

0.16

11

Test material

0.51 µg/mL

100

25*

0.25

6

29*

0.29

7

100

8

0.08

2

11

0.11

5

1.5 µg/mL

100

1

0.01

1

5

0.05

5

100

9

0.09

7

13

0.13

11

5.1 µg/mL

100

13*

0.13

4

19*

0.19

9

100

7

0.07

5

13

0.13

10

15 µg/mL

100

7

0.07

6

8

0.08

7

100

4

0.04

4

7

0.07

5

* Pulverised cells/and or damaged cells with more than ten chromosome aberrations per cell.

 

Table 8: 24-hour assay with metabolic activation (individual results)

Treatment

No. Cells Analysed

% Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Cyclophosphamide

50 µg/mL

25

1.1

20

3

0

3

10

2

1

1

3

0

0

4

1

25

1.9

12

1

0

1

13

4

1

2

10

0

2

0

2

Benzo(a)pyrene

15 µg/mL

25

5.0

0

1

0

0

0

0

0

0

0

0

0

0

1

25

8.5

0

1

0

0

1

0

0

0

0

0

0

0

0

DMSO

1.00%

100

4.8

3

3

0

0

0

0

1

0

0

0

0

1

1

100

7.7

1

5

0

0

0

0

0

1

0

0

0

1

1

Test material

1.5 µg/mL

100

4.9

1

0

0

0

0

0

0

0

0

0

0

0

0

100

5.0

1

3

1*

0

0

0

0

0

0

0

0

1

0

5.1 µg/mL

100

3.8

2

6

0

0

0

0

0

0

0

0

0

1

2

100

5.9

2

2

0

0

0

0

0

0

0

0

0

0

0

15 µg/mL

100

5.1

1

5

0

0

0

0

0

0

0

0

0

1

1

100

3.7

3

4

0

0

0

0

0

0

0

0

0

1

0

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+
* 7 pairs of double minutes in one cell

 

Table 9: 24-hour assay with metabolic activation (summary of results)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Cyclophosphamide

50 µg/mL

25

43

1.72

64

48

1.92

68

25

64*

2.56

92

66*

2.64

92

Benzo(a)pyrene

15 µg/mL

25

1

0.04

4

2

0.08

8

25

2

0.08

8

2

0.08

8

DMSO

1.00%

100

7

0.07

6

9

0.09

8

100

7

0.07

2

9

0.09

4

Test material

1.5 µg/mL

100

1

0.01

1

1

0.01

1

100

5

0.05

4

6

0.06

5

5.1 µg/mL

100

10

0.10

5

13

0.13

6

100

4

0.04

3

4

0.04

3

15 µg/mL

100

7

0.07

6

9

0.09

7

100

8

0.08

6

9

0.09

7

* Pulverised cells/and or damaged cells with more than ten chromosome aberrations per cell.

 

Conclusions:
The test material was evaluated for its ability to induce chromosomal damage in vitro using CHO cells. The test material was tested with and without metabolic activation. The positive controls were found to be valid under the conditions of the test. Under the conditions of the test, there were no increases in the percentage of aberrant cells (including and excluding gaps) with or without metabolic activation.
The test material was therefore concluded to not be clastogenic under the conditions of the test.
Executive summary:

The clastogenicity of the test material was evaluated in an in vitro mammalian chromosome aberration test conducted to a method comparable to OECD 473. Chinese hamster ovary cells were exposed to the test material in the presence and absence of metabolic activation (S9) up to cytotoxic concentrations. Under the conditions of the test, the test material was concluded to be non-clastogenic in the presence and absence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 July 1993 to 22 October 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine biosynthetic pathway - Salmonella
Tryptophan pathway - E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Concentrations of 0.00015 mg/plate to 0.05 mg/plate were chosen for the study. At concentrations above 0.05 mg/plate, the test material was found to be cytotoxic in the range-finding study to strain TA100.
0.00015, 0.0005, 0.0015, 0.005, 0.015 and 0.05 mg/plate were used in the definitive test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
2.5 mL aliquots of molten top agar containing either 50 µM biotin and 50 µM histidine (for Salmonella), or 50 µM biotin and 50 µM tryptophan (for E.coli) were added to 100 µL of bacterial culture (between 1E+09 and 2E+09 bacteria/mL), 100 µL of the appropriate concentration of test substance, or 100 µL of positive or negative (vehicle) control, and 500 µL of S9 mix or PBS (for the non-activated assay). The tubes were mixed and poured onto plates containing 25-30 mL of minimal glucose agar made. Test plates were allowed to harden, then were incubated at 37 °C for approximately 3 days. The plates were counted immediately after removal from incubation or stored at 4 °C before counting.
- Cell density: Between 1E+09 and 2E+09 bacteria/mL

DURATION
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): biotin and histidine (for Salmonella), or biotin and tryptophan (for E.coli)

