Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Genetic toxicity in vitro: Bacterial reverse mutation assay: Key study: OECD guideline 471. GLP study. Under these experimental conditions, no mutagenic activity was revealed.  

 

- Genetic toxicity in vitro: Chromosomal aberrations in mammalian cells: Key study: OECD guideline 473 and EU Method B.10. GLP study.

It is concluded that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

 

- Genetic toxicity in vitro: Gene mutation in mammalian cells: Key study: OECD guideline 476. GLP study. Under these experimental conditions, the test item induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture without metabolic activation, in 5 independent assays following either a short or a continuous treatment. Furthermore, the clear increase in the number of small colonies is rather in favour of a clastogenic activity. In return, neither biologically nor statistically significant mutagenic activity was observed in L5178Y mouse lymphoma cell culture after treatments with the test item in presence of metabolic activation, during two independent assays.

- Genetic toxicity in vitro: Mammalian cell micronucleus test: Key study: OECD guideline 487. GLP study. The test item was considered as not genotoxic in TK6 cells and L5178Y cells. Under these experimental conditions, no difference was observed between results obtained in human lymphoblastoid TK6 cells and mouse lymphoma cells in terms of genotoxicity with a lack of significant increase in the number of micronucleated cells after a short-term treatment without metabolic activation in both cell lines.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK Locus (Trifluorothymidine Resistance)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes.
- Periodically checked for Mycoplasma contamination: yes. Contamination by mycoplasma is checked using Mycoalert mycoplasma detection kit for each batch of the cells. Only the batches, which contain no mycoplasma, are used in the mutagenicity test.
- Periodically "cleansed" against high spontaneous background: yes. In order to maintain a low rate of spontaneous mutation, the L5178Y cells are purged of pre-existing TFT resistant cells according to the following procedure: 10E05 cells per mL are plated in 225 cm2 culture flasks in the RPMI 10 containing THMG (Cole et al., 1986):
9 μg/mL THYMIDINE
15 μg/mL HYPOXANTHINE
0.3 μg/mL METHOTREXATE
22.5 μg/mL GLYCINE
After 24 hours incubation at 37°C, the culture is centrifuged in order to eliminate methotrexate, the pellet is resuspended in RPMI 10 medium containing THG (Thymidine, Hypoxanthine, Glycine) at the same concentrations as those above mentioned. The cells are then incubated at 37°C, humidity near 95% and 5% of CO2 for 48 hours and subcultured 24 hours later at 3.105 cells per mL. After 48 hours of growing in THG medium, the cells are frozen and stored in liquid nitrogen at -196°C.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Without S9 mix (3-hour treatment):
0.15 – 0.12 – 0.1 – 0.08 – 0.06 – 0.05 – 0.04 mM (assay 1)
0.17 – 0.15 – 0.13 – 0.11 – 0.09 – 0.08 mM (assay 2)
0.17 – 0.15 – 0.13 – 0.11 – 0.09 – 0.08 – 0.04 mM (assay 3)

Without S9 mix (24-hour treatment):
0.05 – 0.036 – 0.026 – 0.018 – 0.013 – 0.009 – 0.007 mM (assay 4)
0.040 – 0.036 – 0.033 – 0.030 – 0.027 – 0.025 – 0.012 mM (assay 5)

With S9 mix (3-hour treatment):
0.3 – 0.24 – 0.19 – 0.15 – 0.1 – 0.067 – 0.045 mM (assay 1)
0.6 – 0.5 – 0.41 – 0.34 – 0.29 – 0.24 – 0.2 mM (assay 2)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sodium monobasic phosphate buffer, pH 4
- Justification for choice of solvent/vehicle: Previous solubility assays were performed using 3 different solvents. When it was dissolved at 50 mg/mL in distilled water, the test item induced a homogeneous suspension which rapidly sedimented. In ethanol at 200 mg/mL, the test item induced a non homogeneous pasty suspension. Finally, at 100 mg/mL in DMSO, the test item induced a homogeneous suspension with very small grains that slowly sedimented in the tube. Concurrently, another solvent, i.e. a sodium monobasic phosphate buffer (pH 4) was assayed. It is noteworthy that the test item remains stable for pH < 7 in solution or culture medium.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
For a final volume of 20 mL of RPMI medium with 5% inactivated horse serum containing a suspension of 1 x 10E07 cells in 50 mL sterile centrifugation tubes, an appropriate volume (depending on the solvent used) of the test item in solution at each different concentration was added as well as 1 mL of 150 mM KCl. The treatment was performed under protection from light with stirring by means of a roller (20 rpm) for 3 hours at 37°C. At the end of the treatment period, the cells were washed twice with RPMI 10 medium and counted using a hemocytometer. The cells were then plated in RPMI 20 medium at a mean of 1.6 cells per well (2 plates of 96 wells per dose) for plating efficiency. The remaining cells were diluted at 2x10E05 cells per mL in RPMI 10 and cultured at 37°C.

In order to maintain exponential growth during all the expression time, on day 1 after treatment, the cells were counted and diluted in RPMI 10 medium to provide a cell density of 2x10E05 cells per mL. On day 2 (48 hours after the end of treatment), the cultures were counted again and part of the cells were plated in RPMI 20 at 1.6 cells per well (2 x 96-well microtiter plates per dose) to determine both the relative total growth and the plating efficiency at day 2, while the remaining cells were incubated in selective medium containing TFT (3 μg/mL) at 2 x10E03 cells per well (4*96-well microtiter plates per dose) to determine mutation frequency.

