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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2013 - 15 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver)
Test concentrations with justification for top dose:
3h, ± S9 mix: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 µg/mL
3h, + S9 mix: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 µg/mL
24h, - S9 mix. 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was non-soluble in Distilled water or Dimethyl sulfoxide at 500 mg/mL concentration. However, a proper formulation could be made at the same concentration using Acetone or Ethanol (96%) as vehicle. Due to the better biocompatibility to the test system, Ethanol (96%) was selected for vehicle of the study.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(Untreated)
Negative solvent / vehicle controls:
yes
Remarks:
(Ethanol 96%, DMSO)
Remarks:
Test item vehicle: Ethanol (10 µL/mL), positive control vehicle: DMSO (10 µL/mL)
Positive controls:
yes
Remarks:
(Without metabolic activation)
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.15 µg/mL (3h treatment), 0.10 µg/mL (24h treatment), in DMSO
Positive controls:
yes
Remarks:
(With metabolic activation)
Positive control substance:
cyclophosphamide
Remarks:
4 µg/mL in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (RPMI-5 medium)

DURATION
- Exposure duration: 3h (with and without S9-mix), 24h (without S9-mix)
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 3 days
- Selection time (plating for -trifluorothymidine (TFT) resistance): Two weeks

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by relative survival

OTHER EXAMINATIONS:
- Determination of Survival or Viability:
After the exposure period and the expression time, cells were also diluted to be plated for survival and viability respectively. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Parameter calculated:
- Relative survival
- Mutant Frequency
- Small and large colony mutant frequencies

Others:
The number of viable cells in the individual samples was counted manually using a haemocytometer.
Measurement of pH and osmolality was performed after the treatment period.
Evaluation criteria:
The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore 2006):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(Assay 1: ±S9mix, 3h: >= 100 µg/L; Assay 3: +S9mix, 3h: >= 95 µg/L; -S9mix, 24h: >= 55 µg/L)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH after treatment.
- Effects of osmolality: There were no large changes in osmolality after treatment.
- Water solubility: No insolubility was detected in the final treatment medium at the end of the treatment (although discoloured medium was detected at some concentrations)

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed to select dose levels for the main assays. During the preliminary test, a 3 hour treatment in the presence and absence of S9-mix and a 24 hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity. The highest concentration tested in the preliminary experiment was 2500 μg/mL. Insolubility and cytotoxicity were observed in the preliminary experiment. Concentrations for the main experiments were selected to cover the range from cytotoxicity to little or no cytotoxicity according to the instructions of the relevant OECD guideline.

ADDITIONAL INFORMATION:
No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations.
No significant dose-response to the treatment was indicated by the linear trend analysis.

OBSERVATION:
During Assay 2, a contamination was detected in the samples, therefore it was considered invalid and terminated. An additional experiment (Assay 3) was performed using the same experimental conditions as in Assay 2 to provide valid and interpretable data.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Mutagenicity Results of Assay 1

S9 mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)♦

Mutation frequency

+

3

140 µg/mL

ND

ND

ND

ND

ND

130 µg/mL

ND

ND

ND

ND

ND

120 µg/mL

ND

ND

ND

ND

ND

110 µg/mL

ND

ND

ND

ND

ND

100 µg/mL

NE

NE

NE

NE

NE

90 μg/mL

642/768

64/768

62/768

0.047

125.0

80 µg/mL

640/768

71/768

57/768

0.056

124.5

60 µg/mL

640/768

74/768

54/768

0.087

122.7

40 µg/mL

641/768

74/768

53/768

0.009

128.8

20 µg/mL

652/768

68/768

48/768

0.108

143.1

10 µg/mL

654/768

65/768

49/768

0.785

105.8

5 µg/mL

636/768

75/768

57/768

0.003

133.5

Vehicle control

640/768

71/768

57/768

--

131.8

Vehicle control for CP

645/768

64/768

59/768

--

118.3

Untreated control

642/768

64/768

62/768

--

135.2

Positive control
(CP: 4 μg/mL)

277/768

204/768

287/768

♦♦
3.55E-13

1076.7*

In linear trend analysis β2/var (β) = 0.002, not significant.

S9 mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn) ♦

Mutation frequency

-

3

140 µg/mL

ND

ND

ND

ND

ND

130 µg/mL

ND

ND

ND

ND

ND

120 µg/mL

ND

ND

ND

ND

ND

110 µg/mL

ND

ND

ND

ND

ND

100 µg/mL

ND

ND

ND

ND

ND

90 μg/mL

671/768

38/768

59/768

0.710

111.7

80 µg/mL

672/768

42/768

54/768

0.229

100.8

60 µg/mL

652/768

28/768

88/768

0.818

112.6

40 µg/mL

683/768

38/768

47/768

0.139

79.0

20 µg/mL

673/768

39/768

56/768

<0.001

87.4

10 µg/mL

679/768

47/768

42/768

0.094

80.6

5 µg/mL

672/768

36/768

60/768

0.254

101.5

Vehicle control

672/768

52/768

44/768

--

88.0

Vehicle control for NQO

670/768

39/768

59/768

--

100.8

Untreated control

653/768

40/768

75/768

--

119.8

Positive control
(NQO: 0.15 μg/mL)

407/768

199/768

162/768

♦♦
2.54E-12

749.6*

In linear trend analysis β2/var (β) =1.199, not significant.