NUMBER OF REPLICATIONS: Performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: bacterial lawn
Rationale for test conditions:
Concentrations were determined from the range finding study. Further repititions were performed based on observed toxicity during the definitive tests.
Evaluation criteria:
The bacterial plates were scored on an electronic colony counter, or they were counted manually. The mean number of revertants per plate and the standard deviation was calculated for each concentration and strain. A test result was considered positive if for any strain a significant increase over the negative control in the number of revertant per plate was observed which was concentration-dependent. A significant increase was a two-fold increase when the background was 50 revertants/plate or greater; a three-fold increase when the background was between 10 and 49 revertants/plate; a four-fold increase when the background was less than 10 revertants/plate.
Statistics:
The mean and standard deviation were calcualted for each concentration and strain.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The initial range finding study (TA100 tested in the absence of metabolic activation) indicated that the test material was toxic to bacteria at 0.05 mg/plate and above. The bacterial lawn was not visible at 1.5 and 5 mg/plate. At lower concentrations, 0.015 mg/plate and below, the bacterial lawn was normal and the number of revertant colonies were similar to the control. Based on the range finding study, experiment 1 was conducted with a concentration range between 0.00015 and 0.05 mg/plate.

HISTORICAL CONTROL DATA
- Positive historical control data: The postive control values were within established historical parameters.
- Negative (solvent/vehicle) historical control data: The negative control values were within established historical parameters.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the first definitive test, the test material was found to be cytotoxic at concentrations of 0.05 mg/plate for the strain TA100. Although the test material was non-mutagenic to TA100 in this experiment, these data were used as range-finding information for the other four strains.
A subsequent non-activated experiment (Experiment 2) was conducted with strains TA98, TA1535, TA1537 and WP2 utilising a maximum concentration of 0.2 mg/plate. No increase in revertant colonies was observed in this experiment. No cytotoxicity was observed, indicating that the limit of testing hadn’t been reached.
In Experiment 3, all 5 strains were tested up to a maximum concentration of 0.8 mg/plate. The test material was not mutagenic in strain TA100, and cytotoxicity was observed. The bacterial lawn was significantly reduced at 0.8 and 0.4 mg/plate. In the presence of metabolic activation, the test material was non-mutagenic. Cytotoxicity was observed at 0.2, 0.4 and 0.8 mg/plate. Cytotoxicity in strain TA1535 were similar to those found with TA100. In the absence of metabolic activation, 0.8 and 0.4 mg/plate were also toxic to TA1535. The number of revertant colonies was found to be reduced at these concentrations in the presence of metabolic activation. The test material was not mutagenic to TA1535. The test material was not mutagenic in all other strains tested in this experiment, however these strains were not tested up to limit concentrations (5 mg/plate or cytotoxic concentrations). Toxicity was evident in the bacterial lawn density of TA98, however this did not percent evaluation for revertant colonies.
In Experiment 4, conducted in the absence of metabolic activation, the test material was determined to be non-mutagenic to TA1535. 0.4 mg/plate was determined to be extremely toxic to the bacterial cultures. The test material was tested up to the maximum concentration in all other strains. Toxicity was evident in the bacterial lawn density of TA98, however this did not percent evaluation for revertant colonies.
Experiment 5 was conducted in the presence of metabolic activation. The test material was found to be non-mutagenic in all strains tested up to 5 mg/plate.
Experiment 6 was conducted to confirm the negative results observed with TA1537 and WP2. The test material was not mutagenic up to the maximum concentration 5 mg/plate.

Table 1: Range Finding Study

S9

Number of revertants per plate
(Individual Plates)

Mean Revertants/Plate
Mean (SD)

TA100

DMSO Control

-

104

153

140

132 (25)

Positive Control (ENNG) 9 µg/plate

-

1034

975

547

852 (266)

Test compound 5 mg/plate

-

x

x

x

x

Test compound 1.5 mg/plate

-

x

x

x

x

Test compound 0.5 mg/plate

-

x

x

x

x

Test compound 0.15 mg/plate

-

x

x

x

x

Test compound 0.05 mg/plate

-

x

x

x

x

Test compound 0.015 mg/plate

-

122t

86t

162t

123 (38)

Test compound 0.005 mg/plate

-

138t

144t

133t

138 (6)

Test compound 0.0015 mg/plate

-

145t

108t

152t

135 (24)

X - cytotoxic
t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring
ENNG = N-ethyl-N'nitro-N-nitrosaguanidine

 

Table 2: Results Experiment 1

 

S9

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

TA98

TA100

TA1535

TA1535

WP2

DMSO Control

-

32

30

26

29

(3)

168

165

185

173
(11)

29

21

15

22
(7)

15

12

15

14
(2)

44

43

28

38
(9)

+

45

30

29

35

(9)

168

197

204

190
(19)

17

21

20

19
(2)

8

11

20

14
(8)

52

60

49

54
(6)

Positive Control

-

391

389

401

394

(6)

1288

1207

1504

1333
(154)

236

233

229

233
(4)

399

388

229

418
(43)

700

722

857

760
(85)

Positive Control

+

351

240

314

302
(57)

1121

1098

956

1058
(89)

129

124

258

137
(18)