DURATION
- Exposure duration:
Without S9 mix: 3-hour treatment and 24-hour treatment
With S9 mix: 3-hour treatment
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): The cells were incubated at 37°C in a 5% CO2 incubator, with a humidity level close to 95% for 10-14 days, depending on the endpoint to be assessed. After the incubation time, plates containing cells in non-selective medium were scored for negative wells (i.e. wells without any viable colony) for assessing survival rate (RS0) and relative total growth. Plates containing TFT-selective medium were scored independently for "large" and "small" TFT resistant colonies using a binocular magnifying glass for determining mutation frequency

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicates

DETERMINATION OF CYTOTOXICITY
- Method: platting efficiency.
Short treatment (3 hours) and continuous treatment (24 hours)
Prior to the mutagenesis assay, a preliminary test was carried out to determine the cytotoxicity of the test item to L5178Y cells.
Short treatment: For a final volume of 10 mL of RPMI medium with 5% inactivated horse serum containing a suspension of 5 x 10E06 cells in 50 mL sterile centrifugation tubes, an appropriate volume of the test item in solution at each different concentration was added as well as 0.5 mL of 150 mM KCl.
Continuous treatment: For a final volume of 20 mL of RPMI 10 medium containing a suspension of 2.5 x 10E06 cells in 75 cm2 flasks, an appropriate volume of the test item in solution at each different concentration was added (KCl solution is not used in the continuous treatment).

The treatment was performed under the same conditions as in the mutation assay. After the 3-hour or 24-hour treatment, the surviving cells were counted by means of a hemocytometer. Part of the surviving cells were then re-plated in RPMI 20 medium (mean of 1.6 cells per well, 2 x 96-well microtiter plates per dose) and incubated at 37°C, humidity near 95% and 5% CO2. In parallel, the remaining cells were incubated at a density of 2x10E05 cells/mL in RPMI 10 medium for 48 hours. After 24-hour incubation (day 1), cell counting was performed and if necessary, the cell density was adjusted
at 2x10E05 cells/mL. Cell counting was repeated 24 hours later (day 2). The purpose of these cell countings was to detect a possible toxicity of the test item on cell growth (Turner et al., 1984). New plating efficiencies (2 x 96-well microtiter plates per dose at 1.6 cells per well) were concurrently performed in order to calculate the relative total growth (RTG) as described above. After 10 to 14 days, the number of empty wells containing no cell colonies was counted in order to calculate plating efficiencies
Evaluation criteria:
Each well of the mutation plates (in selective medium containing TFT) was scored as containing either a small colony, a large colony or no colony according to the following criteria:
SMALL COLONIES: colonies having a diameter less than 25% of the diameter of the well. A small colony should also have a dense colonal morphology and a clear contour.
LARGE COLONIES: colonies having a diameter greater than 25% of the diameter of the well. A large colony should show less densely packed cells, and blurred contour.
Any well containing one or more small colonies was scored as positive for small colony.
Any well containing one or more large colonies was scored as positive for large colony.
Any well containing a combination of large and small colonies was scored at the same time as large colony and small colony.
An empty well was one which contains no cell growth.
Statistics:
Statistical evaluation of data for the total number of mutants and for small colony mutants was performed using the method proposed by Robinson et al. (1990).
Briefly, the statistical analysis procedure includes the following steps:
• Test for consistency of duplicate cultures at each dose level for a single experiment. The limit is 10.8 times the current heterogeneity factor (H) (10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinite degrees of freedom. If the current heterogeneity factor is higher than 10.8 for survival heterogeneity factor (Hs) or mutation heterogeneity factor (Hm), this dose level is excluded from further consideration.
In case of heterogeneity in the negative control, the statistical evaluation of data is nevertheless performed.
• The new heterogeneity factor (Hexp) is calculated for doses not excluded. If it exceeds the value for that number of degrees of freedom in the experiment, the experiment may be discarded.
• For each dose level, mutant frequency (MF), log mutant frequency (LMF), the variance (V) of LMF and the weight (W) of MF are calculated for non-excluded dose levels (the value of MF could be slightly different in statistically evaluation compared to recapitulative tables because MF is calculated on pooled independent cultures, both series A and B).
• Each treatment is compared with the control by means of one-sided Dunnett's test for multiple comparisons with the same control.
• Test for linear trend of mutant frequency with dose is performed. The slope b and its variance var (b) are calculated to form the test statistic b2/var (b) that should be compared with tabulated critical values of chi2 with 1 degree of freedom.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at the highest concentrations)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at the highest concentrations)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS WITH METABOLIC ACTIVATION (assays 1 and 2)
During two independent assays performed with metabolic activation, no significant increase in the mutation frequency of total induced mutants (small and large colonies) or in the mean number of small colonies and in the mutation frequency of small colony mutants was noted at any concentrations tested in the presence of the test item.

RESULTS WITHOUT METABOLIC ACTIVATION (assays 1 and 2)
In return, in both independent assays performed without metabolic activation using a short-term treatment of 3 hours, statistically and biologically significant increases in the mutation frequency of total induced mutants were noted at 1 or 3 non toxic concentrations tested, with a dose-effect relationship associated with a statistically significant linear trend. The test item was thus considered as mutagenic in the 3-hour treatment without metabolic activation.

EFFECTS OF pH (assays 3, 4 and 5)
3-hour treatment (assay 3)
In order to check the possible implication of a potential degradation of the test item, a 3rd assay under the same experimental conditions but at lower pH-values (in the acceptable range of pH for this test system) was performed. A statistically and biologically significant increase in the mutation frequency of total induced mutants was noted at the highest non-toxic concentration tested of 0.17 mM, associated with a statistically significant linear trend. The mutagenic effect previously observed in two independent assays was confirmed when using experimental conditions ensuring the maintenance of pH<7 all along the treatment period.