Mutagenicity Results of Assay 3

S9-mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)♦

Mutation frequency

+

3

110 µg/mL

ND

ND

ND

ND

ND

100 µg/mL

NE

NE

NE

NE

NE

95 µg/mL

NE

NE

NE

NE

NE

90 μg/mL

NE

NE

NE

NE

NE

85 µg/mL

646/768

67/768

55/768

0.014

120.7

80 µg/mL

655/768

52/768

61/768

<0.001

123.5

70 µg/mL

646/768

59/768

63/768

<0.001

124.2

60 µg/mL

646/768

60/768

62/768

0.276

109.1

40 µg/mL

661/768

50/768

57/768

0.464

103.9

20 µg/mL

661/768

51/768

56/768

0.531

103.2

10 µg/mL

667/768

51/768

50/768

1.148

94.2

62.5 µg/mL

671/768

51/768

46/768

1.564

88.9

Vehicle control

649/768

57/768

62/768

--

124.4

Vehicle control for CP

654/768

66/768

48/768

--

105.1

Untreated control

658/768

57/768

53/768

--

100.4

Positive control
(CP: 4 μg/mL)

183/768

240/768

345/768

♦♦
1.58E-15

1571.2*

In linear trend analysis β2/var (β) = 1.619, not significant.

S9-mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn) ♦

Mutation frequency

-

24

80 µg/mL

ND

ND

ND

ND

ND

75 µg/mL

ND

ND

ND

ND

ND

70 µg/mL

ND

ND

ND

ND

ND

65 µg/mL

ND

ND

ND

ND

ND

60 µg/mL

ND

ND

ND

ND

ND

55 µg/mL

NE

NE

NE

NE

NE

50 μg/mL

684/768

44/768

40/768

0.270

80.2

45 μg/mL

678/768

51/768

39/768

0.245

80.9

40 μg/mL

663/768

51/768

54/768

0.043

97.5

30 μg/mL

672/768

68/768

28/768

0.058

86.7

20 μg/mL

683/768

47/768

38/768

0.049

87.0

10 μg/mL

670/768

49/768

49/768

0.001

93.2

5 μg/mL

668/768

65/768

35/768

<0.001

91.9

Vehicle control

674/768

55/768

39/768

--

92.4

Vehicle control for NQO

678/768

65/768

25/768

--

87.0

Untreated control

681/768

53/768

34/768

--

82.1

Positive control
(NQO: 0.1 μg/mL)

384/768

269/768

115/768

♦♦
2.27E-13

830.8*

In linear trend analysis β2/var (β) =0.359, not significant.

* = Statistically significant.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.48(at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05.

Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope

+ = in the presence of S9-mix

- = in the absence of S9-mix

Vehicle control = Ethanol

Vehicle control for CP = DMSO

CP = Cyclophosphamide

Vehicle control for NQO = DMSO

NQO = 4-Nitroquinoline-N-oxide

DMSO = Dimethyl sulfoxide

ND = no data (No cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period.)

NE = not evaluated (Due to the high level of cytotoxicity)

Note: Mutation frequency refers to 106viable cells

The overall study was considered to be valid:

The spontaneous mutation frequency of the negative (vehicle) control was in the recommended range (50-170 mutants per 106 viable cells) in each experiment.

The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.

The plating efficiencies for the negative (vehicle) control at the end of the expression period (PEviability) were within the acceptable range (65-120 %) in all assays.

The number of test concentrations evaluated for each treatment was at least seven in each case,.

The tested concentration range in the study was considered to be adequate as the highest examined concentration produced approximately 80-90% toxicity (i.e. approximately 10-20% relative survival or relative total growth) and lower test concentrations were evenly spaced by a factor of no more than two. For determination of the cytotoxicity, the relative total growth was taken into consideration, as the test item showed late phase cytotoxicity during the expression period.

Applicant's summary and conclusion

Conclusions:
No mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Ethanol was used as vehicle of the test item in this study. The test item was examined up to 2500 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 1, 3-hour treatment without metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 3-hour treatment with metabolic activation: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 24-hour treatment without metabolic activation: 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 μg/mL

No insolubility was detected in the final treatment medium at the end of the treatment. There were no large changes in pH or osmolality after treatment. Cytotoxicity was observed in both assays (Assay 1: ± S9mix, 3h: >= 100 µg/L; Assay 3: + S9mix, 3h: >= 95 µg/L; - S9mix, 24h: >= 55 µg/L). No biologically relevant or statistically significant increase in the mutation frequency was observed. No dose response to the treatment was indicated by the linear trend analysis. The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases and the study was considered to be valid. In conclusion, no mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.