429

327

158

385
(53)

692

506

530

576
(101)

Test

compound

0.05

mg/plate

-

10

20

16t

15
(5)

x

z

x

x

12

15

16t

14
(2)

5

5

16t

6
(2)

47

50

53

50
(3)

Test

compound
0.015
mg/plate

-

21

25

26

24
(3)

183

157

145t

162
(19)

15

15

17

16
(1)

6

5

17

7
(2)

45

57

49

50
(6)

Test
compound
0.005
mg/plate

-

22

32

21

25
(6)

181

187

173

180
(7)

10

15

14

13
(3)

10

13

14

13
(3)

43

37

45

42
(4)

Test
compound
0.0015
mg/plate

-

25

24

32

27
(4)

156

172

205

178
(25)

11

16

15

14
(3)

19

12

15

16
(4)

43

50

40

44
(5)

Test
compound
0.0005
mg/plate

-

26

26

30

27
(2)

182

148

157

162
(18)

12

15

10

12
(3)

13

21

10

16
(5)

49

50

40

46
(6)

Test
compound
0.00015
mg/plate

-

38

21

23

27
(9)

193

182

156

177
(19)

12

15

12

13
(2)

12

19

12

15
(4)

54

39

53

49
(8)

Test
compound

0.05
mg/plate

+

42

43

48t

44
(3)

187

181

164t

177
(12)

18

12

11t

14
(4)

4

9

11t

5
(4)

72

66

66

68
(3)

Test
compound
0.015
mg/plate

+

54

54

52

53
(1)

195

188

171

185
(12)

12

16

16

15
(2)

14

15

16

15
(2)

70

60

45

58
(13)

Test
compound
0.005
mg/plate

+

35

40

40

38
(3)

175

205

183

188
(16)

20

20

12

17
(5)

16

17

12

17
(5)

66

55

48

56
(9)

Test
compound 0.0015
mg/plate

+

33

30

29

31
(2)

211

213

194

206
(10)

20

16

11

16
(5)

8

14

11

16
(5)

52

55

55

54
(2)

Test
compound
0.0005
mg/plate

+

33

43

49

42
(8)

171

182

196

183
(13)

13

15

21

16
(4)

15

14

21

16
(4)

72

65

63

67
(5)

Test
compound
0.00015
mg/plate

+

30

35

53

39
(12)

220

211

192

208
(14)

13

21

21

18
(5)

12

20

21

18
(5)

64

77

76

72
(7)

TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate; +S9 2-aminoanthracene (2AA) 0.5 µg/plate

TA100 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 9 µg/plate; +S9 2-aminoanthracene (2AA) 1 µg/plate

TA135 Positive control -S9 Sodium Azide 0.5 µg/plate; +S9 2-aminoanthracene (2AA) 2 µg/plate

TA137 Positive control -S9 9-Aminoacridine 80 µg/plate; +S9 2-aminoanthracene (2AA) 3 µg/plate

WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate; +S9 2-aminoanthracene (2AA) 20 µg/plate

X - cytotoxic

t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

 

Table 3: Results from Experiment 2

 

S9

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

TA98

TA1535

TA1537

WP2

DMSO Control

-

33

34

25

31

(5)

15

14

14

14

(1)

20

32

26

26

(6)

44

70

44

53

(15)

Positive Control

-

820

750

736

769

(45)

236

282

183

234

(50)

616

570

554

580

(32)

603

692

654

650

(45)

Test compound

0.20 mg/plate

-

25

31

13t

23

(9)

8

5

8t

7

(2)

24

18

19

20

(3)

60

63

50

58

(7)

Test compound

0.10 mg/plate

-

29

17

17

21

(7)

15

9

7t

10

(4)

15

18

9

17

(2)

59

53

59

57

(3)

Test compound

0.05 mg/plate

-

16

20

22

19

(3)

13

9

14

12

(3)

15

14

18

16

(2)

51

63

53

56

(6)

Test compound

0.025 mg/plate

-

22

21

24

22

(2)

8

10

12

10

(2)

27

22

17

22

(5)

63

52

46

54

(9)

Test compound

0.0125 mg/plate

-

19

18

30

22

(7)

9

6

14

10

(4)

14

22

12

16

(5)

51

58

43

51

(8)

Test compound

0.0063 mg/plate

-

31

18

29

26

(7)

11

15

13

13

(2)

17

23

16

19

(4)

71

48

53

57

(12)

TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate

TA135 Positive control -S9 Sodium Azide 0.5 µg/plate

TA137 Positive control -S9 9-Aminoacridine 80 µg/plate

WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate

t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

 

Table 4: Results from Experiment 3

 

S9

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

TA98

TA100

TA1535

TA1537

WP2

DMSO Control

-

16

18

21

18
(3)

94

96

118

103
(13)

13

10

8

10
(3)

11

11

11

11
(0)

29

24

nd

27
(4)

+

30

54

29

38
(14)

143

150

138

144
(6)

13

9

9

10
(2)

21

11

17

16
(5)