24-hour treatment (assay 4)
In order to confirm, or not, these effects when using an extended time of exposure, i.e. 24-hour treatment, a complementary assay under this condition was carried out. A statistically and biologically significant increase in the mutation frequency of total induced mutants was noted at the highest concentration tested of 0.036 mM. However this concentration induced a very strong toxicity, and no concentration inducing an adjusted RTG comprised between 10 and 20% was available. It was thus decided to reiterate this assay but with a narrower range of concentrations in order to definitively conclude.

24-hour treatment (assays 5)
Statistically and biologically significant increases in the mutation frequency of total induced mutants were noted at both two highest concentration tested of 0.030 and 0.027 mM (that induced adjusted RTG acceptable or very close to the lowest accepted threshold for the mutagenicity assessment) with IMFs of +250.1 and +145.9 x10-6 mutants (i.e. clearly higher than +126 x10-6 mutants).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without metabolic activation after a 3-hour treatment, the test item revealed a high level of cytotoxicity in L5178Y cells at the 5 highest concentrations tested from 10 to 0.625 mM with no cell growth. The two inferior concentrations of 0.313 and 0.156 mM induced a very strong level of cytotoxicity with adjusted RTGs of 0.05 and 2.8%, respectively. The concentration of 0.15 was retained as the maximum concentration to be tested for the first mutagenicity test without S9-mix following a 3-hour treatment, and a large and narrowed range of concentrations was tested.

With metabolic activation, a very strong cytotoxicity in L5178Y cells at the 4 highest concentrations tested from 10 to 1.25 mM with no cell growth. The two inferior concentrations of 0.625 and 0.313 mM induced a very strong level of cytotoxicity with adjusted RTGs of 0.05 and 0.5%, respectively. The concentration of 0.156 mM induced a moderate cytotoxicity with an adjusted RTG of 30.9%. The concentration of 0.3 mM was choosen as the maximum concentration to be tested in the first assay with S9-mix.

Without metabolic activation after a 24-hour treatment, all the concentrations tested ranging from 0.156 to 10 mM induced a potent cytotoxicity in L5178Y cells, with no cell growth for concentrations ranging from 10 to 0.625 mM. So, no toxicity data were available at these concentrations. The assay was thus reiterated with lower concentrations (i.e. from 0.15 to 0.04 mM). In the new assay, a potent cytotoxicity was noticed on the whole range of concentrations with 0.1 to 7.4% of adjusted RTG.

Table 1: Assay 1 without S9

 

ASSAY 1

WITHOUT S9

3-Hour

treatment

 

Concentrations in mM

0

0.04

0.05

0.06

0.08

0.10

0.12

0.15

MMS

10 μg/mL

Adjusted RTG

( Relative total growth )

100

135.2

83.6

98.4

50.0

45.6

30.8

14.3

83.6

MUTATION FREQUENCY x 10-6 cells

128.0

107.5

180.4

164.3

227.7

263.1

307.7

307.8

591.3

Mean Induction Ratio

-

0.8

1.4

1.3

1.8

2.1

2.4

2.4

4.6

Induced Mutation Frequency x 10-6 cells

-

-20.5

52.4

36.3

99.7

135.1

179.7

179.8

463.3

Statistical significance (Dunnett's test)

-

N.S.

N.S.

N.S.

<0.05

*

*

<0.05

<0.05

 

Table 2: Assay 2 without S9

 

ASSAY 2

WITHOUT S9

3-Hour

treatment

 

Concentrations in mM

0

0.08

0.09

0.11

0.13

0.15

0.17

MMS

10 μg/mL

Adjusted RTG

( Relative total growth )

100

54.7

47.4

30.0

25.0

14.6

6.1

94.9

MUTATION FREQUENCY x 10-6 cells

114.8

168.0

187.7

216.4

221.0

302.2

**

540.0

Mean Induction Ratio

-

1.5

1.6

1.9

1.9

2.6

**

4.7

Induced Mutation Frequency x 10-6 cells

-

53.3

73.0

101.6

106.3

187.4

**

425.3

Statistical significance (Dunnett's test)

-

N.S.

<0.05

<0.05

<0.05

*

**

<0.05

 

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

**: Adjusted RTG being inferior to the limits accepted for mutagenicity analysis, this concentration was not taken into account in the interpretation of the results

 

Table 3: Assay 1 with S9

 

ASSAY 1

WITH S9

3-Hour

treatment

 

Concentrations in mM

0

0.045

0.067

0.10

0.15

0.19

0.24

0.30

CPA

2 μg/mL

Adjusted RTG

( Relative total growth )

100

110.8

109.6

142.0

118.1

95.8

77.6

27.0

92.9

MUTATION FREQUENCY

x 10-6 cells

122.1

117.4

117.1

79.3

92.9

95.3

78.1

146.0

428.6

Mean Induction Ratio

-

1.0

1.0

0.6

0.8

0.8

0.6

1.2

3.5

Induced Mutation Frequency

x 10-6 cells

-

-4.8

-5.1

-42.8

-29.2

-26.9

-44.0

23.9

306.5

Statistical significance (Dunnett's test)

-

N.S.

N.S.

N.S.

*

*

*

N.S.

