Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 April 2011 - 28 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(21 July 1997)
Deviations:
no
Qualifier:
according to
Guideline:
other: UKEMS Guidelines (1990)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Date of inspection: 8 March 2011)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ammonium perrhenate

- Substance type: White powder

- Physical state: Powder

- Analytical purity: 100%

- Lot/batch No.: 11311

- Expiration date of the lot/batch: 28 March 2012

- Stability under test conditions: Not tested.

- Storage condition of test material: Stored at 15 - 25°C, protected from light.

Method

Target gene:
tk (thymidine kinase) gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 growth media

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: yes

- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver post mitochondrial fraction (S 9) obtained from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Range-finding test: 83.84, 167.7, 335.4, 670.8, 1342, 2683 µg/mL
Experiment 1: 400, 800, 1200, 1600, 2000, 2400, 2683 µg/mL.
Experiment 2: 500, 1000, 1400, 1700, 2000, 2300, 2683 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Purified water

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Ammonium perrhenate was soluble in water for irrigation (purified water) at a concentration of at least 32.15 mg/mL (the solubility limit in culture medium was at least 3215 µg/mL, and no precipitation was observed at this concentration approximately 24 hours after test article addition).
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(Concurrent vehicle)
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10 fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation system

Migrated to IUCLID6: 15, 20 µg/mL
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation system

Migrated to IUCLID6: 2, 3 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In RPMI 1640 growth media

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days
- Incubation time for viability test: 8 days
- Incubation time for TFT (5 trifluorothymidine) resistance: 12 days

SELECTION AGENT (mutation assays): 5 trifluorothymidine

NUMBER OF REPLICATIONS: Duplicate (single cultures only for positive control)

NUMBER OF CELLS EVALUATED: 192 wells averaging 1.6 cells/well (viability test); 384 wells at 2 x 1E+03 cells/well (TFT resistance) in 96-well microtitre plates.

DETERMINATION OF CYTOTOXICITY
- Method: Relative Total Growth (RTG)

OTHER EXAMINATIONS:
Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment.

Evaluation criteria:
The test article was considered to be mutagenic in this assay if:
1. The Mutant frequency of any test concentration exceeded the sum of the mean control mutant frequency plus Global Evaluation Factor (GEF, 126 mutants per 1E+06 viable cells).
2. The linear trend test was positive.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results that only partially satisfied the assessment criteria described above were considered on a case by case basis.
Statistics:
Test for linear trend.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(Without S9 mix) maximum cytotoxicity as %RTG of 76% at 2683 µg/mL. (With S9 mix) no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(Without S9 mix) maximum cytotoxicity as %RTG of 72% at 2683 µg/mL. (With S9 mix) maximum cytotoxicity as %RTG of 72 at 1000 µg/mL (not dose-dependent)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A summary of the results for Experiments 1 and 2 is shown in Table 2 in 'Any other information on results incl. tables'.
In the absence and presence of S-9, no increases in mutant frequency that exceeded the GEF of 126 mutants per 1E+06 viable cells were observed at any concentration analysed in Experiments 1 and 2. A weak linear trend was observed in the absence of S-9 in Experiment 1. This was primarily attributable to the fact that the mean mutant frequencies at 400, 800 and 1200 µg/mL were slightly below the mean vehicle control mutant frequency and the mutant frequency at 2683 µg/mL was approximately 1.6-fold greater than at 800 µg/mL. However, no increases in mutant frequency of greater than 14 mutants per 1E+06 viable cells above the concurrent mean vehicle control value (i.e. considerably below the GEF of 126) were observed at any concentration analysed under this treatment condition, therefore the observation was not considered biologically relevant.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range Finder Experiment at the highest concentration tested (2683 µg/mL), compared to the concurrent vehicle controls.

- Water solubility: From the preliminary solubility data, Ammonium perrhenate was soluble in water for irrigation (purified water) at a concentration of at least 32.15 mg/mL (the solubility limit in culture medium was at least 3215 µg/mL).

- Precipitation: No precipitation was observed at the concentration of 3215 µg/mL approximately 24 hours after test article addition.

- Other confounding effects: Not reported.

RANGE-FINDING/SCREENING STUDIES:
The highest concentration tested (2683 µg/mL) gave 45% and 73% RTG in the absence and presence of S-9, respectively (see Tabe 1, in 'Any other information on results incl. tables').