34

32

30

32
(2)

Positive Control

-

415

657

520

531
(121)

693

784

804

760
(59)

253

123

234

203
(70)

1038

973

930

980
(54)

340

493

409

414
(77)

Positive Control

+

437

489

525

484
(44)

867

860

765

831
(57)

125

179

nd

152
(38)

89

73

79

80
(8)

1038

704

960

901
(175)

Test compound

0.8

mg/plate

-

13

14

10t

12
(2)

x

x

x

x

x

x

x

x

17

8

8

11
(5)

23

16

15

18
(4)

Test compound

0.4

mg/plate

-

10

8

15t

11
(4)

x

x

x

x

x

x

x

x

16

17

15

16
(1)

25

26

22

24
(2)

Test compound

0.2

mg/plate

-

11

14

18t

14
(4)

36

26

33t

32
(5)

2

2

6t

3
(2)

16

11

17

15
(3)

19

22

27

23
(4)

Test compound

0.1

mg/plate

-

12

16

11

13
(3)

33

37

36t

35
(2)

7

4

10

7
(3)

8

15

13

12
(4)

28

26

21

25
(4)

Test compound

0.05

mg/plate

-

17

17

20

18
(2)

48

60

47

52
(7)

12

6

9

9
(3)

14

23

18

18
(5)

26

14

22

21
(6)

Test compound

0.025

mg/plate

-

16

21

20

19
(3)

60

48

85

64
(19)

9

9

11

10
(1)

7

13

16

12
(5)

21

33

26

27
(6)

Test compound

0.8

mg/plate

+

18

29

23t

23
(6)

28

32

32t

31
(2)

4

11

4t

6
(4)

18

19

29

22
(6)

26

29

28

28
(2)

Test compound

0.4

mg/plate

+

31

32

28

30
(2)

35

53

47t

45
(9)

12

7

5t

8
(4)

17

25

14

19
(6)

32

40

34

35
(4)

Test compound

0.2

mg/plate

+

33

25

45

34
(10)

50

55

57t

54
(4)

8

7

10

8
(2)

13

23

26

21
(7)

30

38

28

32
(5)

Test compound

0.1

mg/plate

+

38

36

43

39
(4)

97

123

130

117
(17)

20

13

14

16
(4)

21

19

20

20 910

35

37

34

35
(2)

Test compound

0.05

mg/plate

+

40

38

29

36
(6)

108

122

90

107
(16)

11

15

7

11
(4)

20

23

20

21 92)

28

26

39

31
(7)

Test compound

0.025

mg/plate

+

47

34

40

40
(7)

143

145

120

136
(14)

14

10

13

12
(2)

9

15

16

13 94)

32

36

26

31
(5)

TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate; +S9 2-aminoanthracene (2AA) 0.5 µg/plate

TA100 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 9 µg/plate; +S9 2-aminoanthracene (2AA) 1 µg/plate

TA135 Positive control -S9 Sodium Azide 0.5 µg/plate; +S9 2-aminoanthracene (2AA) 2 µg/plate

TA137 Positive control -S9 9-Aminoacridine 80 µg/plate; +S9 2-aminoanthracene (2AA) 3 µg/plate

WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate; +S9 2-aminoanthracene (2AA) 20 µg/plate

X - cytotoxic

t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

nd - not done, only two plates poured

 

Table 5: Results from Experiment 4

 

S9

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

TA98

TA1535

TA1537

WP2

DMSO Control

-

24

21

26

24

(3)

13

13

11

12

(1)

13

8

14

12

(3)

414

35

36

37

(3)

Positive Control

-

479

455

478

471

(14)

274

250

220

248

(27)

22

246

491

253

(235)

236

184

199

206

(27)

Test compound

5

mg/plate

-

ND

ND

ND

ND

ND

ND

ND

ND

17

10

24

17

(7)

48

22

27

32

(14)

Test compound

3.2

mg/plate

-

14

15

16t

15

(1)

ND

ND

ND

ND

16

19

12

16

(4)

36

42

48

42
(6)

Test compound

1.6

mg/plate

-

21

19

18t

19

(2)

ND

ND

ND

ND

16

11

7

11

(5)

31

36

46

38

(8)

Test compound

0.8

mg/plate

-

14

11

16t

14

(3)

ND

ND

ND

ND

11

15

9

12

(3)

46

34

36

39

(6)

Test compound

0.4

mg/plate

-

21

15

18t

18

(3)

x

x

x

x

12

14

12

13

(1)

41

42

44

42

(2)

Test compound

0.2

mg/plate

-

9

9

15

11

(3)

5

5

6t

5

(1)

18

18

15

17

(2)

33

27

39

33

(6)

Test compound

0.1

mg/plate

-

19

21

22

21

(2)

2

7

10t

6

(4)

12

13

12

12

(1)

ND

ND

ND

ND

Test compound

0.05

mg/plate

-

15

21

18

18

(3)

6

8

14

9 (4)

ND

ND

ND

ND

ND

ND

ND

ND

Test compound

0.025

mg/plate

-

29

26

27

27

(2)