<0.05

Table 4: Assay 2 with S9

ASSAY 2

WITH S9

3-Hour

treatment

 

Concentrations in mM

0

0.20

0.24

0.29

0.34

0.41

0.50

0.60

CPA

2 μg/mL

Adjusted RTG

( Relative total growth )

100

46.8

29.4

28.0

2.4

1.8

0.8

0.5

95.8

MUTATION FREQUENCY x 10-6 cells

154.2

219.1

230.9

172.8

185.7

**

**

**

690.9

Mean Induction Ratio

-

1.4

1.5

1.1

1.2

**

**

**

4.5

Induced Mutation Frequency x 10-6 cells

-

64.9

76.7

18.6

31.5

**

**

**

536.7

Statistical significance (Dunnett's test)

-

N.S.

N.S.

N.S.

*

**

**

**

<0.05

 N.S.: Not statistically significant (with a = 0.05 critical value); CPA: cyclophosphamide

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

**: Adjusted RTG being inferior to the limits accepted for mutagenicity analysis, this concentration was not taken into account in the interpretation of the results

Table 5: Assay 3 without S9 (3 -hour treatment, pH < 7)

ASSAY 3

WITHOUT S9

3-Hour

treatment

 

Concentrations in mM

0

0.04

0.08

0.09

0.11

0.13

0.15

0.17

MMS 10 μg/mL

Adjusted RTG

(Relative total growth )

100

96.0

68.1

62.7

57.4

39.5

33.9

22.2

60.8

MUTATION FREQUENCY x 10-6 cells

137.8

146.7

149.3

150.7

181.6

242.0

215.3

336.5

765.3

Mean Induction Ratio

-

1.1

1.1

1.1

1.3

1.8

1.6

2.4

5.6

Induced Mutation Frequency x 10-6 cells

-

8.9

11.5

13.0

43.8

104.2

77.5

198.7

627.5

Statistical significance (Dunnett's test)

-

*

N.S.

N.S.

*

<0.05

N.S.

<0.05

<0.05

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

Table 6: Assay 4 without S9 (24 -hour treatment, pH < 7)

ASSAY 4

WITHOUT S9

24-Hour

treatment

 

Concentrations in mM

0

0.007

0.009

0.013

0.018

0.026

0.036

MMS

2 μg/mL

Adjusted RTG

(Relative total growth )

100

118.0

120.7

117.3

90.6

57.4

7.5

127.1

MUTATION FREQUENCY x 10-6 cells

185.9

189.5

152.7

160.8

206.2

261.4

513.6

656.3

Mean Induction Ratio

-

1.0

0.8

0.9

1.1

1.4

2.8

3.5

Induced Mutation Frequency x 10-6 cells

-

3.6

-33.2

-25.1

20.3

75.5

327.7

470.4

Statistical significance (Dunnett's test)

-

N.S.

N.S.

N.S.

N.S.

N.S.

<0.05

<0.05

 

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

Table 7: Assay 5 without S9 (24 -hour treatment, pH < 7)

ASSAY 5

WITHOUT S9

24-Hour

treatment

 

Concentrations in mM

0

0.012

0.025

0.027

0.030

0.033

MMS

2 μg/mL

Adjusted RTG

(Relative total growth )

100

66.8

27.6

22.2

9.8

6.2

184.5

MUTATION FREQUENCY x 10-6 cells

127.2

215.1

218.8

273.2

377.3

**

365.2

Mean Induction Ratio

-

1.7

1.7

2.1

3.0

**

2.9

Induced Mutation Frequency x 10-6 cells

-

87.9

91.5

145.9

250.1

**

237.9

Statistical significance (Dunnett's test)

-

<0.05

<0.05

*

<0.05

**

<0.05

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

Conclusions:
Under these experimental conditions, the test item induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture without metabolic activation, in 5 independent assays following either a short or a continuous treatment. Furthermore, the clear increase in the number of small colonies is rather in favour of a clastogenic activity. In return, neither biologically nor statistically significant mutagenic activity was observed in L5178Y mouse lymphoma cell culture after treatments with the test item in presence of metabolic activation, during two independent assays.
Executive summary:

The search for any mutagenic activity of the test item was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 476, in 2 independent assays with metabolic activation, 3 independent assays without metabolic activation following a short treatment and 2 independent assays without metabolic activation following a continuous treatment. The acceptance criteria for the assay were fulfilled. The current study was considered as valid. Under these experimental conditions, the test item induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture without metabolic activation, in 5 independent assays following either a short or a continuous treatment. Furthermore, the clear increase in the number of small colonies is rather in favour of a clastogenic activity. In return, neither biologically nor statistically significant mutagenic activity was observed in L5178Y mouse lymphoma cell culture after treatments with the test item in presence of metabolic activation, during two independent assays.

Endpoint:
in vitro gene mutation study in bacteria
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sodium monobasic phosphate buffer, pH 4
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9-mix: TA1535, TA100
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9-mix: TA1537
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9-mix: TA98
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9-mix: TA102
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
With S9-mix: TA1535, TA1537, TA98, TA100
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9-mix: TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Without metabolic activation: 0.1 mL of a bacterial suspension from a culture agitated overnight at 37°C and 0.1 mL of the test item at the relevant initial concentration were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. Three plates were used per dose. The plates were then incubated at 37°C for approximately 48 h. At the end of the expression time, colonies of revertants were counted for each plate.

With metabolic activation: The method was the same as the one described without metabolic activation except that immediately before spreading
in the plates, 0.5 mL of the S9 mix metabolic activation system was added in soft agar.

Repeat test
Without metabolic activation:
The test was subsequently repeated in an independent assay. The same method was used but the test dose range was modified, depending on the results obtained during the first test.