COMPARISON WITH HISTORICAL CONTROL DATA: Not reported.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Not reported.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Experiment 1

Any other information on results incl. tables

Table 1 RTG Values (%RTG)- 3 hour Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RTG

+S-9

% RTG

0

100

100

83.84

85

103

167.7

61

104

335.4

87

118

670.8

69

100

1342

70

97

2683

45

73

 

Table 2 Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RTG

MF§

 

% RTG

MF§

0

 

100

61.31

 

0

 

100

69.15

 

400

 

105

49.95

 

400

 

112

50.51

 

800

 

96

47.20

 

800

 

94

59.83

 

1200

 

93

50.72

 

1200

 

90

55.14

 

1600

 

91

62.35

 

1600

 

98

61.10

 

2000

 

82

66.74

 

2000

 

124

44.85

 

2400

 

82

61.60

 

2400

 

120

49.31

 

2683

 

76

75.08

 

2683

 

120

56.23

 

Linear trend

*

Linear trend

NS

MMS

 

 

 

 

B[a]P

 

 

 

 

15

 

37

590.87

 

2

 

75

301.69

 

20

 

37

604.04

 

3

 

61

636.83

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3 Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RTG

MF§

 

%RTG

MF§

0

 

100!

68.73!

 

0

 

100

85.24

 

500

 

122

83.73

 

500

 

116

94.66

 

1000

 

96

100.10

 

1000

 

72

113.17

 

1400

 

97

113.58

 

1400

 

82

81.48

 

1700

 

95

107.26

 

1700

 

94

75.02

 

2000

 

147

102.24

 

2000

 

54

140.27

 

2300

 

79

103.34

 

2300

 

96

98.96

 

2683

 

72

86.51

 

2683

 

83

94.35

 

Linear trend

NS

Linear trend

NS

MMS

 

 

 

 

B[a]P

 

 

 

 

15

 

49

577.84

 

2

 

84

360.66

 

20

 

39

527.73

 

3

 

25

676.59

 

§ 5‑TFT resistant mutants/106viable cells 2 days after treatment

%RTG % Relative total growth

NS Not significant

! Based on one replicate only

*, **, *** Test for linear trend: χ2(one-sided), significant at 5%, 1% and 0.1% level respectively

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with or without metabolic activation

It is concluded that Ammonium perrhenate did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to 2683 µg/mL, equivalent to 10 mM in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).
Executive summary:

Ammonium perrhenatewas assayed for its ability to induce mutation at the tk locus (5‑trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S‑9). The test article was formulated in water for irrigation (purified water).

A 3 hour treatment incubation period was used for all experiments performed in the absence and presence of S‑9.

In the cytotoxicity Range-Finder Experiment, 3 hour treatment, six concentrations were tested in the absence and presence of S‑9 ranging from 83.84 to 2683  µg/mL (equivalent to 10 mM at the highest concentration tested).The highest concentration tested (2683 µg/mL), gave 45% and 73% relative total growth (RTG) in the absence and presence of S-9, respectively.

In Experiment 1 seven concentrations, ranging from 400 to 2683 µg/mL, were tested in the absence and presence of S‑9. The highest concentration analysed was 2683 µg/mL in the absence and presence of S‑9, which gave 76% and 120% RTG, respectively.

In Experiment 2 seven concentrations, ranging from 500 to 2683 µg/mL, were tested in the absence and presence of S‑9. The highest concentration analysed was 2683 µg/mL in the absence and presence of S‑9, which gave 72% and 83% RTG, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S‑9. Mutant frequencies in negative control cultures were considered acceptable. Clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S‑9) and Benzo[a]pyrene (with S‑9). Therefore the study was accepted as valid.

In the absence and presence of S-9, no increases in mutant frequency that exceeded the Global Evaluation Factor (GEF,126 mutants per 106 viable cells) were observed at any concentration analysed in Experiments 1 and 2. A weak linear trend was observed in the absence of S-9 in Experiment 1.This was primarily attributable to the fact that the mean mutant frequencies at 400, 800 and 1200 µg/mL were slightly below the mean vehicle control mutant frequency and the mutant frequency at 2683 µg/mL was approximately 1.6-fold greater than at 800 µg/mL. However, no increases in mutant frequency of greater than 14 mutants per 106 viable cells above the concurrent mean vehicle control value (i.e. considerably below the GEF of 126) were observed at any concentration analysed under this treatment condition, therefore the observation was not considered biologically relevant.

It is concluded that Ammonium perrhenate did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to 2683 µg/mL, equivalent to 10 mM, in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).