6

15

4

8 (6)

ND

ND

ND

ND

ND

ND

ND

ND

Test compound

0.0125

mg/plate

-

ND

ND

ND

ND

15

9

11

12 (3)

ND

ND

ND

ND

ND

ND

ND

ND

TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate

TA135 Positive control -S9 Sodium Azide 0.5 µg/plate

TA137 Positive control -S9 9-Aminoacridine 80 µg/plate

WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate

t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

 

Table 6: Results from Experiment 5

 

S9

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

TA98

TA1535

TA1537

WP2

DMSO Control

-

ND

ND

ND

ND

ND

ND

ND

ND

6

17

12

12
(6)

44

36

40

40
(4)

+

37

43

40

40
(3)

15

13

16

15
(2)

15

26

25

22
(6)

30

30

34

31
(2)

Positive Control

-

ND

ND

ND

ND

ND

ND

ND

ND

324

405

436

388
(58)

636

721

694

684
(43)

Positive Control

+

864

713

761

779

(77)

169

181

178

176
(6)

279

307

321

302
(210

194

196

132

174
(36)

Test compound 5.0 mg/plate

-

ND

ND

ND

ND

ND

ND

ND

ND

9

12

13

11
(2)

24

22

26

24
(2)

Test compound 3.2 mg/plate

-

ND

ND

ND

ND

ND

ND

ND

ND

15

17

18

17
(2)

33

37

32

34
(3)

Test compound 1.6 mg/plate

-

ND

ND

ND

ND

ND

ND

ND

ND

6

3

13

7
(5)

54

26

30

37
(15)

Test compound 0.8 mg/plate

-

ND

ND

ND

ND

ND

ND

ND

ND

7

11

14

11
(4)

43

23

24

30
(11)

Test compound 0.4 mg/plate

-

ND

ND

ND

ND

ND

ND

ND

ND

6

14

16

12
(5)

18

35

45

33
(14)

Test compound 0.2 mg/plate

-

ND

ND

ND

ND

ND

ND

ND

ND

6

10

13

10
(4)

32

22

23

26
(6)

Test compound 5.0 mg/plate

+

26

15

20

20

(6)

4

10

4t

6
(3)

28

24

27

26
(2)

41

38

39

39
(2)

Test compound 3.2 mg/plate

+

50

44

51

48

(4)

11

3

11t

8
(5)

18

17

29

21
(7)

21

35

35

30
98)

Test compound 1.6 mg/plate

+

27

26

25

26

(1)

8

10

15

11
(4)

22

12

15

16
(5)

16

35

35

29
(11)

Test compound 0.8 mg/plate

+

45

34

41

40

(6)

6

6

10

7
(2)

25

16

15

19
(6)

40

24

36

33
(8)

Test compound 0.4 mg/plate

+

45

41

57

48

(8)

12

9

11

11(2)

52

39

36

42
(9)

37

40

32

36
(4)

Test compound 0.2 mg/plate

+

46

27

38

34

(11)

19

11

6

12

(7)

25

31

28

28
(3)

37

43

45

42
(4)

Test compound 0.1 mg/plate

+

38

38

46

41

(5)

12

10

18

13

(4)

19

19

20

19
(1)

ND

ND

ND

ND

TA98 Positive control -S9 2-Nitrofluorene (2NF) 3 µg/plate; +S9 2-aminoanthracene (2AA) 0.5 µg/plate

TA100 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 9 µg/plate; +S9 2-aminoanthracene (2AA) 1 µg/plate

TA135 Positive control -S9 Sodium Azide 0.5 µg/plate; +S9 2-aminoanthracene (2AA) 2 µg/plate

TA137 Positive control -S9 9-Aminoacridine 80 µg/plate; +S9 2-aminoanthracene (2AA) 3 µg/plate

WP2 Positive control -S9 N-ethyl-N'nitro-N-nitrosaguanidine (ENNG) 2 µg/plate; +S9 2-aminoanthracene (2AA) 20 µg/plate

X - cytotoxic

t: Chemical demonstrated some cytotoxicity, but didn't prevent scoring

ND = Not determined

 

Table 7: Results from Experiment 6

 

S9

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

Number of revertants per plate
 (Individual Plates)

Mean Revertants/Plate
Mean (SD)

TA1537

WP2

DMSO Control

+

16

15

19

17 (2)

39

28

44

37 (8)

Positive Control

+

183

276

180

213 (55)

196

217

202

205 (11)

Test compound 5.0 mg/plate

+

10

16

21

16 (6)

27

26

29

27 (2)

Test compound 3.2 mg/plate

+

12

20

7

13 (7)

30

25

20

25 (5)

Test compound 1.6 mg/plate

+

11

42

26

26 (16)

31

34

32

32 (2)

Test compound 0.8 mg/plate

+

12

17

10

13 (4)

34

33

30

32 (2)

Test compound 0.4 mg/plate

+

39

39

37

38 (1)

35

32

40

35 (4)