With metabolic activation:
According to the results obtained in the first assay in the presence of metabolic activation, the second assay can either be performed using a standard plate-incorporation technique, or be extended by use of the pre-incubation modification of the assay. As a negative response was observed in the first assay, the method used in a second assay was the pre-incubation test. On a technical point of view, this method is the same as the one using agar plate incorporation with, however, the following modifications: The following solutions were added in this order: the bacterial strain to be tested (100 μL), S9-mix (500 μL) and at the end the test item solution (100 μL as an aqueous solvent was used). The mixture was preincubated with stirring at 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.

NUMBER OF REPLICATIONS: Two independent assays using triplicates

DETERMINATION OF CYTOTOXICITY
- Method: Examination of the background lawn.



Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Moderate to acceptable precipitates were observed in all strains at the highest dose studied, both with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The examination of the background lawn demonstrated that the test substance induced no toxicity.

In two independent assays performed either with or without metabolic activation (the second assay with S9 -mix was performed according to the pre-incubation protocol), no statistically or biologically significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested.

Table 1: Assay 1

 

TA 1535

TA 1537

TA 98

TA 100

TA102

DOSE μg/plate

revertants /plate

Induction Ratio (a)

DOSE μg/plate

revertants /plate

Induction Ratio (a)

DOSE

μg/plate

revertants /plate

Induction Ratio (a)

DOSE μg/plate

revertants /plate

Induction Ratio (a)

DOSE

μg/plate

revertants /plate

Induction Ratio (a)

Positive control

(b)

650.7

74.8

(b)

738.7

113.6

(b)

624.0

42.2

(b)

1189.3

11.9

(b)

1968.0

8.7

TEST ITEM

WITHOUT

S9-mix

 

0

8.7

-

0

6.5

-

0

14.8

-

0

100.3

-

0

226.3

-

50

11.0

1.3

50

8.0

1.2

50

16.0

1.1

50

89.3

0.9

50

240.0

1.1

150

11.3

1.3

150

5.7

0.9

150

16.7

1.1

150

88.3

0.9

150

256.7

1.1

500

11.3

1.3

500

7.3

1.1

500

11.0

0.7

500

77.3

0.8

500

237.3

1.0

1500

7.3

0.8

1500

4.7

0.7

1500

13.0

0.9

1500

64.7

0.6

1500

181.3

0.8

5000

11.3

1.3

5000

13.3

2.0

3000

6.3

0.4

5000

55.7

0.6

3000

93.0

0.4

-

-

-

-

-

-

5000

17.3

1.2

-

-

-

5000

112.0

0.5

 

 

Positive control

(c)

826.0

62.6

(c)

664.0

97.6

(c)

3634.7

114.3

(c)

3274.7

29.1

(c)

1050.7

3.0

TEST ITEM

WITH

S9-mix

WITHOUT

PRE-INCUBATION

 

0

13.2

-

0

6.8

-

0

31.8

-

0

112.5

-

0

348.7

-

50

8.7

0.7

50

11.3

1.7

50

27.0

0.8

50

101.0

0.9

50

282.7

0.8

150

10.0

0.8

150

6.7

1.0

150

18.7

0.6

150

90.3

0.8

150

326.0

0.9

500

9.0

0.7

500

10.0

1.5

500

18.7

0.6

500

92.3

0.8

500

362.7

1.0

1500

12.3

0.9

1500

6.0

0.9

1500

21.0

0.7

1500

97.3

0.9

1500

310.7

0.9

5000

15.3

1.2

5000

11.7

1.7

3000

7.0

0.2

5000

79.3

0.7

3000

292.0

0.8

-

-

-

-

-

-

5000

14.7

0.5

-

-

-

5000

222.0

0.6

(a) Induction Ratio = number of revertants in the treated / number of revertants in the control

Reference positive compounds (μg/plate):

(b) TA1535 and TA100: Sodium azidew 1; TA1537: 9-amino-acridined 50; TA98: 2-nitrofluorened 2; TA102: Mitomycin Cw 0.125

(c) TA1535, TA1537, TA98, TA100: 2-anthramined 2; TA102: benzo(a)pyrened 2

Solvents used for positive controls: d DMSO; w distilled water

Table 2: Assay 2

 

TA 1535

TA 1537

TA 98

TA 100

TA102

DOSE

μg/plate

revertants /plate

Induction Ratio (a)

DOSE

μg/plate

revertants /plate

Induction Ratio (a)

DOSE

μg/plate

revertants /plate

Induction Ratio (a)

DOSE

μg/plate

revertants /plate

Induction Ratio (a)

DOSE

μg/plate

revertants /plate

Induction Ratio (a)

Positive control

(b)

786.7

68.4

(b)

806.7

118.6

(b)

369.3

25.0

(b)

690.7

6.4

(b)

1886.7

6.9

TEST ITEM

WITHOUT

S9-mix

 

0

11.5

-

0

6.8

-

0

14.8

-

0

108.3

-

0

271.7

-

50

12.3

1.1

50

4.7

0.7

50

16.3

1.1

50

96.7

0.9

50

234.0

0.9

150

9.7

0.8

150

5.3

0.8

150

16.7

1.1

150

100.7

0.9

150

275.3

1.0

500

13.3

1.2

500

6.7

1.0

500

16.3

1.1

500

110.0

1.0

500

283.3

1.0

1500

8.3

0.7

1500

4.7

0.7

1500

10.7

0.7

1500

67.3

0.6

1500

218.7

0.8

5000

7.3

0.6

5000

5.0

0.7

3000

12.7

0.9

5000

71.7

0.7

3000

180.7

0.7

-

-

-

-

-

-

5000

11.0

0.7

-

-

-

5000

206.0

0.8

 

