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:
2009-07-03 to 2009-10-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Remarks:
GLP study conducted according to current accepted guidelines. The justification for read-across from MnCl2 to manganese dinitrate is based on the fact that in an in vitro system, in the case of both substances, the aqueous solution surrounding the test cells will consist simply of Mn2+ cations with the anions not likely to play any part in the toxicity.
Justification for type of information:
See the read-across report attached in Section 13
Cross-reference
Reason / purpose:
other: Read-across target
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
See the read-across report attached in Section 13
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Test material-induced toxicity was noted at the highest dose level employed in the test.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material : Manganese chloride, MnCl2 (Eramet)
- Molecular formula : MnCl2
- Substance type: Light pink solid flakes
- Physical state: Solid
- Analytical purity: >95%
- Lot/batch No.: 104896-05
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
TK locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Please see table 1 under section Any other information on materials and methods incl. tables for dosing regime
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: R0 medium
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
In the absence of metabolic activation. 400 µg/mL and 150 µg/mL for the 4 hour and 24 hour exposures respectively.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 or 24 hours
- Generation time : 12 hours
- Selection time (if incubation with a selection agent): 2 days


SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine (TFT) selective medium
STAIN : MTT


NUMBER OF REPLICATIONS:Each dose level was performed in duplicate


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Plate scoring: Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgement. Large colonies were defined as those that covered approximately 1/4 to 3/4 of the surface of the well and were generally no more than one or two cells thick. Generally all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies normally are more than two cells thick. 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates to visualise the mutant colonies. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour.

% Relative Suspension Growth (%RSG), Day 2 Viability (%V), Relative Total Growth (RTG) and Mutation Frequency (MF) were all calculated to assess the mutagenic potential. Please refer to section: Any other information on materials and methods incl. tables under the appropriate headings for full details.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency over the concurrent vehicle mutant frequency value.
Statistics:
For a response to be considered positive, the induced mutation frequency value must exceed the set Global Evaluation Factor (GEF) value at 126 x 10E-6 for the microwell method. Any test material dose level that exhibits a mutation frequency value that is greater than the corresponding vehicle control by the GEF is considered positive. If a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. When a test material induced modest reproducible increases in the mutation frequencies that did not exceed the GEF value, then scientific judgement was applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Small significant increases designated by the UKEMS statistical package were reviewed using the criteria set out above and disregarded at the discretion of the Study Director.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Test material-induced toxicity was noted at the highest dose level employed in the test.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of the test material was observed at and above 10 µg/mL in the 4-hour exposure groups in the absence of metabolic activation and at and above 20 µg/mL in the 4-hour exposure group in the presence of metabolic activation.


RANGE-FINDING/SCREENING STUDIES: All three exposure groups employed in the screening test exhibited a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. A precipitate of the test material was observed at and above 78.75 µg/mL in the 4-hour exposure group in the absence of metabolic activation, at and above 39.98 µg/mL in the 4-hour exposure group in the presence of metabolic activation, and at and above 19.69 µg/mL in the 24-hour exposure group in the absence of metabolic activation. In the mutagenicity experiments the maximum dose level was limited by test-material-induced toxicity.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 2: Results from the preliminary toxicity test

 

Dose (µg/mL)

%RSG (-S9) 4-Hour Exposure

%RSG (=S9) 4-Hour Exposure

%RSG (-S9) 24-Hour Exposure

0

100

100

100

4.92

102

89

33

9.84

96

100

11

19.69

89

89

1

39.38

72

75

0

78.75

2

57

0

157.5

9

1

0

315

1

0

0

630

0

0

0

1260

0

0

0

 

Table 3: Summary of results for main experiment, 4 hour exposure

 

Treatment (µg/mL)

4-Hours –S9

Treatment (µg/mL)

4-Hours +S9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1.00

81.37

0

100

1.00

74.20

2.5†

97

 

 

20

91

1.04

64.08

5

95

1.06

73.26

40

68

0.86

78.58

10

91

0.94

96.72

60

43

0.41

116.75

20

101

1.24

90.28

80

37

0.32

96.12

40

44

0.46

104.53

100

32

0.22

104.65

60

19

0.08

128.81

120

26

0.15

104.12

80

17

0.13

91.23

140

25

0.18

100.39

120†

14

 

 

160‡

13

0.04

42.87

Linear trend

NS

Linear trend

EMS

 

 

 

CP

 

 

 

400

71

0.62

656.51

2

55

0.22

1662.80

† Not plated for viability or 4-TFT resistance

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

NS Not significant

‡ Treatment excluded from test statistics due to toxicity

 

Table 4: Summary of results for main experiment, 24 hour exposure

Treatment (µg/mL)

4-Hours –S9

%RSG

RTG

MF§

0

100

1.00

103.16

0.31

97

0.99

91.33

0.63

105

0.97

79.60

1.25

95

1.07

50.22

2.5

76

0.81

100.92

5

41

0.38

173.75*

7.5

14

0.11

372.63*

10†

7

 

 

15†

4

 

 

Linear trend

***

EMS

 

 

 

150

54

0.32

1211.25

† Not plated for viability or 4-TFT resistance

* p<0.05

*** p<0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results: Negative with and without metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.The justification for read-across from MnCl2 to manganese dinitrate is based on the fact that in an in vitro system, in the case of both substances, the aqueous solution surrounding the test cells will consist simply of Mn2+ cations with the anions not likely to play any part in the toxicity.