Test compound 0.2 mg/plate

+

37

25

37

33 (7)

32

27

29

29 (3)

Test compound 0.1 mg/plate

+

19

26

21

22 (4)

ND

ND

ND

ND

TA137 Positive control +S9 2-aminoanthracene (2AA) 3 µg/plate

WP2 Positive control +S9 2-aminoanthracene (2AA) 20 µg/plate

ND = Not determined

Conclusions:
Under the conditions of the test, the test material was determined to be non-mutagenic in all strains tested. The assays were conducted in the presence and absence of metabolic activation up to cytotoxic concentrations (where appropriate), or up to limit concentrations.
Executive summary:

The mutagenicity of the test material was evaluated in a bacterial reverse mutation assay (Ames test) conducted to a method equivalent to OECD 471 and EU Method B13/14. The study was conducted with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli WP2 in the presence and absence of S9 metabolic activation up to limit concentrations (5 mg/plate) or up to cytotoxic concentrations.

Under the conditions of the test, the test material was determined to be non-mutagenic in all strains tested. The assays were conducted in the presence and absence of metabolic activation up to cytotoxic concentrations (where appropriate), or up to limit concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 1998 and 3 September 1998
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:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
TK +/- locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MRC Cell Mutation Unit, University of Sussex, Brighton, UK
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cell stocks were stored in liquid nitrogen at -196 °C. Cells were routinely cultured in RPMI 1640 medium supplemented with 10 % donor horse serum and 20 mM Hepes buffer (R10) at 37 °C with 5 % CO2 in air. The cells were subcultured accordingly.
- Properly maintained: yes
- Periodically 'cleansed' against high spontaneous background: yes (before stocks were frozen, they were cleansed of homozygous (TK-/-) mutants by culturing in THMG medium for 24 hours).
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The molecular weight of the test material was calculated to be 315, therefore the maximum dose level was 3150 µg/mL equivalent to 10 mM. Test concentrations for the definitive test were based on the results on the range finding tests.
The following concentrations were used in the definitive tests:
Experiment 1
With and without S9: 0, 6, 12, 24, 36 and 42 µg/mL
Experiment 2
With and without S9: 0, 6, 12, 24, 36, 48 and 60 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5E+05 cells/mL
DURATION
- Exposure duration: 3 hours in the presence and absence of metabolic activations, and a continuous 24-hour exposure in the absence of metabolic activation.
- Expression time (cells in growth medium): 2 days
STAIN : 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for roughly two hours. MTT stain assisted in the scoring of viable mutant colonies.
SELECTION AGENT (mutation assays): Selective medium containing 4 µg/mL 5-trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: Performed in duplicate
DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth and cloning efficiency. At the end of the treatment period for each experiment, the test material was removed by centrifugation and the cells were washed twice in R10 medium and resuspended in R20 medium at a cell density of 2E+05 cells/mL. A sample of cells were removed for dilution to 10 cells/mL and plating in non-selective medium (2 cells/well) to determine Day 0 cloning efficiency and viability (% S) immediately after treatment. The % S value was adjusted to account for immediate post-treatment toxicity as a comparison to the vehicle control using the cell count data. A comparison of each treatment % S value to the vehicle control value was performed to give a Relative Survival value (% RS). The remaining cells were incubated and subcultured daily for the required expression period by counting and dilution to 2E+05 cells/mL. The daily cell counts were used to obtain a relative total growth (RTG) value.
Evaluation criteria:
The normal range for mutant frequency per survivor is 10-125E-06 at the conducting laboratory.
Vehicle controls should be within this range. Experiments with values greater than 150E-06 should be repeated. Positive control chemicals should give a significant increase (at least 3-5 fold).
For a significant positive response, 2 or more of the following criteria should be met:
1. A statistically significant increase in mutant frequency
2. A greater than 3-fold increase in the mutant frequency per survivor over the negative control
3. A dose-related increase in the mutant frequency per survivor
4. An increase in the absolute number of mutants
A test material may be reported as equivocal if only one of the above are met.
Statistics:
Plating efficiency was calculated using the zero term of the Poisson distribution method.
P(0) = Number of negative wells/total wells plated
P.E. % = (-ln P(0) x 100 )/ number cells per well
Mutation frequency per survivor was calculated using the following:
MF (per survivor) = [(-lnP(0) selective medium)/cells per well in selective medium]]/surviving fraction in non-selective medium
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Remarks:
Both vehicle control mutant frequencies were within the range of 10 to 125E-06 viable cells typical for the conducting laboratory.
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The preliminary toxicity test was performed with cell cultures at 5E+05 cells/mL, using a 3 hour exposure time both with and without metabolic activation and a continuous 24 hour exposure without metabolic activation. The dose range initially used was 49.22, 98.44, 196.88, 393.75, 787.5, 1575 and 3150 µg/mL.
After the 3 hour exposure period, there were no viable cells at and above 98.44 µg/mL, the toxicity test was repeated for all three exposure conditions using a revised dose range of 3.13 to 75 µg/mL. The cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 10 cells/mL. The cells were plated for survival in selective media (2 cells/well) in 96 well microtitre plates. The plates were incubated at 37 °C for 10-14 days and the wells were scored for colony formation. The toxicity values obtained using the 96 well plate data was adjusted to account for immediate post-treatment toxicity as a comparison to the vehicle control using cell count data.
Precipitation was noted in the highest dose levels. In both the absence and presence of metabolic activation, marked toxicity was observed with the test material at and above 50 µg/mL, with a rapid onset of toxicity in the 3 hour pulse treatments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Previous 20 studies
Without metabolic activation (S9)
Mean MF 1152.7 (SD 641.7) (Range 362.33-3108.19)
With metabolic activation (S9)
Mean MF 969.0 (SD 355.7) (Range 226.33-1559.97)
- Negative (solvent/vehicle) historical control data:
Without metabolic activation (S9)
Mean MF 100.474 (SD 44.2) (Range 31.91-194.07)
With metabolic activation (S9)
Mean MF 103.1 (SD 32.8) (Range 54.34-194.86)