 

Positive control

 

(c)

246.0

24.6

(c)

225.3

24.5

(c)

1696.0

66.0

(c)

1925.3

17.9

(c)

1168.0

3.0

TEST ITEM

WITH

S9-mix

WITH

PRE-INCUBATION

 

0

10.0

-

0

9.2

-

0

25.7

-

0

107.5

-

0

387.7

-

50

7.7

0.8

50

7.0

0.8

50

23.3

0.9

50

100.3

0.9

50

299.3

0.8

150

13.3

1.3

150

6.3

0.7

150

20.3

0.8

150

93.7

0.9

150

298.0

0.8

500

10.7

1.1

500

4.0

0.4

500

28.0

1.1

500

126.0

1.2

500

313.3

0.8

1500

6.7

0.7

1500

7.3

0.8

1500

12.3

0.5

1500

73.0

0.7

1500

235.3

0.6

5000

8.7

0.9

5000

8.7

0.9

3000

14.3

0.6

5000

99.3

0.9

3000

250.0

0.6

-

-

-

-

-

-

5000

17.3

0.7

-

-

-

5000

258.0

0.7

(a) Induction Ratio = number of revertants in the treated / number of revertants in the control

Reference positive compounds (μg/plate):

(b) TA1535 and TA100: Sodium azidew 1; TA1537: 9-amino-acridined 50; TA98: 2-nitrofluorened 2; TA102: Mitomycin Cw 0.125

(c) TA1535, TA1537, TA98, TA100: 2-anthramined 1; TA102: benzo(a)pyrened 2

Solvents used for positive controls: d DMSO; w distilled water

Conclusions:
Under these experimental conditions, no mutagenic activity was revealed.
Executive summary:

The mutagenic activity of the test substance was assessed by means of the Ames´s test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in two independent assays. The validity criteria for the assay were fulfilled. The study was thus considered as valid. Under these experimental conditions, no mutagenic activity was revealed.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Blood taken from healthy male non-smoking donors was pooled and diluted with tissue culture medium. The cultures were incubated in the presence of PHA before being treated with the test substance.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
First test
In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 24.88, 320.01 and 533.35 μg/mL.
In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 24.88, 192 and 320.01 μg/mL.

Second test
In the absence of S9 mix - 21 hour continuous treatment: 2.5, 41.47 and 320.01 μg/mL.
In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 2.5, 10 and 41.47 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37°C in a 5% CO2 atmosphere for approximately 48 hours. All cultures were centrifuged and resuspended in fresh (pH adjusted) medium just before treatment.

DURATION
- Exposure duration:
First test
In the absence of S9 mix - 3 hour treatment, 18 hour recovery
In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery

Second test
In the absence of S9 mix - 21 hour continuous treatment
In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN (for cytogenetic assays): The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: One hundred metaphase figures were examined from each culture.

DETERMINATION OF CYTOTOXICITY
- Method:
The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity. From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the vehicle control value was the highest concentration selected for metaphase analysis.
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
Key result
Species / strain:
lymphocytes: Blood taken from healthy male non-smoking donors was pooled and diluted with tissue culture medium. The cultures were incubated in the presence of PHA before being treated with the test substance.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
First test:
In the absence of S9 mix, precipitation was observed at concentrations from 320.01 μg/mL.
In the absence of S9 mix, precipitation was observed at concentrations from 192 μg/mL.

Second test:
In the absence of S9 mix, precipitation was observed at concentrations from 69.12 μg/mL.
In the absence of S9 mix, precipitation was observed at concentrations from 115.2 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
All mean values for the vehicle control (water) were within laboratory historical control range, when taken at the 99% confidence limit.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First test:
In the absence of S9 mix following 3 hour treatment, the test substance caused a reduction in the mitotic index to 60% of the vehicle control value at 888.91μg/mL.

In the presence of S9 mix (2% v/v final concentration) following 3 hour treatment, the test substance caused a reduction in the mitotic index to 55% of the vehicle control value at 888.91μg/mL.

Second test:
In the absence of S9 mix following 21 hour continuous treatment, the test substance caused a reduction in the mitotic index to 51% of the vehicle control value at 320.01 μg/mL.

In the presence of S9 mix (5% v/v final concentration) following 3 hour treatment, the test substance caused a reduction in the mitotic index to 80% of the vehicle control value at 1481.52 μg/mL, the highest tested concentration. However, as clear cytotoxicity was observed at lower non-precipitating concentrations, where a reduction in the mitotic index to 51% of the vehicle control value was observed at 41.47 μg/mL.

First test:

In the absence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations 24.88 or 533.35 μg/mL, when compared with the vehicle control. However, at both concentrations,

the mean values (including gaps only) were outside the laboratory historical control range. At the intermediate concentration of 320.01 μg/mL, statistically significant increases (p<0.01: including and excluding gaps) in the proportion of metaphase figures containing chromosomal aberrations were observed. These increases exceeded the laboratory historical control range. As these increases were at a precipitating concentration with no associated evidence of a concentration-response at the higher, more cytotoxic concentration, this

response was considered of little or no biological relevance.