Table 1: Mutant Frequencies from Experiment 1 – without metabolic activation

Treatment
(µg/mL)

Viable

Small Colonies

Large Colonies

Proportion small colony mutants

(µg/mL)

Mutants

MF*

Mutants

MF*

Yv

Nv

Ym

Nm

Ym

Nm

0

142

768

722

768

36.6

694

768

60.0

0.38

6

74

384

357

384

44.3

350

384

56.3

0.44

12

65

384

354

384

45.8

343

384

63.6

0.42

24

86

384

348

384

65.8

356

384

50.6

0.56

36

68

384

357

384

42.1

350

384

53.6

0.44

42

96

384

352

384

62.8

355

384

56.6

0.52

800 EMS

150

384

326

384

174.2

182

384

794.3

0.22

EMS = Ethylmethanesulphonate
* TFT resistant mutants/1E+06 viable cells 2 days after treatment
Yv = Number of wells without colonies, day 2 viability plates
Nv = Number of wells scored, day 2 viability plates
Ym = Number of wells without colonies, mutation plates
Nm = Number of wells scored, mutation plates

 

 

Table 2: Summary of results from Experiment 1 – without metabolic activation

Treatment
(µg/mL)

%S

%RS

%V

RTG

MF

0

68.28

84.40

1.00

100.65

100.65

6

67.24

82.33

1.01

105.06

105.06

12

80.87

88.81

1.07

115.10

115.10

24

71.34

74.81

0.83

121.85

121.85

36

57.33

86.56

0.90

99.93

99.93

42

17.78

69.31

0.28

124.79

124.79

Positive control
(EMS)
800

56.53

47.00

0.39

1202.50

1202.50

%RS = Percent Relative Survival
%S = Percent Survival Day 0
%V = Percent Viability Day 2
RTG = Relative Total Growth
MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment
EMS = Ethylmethanesulphonate
Test for linear trend: Slope 2.887E-07
Variance: 2.911E-013
b²/Sb: 0.286

 

Table 3: Mutant Frequencies from Experiment 1 – with metabolic activation

Treatment
(µg/mL)

Viable

Small Colonies

Large Colonies

Proportion small colony mutants
(µg/mL)

Mutants

MF*

Mutants

MF*

Yv

Nv

Ym

Nm

Ym

Nm

0

153

768

726

768

34.9

685

768

70.9

0.34

6

67

384

359

384

38.6

340

384

69.7

0.36

12

62

384

351

384

49.3

345

384

58.7

0.46

24

72

384

352

384

52.0

343

384

67.5

0.44

36

76

384

357

384

45.0

360

384

39.8

0.53

42

62

384

352

384

47.7

355

384

43.1

0.52

7.5 CP

169

384

270

384

429.1

313

384

249.1

0.62

CP = Cyclophosphamide
* TFT resistant mutants/1E+06 viable cells 2 days after treatment
Yv = Number of wells without colonies, day 2 viability plates
Nv = Number of wells scored, day 2 viability plates
Ym = Number of wells without colonies, mutation plates
Nm = Number of wells scored, mutation plates

 

 

Table 4: Summary of results from Experiment 1 – with metabolic activation

Treatment
(µg/mL)

%S

%RS

%V

RTG

MF

0

73.25

100.00

80.67

1.00

110.11

6

71.88

98.13

87.30

1.11

113.45

12

69.11

94.35

91.18

1.09

113.87

24

73.49

100.32

83.70

0.93

125.94

36

68.85

93.98

81.00

0.97

87.97

42

38.05

51.94

91.18

0.77

94.87

Positive control
(CP)
7.5

44.83

61.20

41.04

0.45

800.90

%RS = Percent Relative Survival
%S = Percent Survival Day 0
%V = Percent Viability Day 2
RTG = Relative Total Growth
MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment
CP = Cyclophosphamide
Test for linear trend: Slope -4.745E-007
Variance: 2.281E-013
b²/Sb: 0.987

 

Table 5: Mutant Frequencies from Experiment 2 – without metabolic activation

Treatment

(µg/mL)

 

Viable

Small Colonies

Large Colonies

Proportion small colony mutants

(µg/mL)

 

Mutants

MF*

Mutants

MF*

Yv

Nv

Ym

Nm

Ym

Nm

0

117

768

723

768

32.1

695

768

53.1

0.38

6

51

384

366

384

23.8

349

384

47.3

0.34

12

52

384

369

384

19.9

346

384

52.1

0.28

24

43

384

365

384

23.2

349

384

43.7

0.35

36

77

384

352

384

54.2

368

384

26.5

0.67

48

113

384

358

384

57.3

363

384

46.0

0.55

60 X

152

384

350

384

100.0

364

384

57.7

0.63

150 EMS

83

384

296

384

169.9

150

384

613.7

0.27

EMS = Ethylmethanesulphonate

X = Excluded from statistical analysis due to toxicity

* TFT resistant mutants/1E+06 viable cells 2 days after treatment

Yv = Number of wells without colonies, day 2 viability plates

Nv = Number of wells scored, day 2 viability plates

Ym = Number of wells without colonies, mutation plates

Nm = Number of wells scored, mutation plates

 

Table 6: Summary of results from Experiment 2 – without metabolic activation

Treatment
(µg/mL)

%S

%RS

%V

RTG

MF

0

73.67

100.00

94.08

1.00

88.66

6

81.76

110.99

100.94

1.11

73.57

12

68.75

93.33

99.97

1.19

74.28

24

29.07

39.47

109.47

1.30

69.22

36

13.41

18.20

80.34

0.64

83.10

48

5.54

7.52

61.16

0.28

106.73

60 X

0.87

1.18

46.34

0.14

163.53

Positive control
(EMS)
150

49.85

67.67

76.59

0.73

1190.43

%RS = Percent Relative Survival
%S = Percent Survival Day 0
%V = Percent Viability Day 2
RTG = Relative Total Growth
MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment
EMS = Ethylmethanesulphonate

X = Treatment excluded from statistical analysis due to toxicity
Test for linear trend: Slope 1.492E-007
Variance: 2.132E-013
b²/Sb: 0.104

 

Table 7: Mutant Frequencies from Experiment 2 – with metabolic activation

Treatment

(µg/mL)

 

Viable

Small Colonies

Large Colonies

Proportion small colony mutants

(µg/mL)

 

Mutants

MF*

Mutants

MF*

Yv

Nv

Ym

Nm

Ym

Nm

0

151

768

689

768

66.7

682

768

73.0

0.48

6

62

384

358

384

38.4

333

384

78.1

0.34

12

61

384

355

384

42.7

337

384

71.0

0.38

24

59

384

350

384

49.5

341

384

63.4

0.44

36

79

384

350

384

58.6

339

384

78.8

0.43

48

126

384

360

384

57.9

344

384

98.7

0.38

60 X

353

384

380

384

124.4

380

384

124.4

0.5

7.5 (CP)

253

384

253

384

1000.0

324

384

407.2

0.69

CP = Cyclophosphamide

X = Excluded from statistical analysis due to toxicity

* TFT resistant mutants/1E+06 viable cells 2 days after treatment

Yv = Number of wells without colonies, day 2 viability plates

Nv = Number of wells scored, day 2 viability plates

Ym = Number of wells without colonies, mutation plates

Nm = Number of wells scored, mutation plates

 

Table 8: Summary of results from Experiment 2 – with metabolic activation

Treatment
(µg/mL)

%S

%RS

%V

RTG

MF

0

93.66

100.00

81.33

1.00

148.71

6

105.22

112.35

91.18

1.06

122.73

12

89.34

95.39

91.99

1.09

119.88

24

78.44

83.75

93.66

1.04

119.48

36

60.85

64.98

79.06

0.85

145.67

48

8.67

9.25

55.72

0.12

163.61

60 X

1.44

1.54

4.21

0.00

250.12

Positive control
(CP)
7.5

24.53

26.19

20.86

0.18

1648.77

%RS = Percent Relative Survival
%S = Percent Survival Day 0
%V = Percent Viability Day 2
RTG = Relative Total Growth
MF = 5-TFT resistant mutants/1E+06 viable cells 2 days after treatment
CP = Cyclophosphamide
Test for linear trend: Slope 2.529E-007
Variance: 3.683E-013
b²/Sb: 0.174

 

Conclusions:
Under the conditions of the test, no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cell were observed and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

The mutagenicity of the test material was evaluated in a study conducted in accordance with OECD 476 and EU Method B.17. Two experiments were conducted up to precipitating cytotoxic concentrations in the presence and absence of metabolic activation for 3-hour and 24-hour exposure times. Under the conditions of the test, no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cell were observed and is therefore considered to be non-mutagenic under the conditions of the test.

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

Additional information

Justification for classification or non-classification

In accordance with Annex I of Regulation EC 1272/2008, the substance does not require classification for germ cell mutagenicity.