In the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the vehicle control. All mean values were within the laboratory historical control range, when taken at the 99% confidence limit, with the exception of 320.01 μg/mL concentration (excluding gaps only), which exceeded historical confidence limits, yet was still within the laboratory historical control range.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Second test:

In both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the vehicle control. All mean values for the vehicle control (water), and all test substance treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Conclusions:
It is concluded that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in human lymphocytes cultured in vitro. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Vehicle and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In order to determine the toxicity of the test substance to cultured human lymphocytes, the mitotic index was assessed for all cultures treated with the test substance and the vehicle control, water (purified in-house by reverse osmosis). Justification for concentration selection was based on microscopically visible precipitate, as concentrations beyond the limit of solubility were selected where precipitate did not interfere with analysis. Otherwise, selection was determined by cytotoxicity.

On the basis of these data, the following concentrations were selected for metaphase analysis:

First test

In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 24.88, 320.01 and 533.35 μg/mL.

In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 24.88, 192 and 320.01 μg/mL.

In the absence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at either 24.88 or 533.35 μg/mL, when compared with the vehicle control. However, at both concentrations, the mean values (including gaps only) were outside the laboratory historical control range. At the intermediate concentration of 320.01 μg/mL, statistically significant increases (p<0.01: including and excluding gaps) in the proportion of metaphase figures containing chromosomal aberrations were observed. These increases exceeded the laboratory historical control range. As these increases were observed at a precipitating concentration with no associated evidence of a concentration-response at the higher, more cytotoxic concentration, this response was considered of little or no biological relevance.

In the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the vehicle control. All mean values were within the

laboratory historical control range, when taken at the 99% confidence limit, with the exception of 320.01 μg/mL concentration (excluding gaps only), which exceeded historical confidence limits, yet was still within the laboratory historical control range.

Second test

In the absence of S9 mix - 21 hour continuous treatment: 2.5, 41.47 and 320.01 μg/mL.

In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 2.5, 10 and 41.47 μg/mL.

In both the absence and presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the vehicle control. All treatment concentrations (including and excluding gaps) were within the laboratory historical control range, when taken at the 99% confidence limit. No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis, in either test.

All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Endpoint:
in vitro cytogenicity / micronucleus study
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
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
without
Test concentrations with justification for top dose:
0.036, 0.050, 0.061, 0.075, 0.10, 0.13, 0.17 and 0.20 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sodium monobasic phosphate buffer (NaH2PO4, pH 4)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: At the treatment day, TK6 and L5178Y cells were suspended at 200 000 and 150 000 cells/mL, respectively, in RPMI 10 medium (containing serum). A volume of 2 mL from this suspension was distributed in each well of 12-wells plate. Cells were thus incubated for 2 hours at 37 °C before treatment, as follows: the test item was added at different concentrations (1 culture per concentration). The plates were gently stirred and then incubated at 37 °C. After a period of 3 hours of treatment, the cells were transferred in 15-mL tubes that are centrifugated for 6 minutes at 1000 rpm, then followed by gentle pouring off to discard the supernatant. The cells were washed 2 times by addition of PBS. After the washing, cells were resuspended in 2 mL of RPMI 10, transferred in a 12-well plate, then were incubated for 1.5 to 2 cell cycles (i.e. around 24 or 21 hours for TK6 and L5178Y cells, respectively). At the end of this period, the cells were transferred into 15-mL tubes. For each culture, the cells were collected by centrifugation for 6 minutes at 1000 rpm and washed twice with PBS. Thereafter, the cells were subjected to hypotonic shock for 4 min using RPMI 1640 containing 1% of pluronic acid and distilled water:RPMI 1640 medium – distilled water (1:1).

DURATION
- Exposure duration: 3 hours

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): 4% dilution of Giemsa reagent in water for 10 minutes.

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: One slide per culture is coded, and the micronuclei of 1000 mononucleated cells per culture are counted (2000 mononucleated cells/concentration).

DETERMINATION OF CYTOTOXICITY
- Method: No preliminary toxicity was performed. Instead, a wide range of concentrations was chosen and the determination of the cytotoxicity was combined to the main assay. Each cell suspension was mixed in Trypan Blue 0.4 % (5:1) and cells were enumerated in a Mallassez cytometer, in order to calculate the population doubling (PD) and the relative PD (RPD).
Evaluation criteria:
The frequency of the number of micronuclei is assessed in mononucleated cells. Micronuclei are identified according to the criteria of Fenech et al. (2000, 2003).
Micronuclei are morphologically identical, but smaller, than nuclei. They also have the following characteristics:
- The diameter of micronuclei usually varies between 1/16th and 1/3rd of the mean diameter of the main nuclei, which corresponds to 1/256th, and 1/9th of the area of one of the main nuclei in a binucleated cell, respectively.
- Micronuclei are non-refractile and they can therefore be readily distinguished from artefact such as staining particles;
- Micronuclei are not linked or connected to the main nuclei;
- Micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary;

Micronuclei usually have the same staining intensity as the main nuclei but occasionally staining may be more intense.
Statistics:
Statistical analysis of the results obtained in the cells treated at each concentration level was performed using the χ2 test in comparison with those in control groups.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the short-term treatment without metabolic activation followed by a 24-hour recovery period in TK6 cells, the test item induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.061 to 0.036 mM. Indeed, 6 to 14 micronucleated mononucleated cells were observed per 2000 cells, vs. 9 in the negative control.

In the short-term treatment without metabolic activation followed by a 21-hour recovery period in L5178Y cells, the test item induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.1 to 0.036 mM. Indeed, 4 to 11 micronucleated mononucleated cells were observed per 2000 cells, vs. 13 in the negative control.

Under these experimental conditions, no difference was observed between results obtained in human lymphoblastoid TK6 cells and mouse lymphoma cells in terms of genotoxicity with a lack of significant increase in the number of micronucleated cells after a short-term treatment without metabolic activation in both cell lines.

The lonely difference between both cell lines was the level of cytotoxicity with a RPD of around 50% at 0.061 mM in TK6 cells and at 0.1 mM in L5178Y cells.

In the toxicity assay in TK6 cells, a very strong cytotoxicity was observed at the 5 highest concentrations ranging from 0.2 to 0.075 mM, with RPD ranging from –101 to 4.4%. The immediately lower concentration of 0.061 mM induced a moderate and acceptable level of toxicity with a RPD of 44.8%, i.e. 55.2% of cytostasis. Under these conditions, the concentration of 0.061 mM was retained as the maximum concentration to be tested in the main mutagenicity assay in TK6 human lymphoblastoid cells.

In the toxicity assay in L5178Y cells, a very strong cytotoxicity was observed at the 3 highest concentrations ranging from 0.2 to 0.13 mM, with RPD ranging from –12.3 to 1.6%. The immediately lower concentration of 0.1 mM induced a moderate and acceptable level of toxicity with a RPD of 48.7%, i.e.
51.3% of cytostasis. Under these conditions, the concentration of 0.10 mM was retained as the maximum concentration to be tested in the main mutagenicity assay in L5178Y mouse lymphoma cells.

Table 1 In vitro mammalian cell micronucleus test

 

 

 

 

Conc. in mM

TK6 CELLS

 

L5178Y CELLS

 

Assay without metabolic activation with 3-hour treatment with 24-27-hour

recovery period

(assay S9- 3h/+24-27h)

 

Assay without metabolic activation with 4-hour treatment with 21-24-hour

recovery period

(assay S9- 3h/+21-24h)

 

Relative Population Doubling (%)

Cytostase (%)

Number of micronucleated cells per 2000 mononucleated cells

p

Relative Population Doubling (%)

Cytostase (%)

Number of micronucleated cells per 2000 mononucleated cells

p

Negative control

0

-

-

9

-

-

-

13

-

Mitomycin C

*

-22.0

122.0

138

<0.001

103.7

-3.7

210

<0.001

Test item

0.1

 

 

 

 

48.7

51.3

11

N.S.

0.075

 

 

 

 

53.1

46.9

7

N.S.

0.06

44.8

55.2

6

N.S.

62.6

37.4

7

N.S.

0.05

70.0

30.0

14

N.S.

98.6

1.4

4

N.S.

0.04

103.4

-3.4

9

N.S.

91.5

8.5

9

N.S.

*: 0.5 and 0.2 μg/mL in TK6 and L5178Y cells, respectively

N.S. not statistically significant

 

Conclusions:
The test item was considered as not genotoxic in TK6 cells and L5178Y cells. Under these experimental conditions, no difference was observed between results obtained in human lymphoblastoid TK6 cells and mouse lymphoma cells in terms of genotoxicity with a lack of significant increase in the number of micronucleated cells after a short-term treatment without metabolic activation in both cell lines.
Executive summary:

The genotoxic activity of the test item was assessed by means of the in vitro micronucleus test in TK6 lymphoblastoid human cells and L5178Y mouse lymphoma cells treated in absence of metabolic activation, with a short-term treatment of 3 hours. Under these experimental conditions, no genotoxic activity was revealed in absence of metabolic activation, with a short-term treatment. Under these experimental conditions, no difference was observed between results obtained in human lymphoblastoid TK6 cells and mouse lymphoma cells in terms of genotoxicity with a lack of significant increase in the number of micronucleated cells after a short-term treatment without metabolic activation in both cell lines. The lonely difference between both cell lines was the level of cytotoxicity with a RPD of around 50% at 0.061 mM in TK6 cells and at 0.1 mM in L5178Y cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

- Genetic toxicity in vitro: Bacterial reverse mutation assay:

Key study: OECD guideline 471. GLP study.

Under these experimental conditions, no mutagenic activity was revealed. 

 

- Genetic toxicity in vitro: Chromosomal aberrations in mammalian cells:

Key study: OECD guideline 473 and EU Method B.10. GLP study.

It is concluded that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

 

- Genetic toxicity in vitro: Gene mutation in mammalian cells:

Key study: OECD guideline 476. GLP study.

Under these experimental conditions, the test item induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture without metabolic activation, in 5 independent assays following either a short or a continuous treatment. Furthermore, the clear increase in the number of small colonies is rather in favour of a clastogenic activity. In return, neither biologically nor statistically significant mutagenic activity was observed in L5178Y mouse lymphoma cell culture after treatments with the test item in presence of metabolic activation, during two independent assays.

 

- Genetic toxicity in vitro: Mammalian cell micronucleus test:

Key study: OECD guideline 487. GLP study.

The test item was considered as not genotoxic in TK6 cells and L5178Y cells. Under these experimental conditions, no difference was observed between results obtained in human lymphoblastoid TK6 cells and mouse lymphoma cells in terms of genotoxicity with a lack of significant increase in the number of micronucleated cells after a short-term treatment without metabolic activation in both cell lines.

Justification for selection of genetic toxicity endpoint

Only in vitro studies are available. Negative results were obtained in the in vitro Ames test and chromosome aberrations assay. Negative results were obtained in the in vitro mammalian cell gene mutation assay with metabolic activation. Positive results were obtained in the in vitro mammalian cell gene mutation assay without metabolic activation (included under endpoint selection).

Justification for classification or non-classification

Based on the results obtained from the in vitro studies, the substance is not classified for mutagenicity.