Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key Study: Ames Read-Across Source MnCl2 – Thompson & Bowles (2009)


The test material has been found to be non-mutagenic under the test conditions reported.


 


Key Study: Chromosome Aberration Read-Across Source MnCl2 – Morris & Durwood (2009)


The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups in the absence or presence of a liver enzyme metabolising system or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.


 


Key Study: Mouse Lymphoma Read-Across Source MnCl2 – Flanders (2009)


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.


 


Supporting Study: Various In Vitro Endpoints Read-Across Source Mn Substances – ATSDR (2008)


The ATSDR document concludes that the results of in vitro studies with manganese compounds are conflicting.


 


In accordance with column 2 of REACH Annex VIII, In vitro cytogenicity study in mammalian cells or  in vitro micronucleus study (required in section 8.4.2. does not usually need to be conducted as adequate data from an in vivo cytogenicity test are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
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
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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to current accepted guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
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
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: 2 µg/mL
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 10^-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.
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.

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

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.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Species / strain:
lymphocytes: human, peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-07-03 to 2009-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to current standard guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human, peripheral
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
See table 1 under section Any other information on materials and methods incl. tables
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Minimal Essential Medium (MEM)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9: 0.4 and 0.2 µg/mL for the 4(20)-hour and 24-hour exposures respectively in MEM
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 S9: 5 µg/mL dissolved in dimethyl sulphoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: Approximately 48 hours incubation at 37 °C 5% CO2 in humidified air.
- Exposure duration: 4 hour exposure (with a further 20 hour incubation once test material had been removed) and 24 hour exposure
- Expression time (cells in growth medium): Typically 17 hours average generation time under experimental exposure conditions.
- Fixation: Mitosis was arrested two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M hypotonic KCl. After 14 minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4 °C for at least four hours to ensure complete fixation.


SPINDLE INHIBITOR (cytogenetic assays): Demecolcin (Colcemid 0.1 µg/mL) two hours before the required harvest time.
STAIN (for cytogenetic assays): To prepare metaphase spreads, lymphocytes were suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.


NUMBER OF REPLICATIONS: Duplicate lymphocyte cultures (A and B) were prepared for each dose level.


NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

- Other: Slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation of the test material. These observations were used to select the dose levels for mitotic index evaluation.
Evaluation criteria:
Coding:
Slides were coded using a computerised random number generator and any supplementary slides were coded manually.
Mitotic index:
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Scoring of chromosome damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted. Where there were approximately 30 to 50 % of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) Scott et al and Savage (1976) in the UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogenticist prior to decoding the slides.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: human, peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material was noted to precipitate at 315 µg/mL in the 4(20)-hour cultures without S9, and above 630 µg/mL in the 4(20)-hour cultures in the presence of S9. No precipitate was observed at the end of the exposure period in the 24-hour cultures.


RANGE-FINDING/SCREENING STUDIES: The preliminary toxicity test dose range was 4.92 to 1260 µg/mL. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test material was observed in the cultures at the end of the exposure, at and above 157.5 µg/mL in the 4(20)-hour exposure in the absence of S9 at and above 78.75 µg/mL in the 4(20)-hour exposure in the presence of s(, and at above 315 µg/mL in the 24 hour continuous exposure group. Haemolysis was observed at and above 315 µg/mL at harvesting in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1260 µg/mL in the 4(20)-hour exposure in the presence of metabolic activation and up to 157.5 µg/mL in the 4(20)-hour exposure in the absence of S9. The maximum dose with metaphases present in the 24-hour continuous exposure was 39.38 µg/mL. The test material induced clear evidence of toxicity in all of the exposure groups.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of test and of the activity of the metabolising system. The test material was found to be toxic to lymphocytes, and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included dose levels that induced approximately 50% mitotic inhibition.

Please refer to attached document, Appendix 1 for full tabulated results

Conclusions:
Interpretation of results: negative with or without metabolic activation

The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups in the absence or presence of a liver enzyme metabolising system or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP study conducted to current accepted guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine synthesis in the Salmonella typhimurium strains and tryptophan synthesis in the E. coli strain used.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: GC base pairing at the primary reversion site.
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: AT base pairing at the primary reversion site.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test material was found to be soluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL. Sterile distilled water was chosen as the solvent.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)


DURATION
- Preincubation period: 10 hours at 36 °C
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not reported
Evaluation criteria:
Several criteria for determining a positive result. Dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however statistical significance will not be the only determining factor for a positive response.
Statistics:
The following was used to statistically evaluate the results from the mutagenicity test. Kirkland DJ (ED) (1989) Statistical Evaluation of Mutagenicity Test Data (UKEMS) sub-committee on Guidelines for Mutagenicity Testing. Report Part III - Cambridge University Press.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: Not reported


RANGE-FINDING/SCREENING STUDIES: The test substance was found to be non-toxic in the range-finding study.


COMPARISON WITH HISTORICAL CONTROL DATA: The historical control values were found to concur with the results from the study for both positive and vehicle controls.


ADDITIONAL INFORMATION ON CYTOTOXICITY: Not reported

Table 1: Range finding study – toxicity assay

 

With (+) or Without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

75

81

78

96

89

77

85

74

70

86P

84P

+

TA100

77

96

84

85

74

75

91

88

74

70P

73P

-

WP2uvrA-

25

23

25

23

27

30

23

35

31

22P

27P

+

WP2uvrA-

37

24

27

34

29

25

36

32

38

30P

24P

P - Precipitate

 

Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls), Experiment 1

 

Number of Revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

93

15

22

14

10

96 (95)

19 (18)

20 (22)

16 (17)

13 (12)

97

21

24

20

13

 

Table 3: Spontaneous Mutation Rates (Concurrent Negative Controls), Experiment 2

 

Number of Revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

123

22

33

22

12

123 (118)

25 (23)

25 (29)

25 (24)

12 (12)

109

21

29

24

13

 

Table 4: Test Results, Experiment 1 – Without Metabolic Activation

 

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

95

111  (102)

99    8.3#

16

20   (18)

19    2.1

23

25    (23)

20     2.5

26

21   (23)

23    2.5

15

13    (14)

13    1.2

50

112

96    (104)

104   8.0

16

20   (19)

20    2.3

18

25     (22)

23      3.6

21

22    (22)

24     1.5

14

13    (12)

10     2.1

150

115

110   (109)

102    6.6

21

13   (18)

21    4.6

26

15    (20)

19     5.6

16

20    (21)

27     5.6

12

11    (12)

13     1.0

500

103

92    (95)

90     7.0

18

19   (17)

14    2.6

21

23     (25)

30      4.7

21

21     (21)

20      0.6

15

15    (12)

15     0.0

1500

110 P

95 P  (103)

104P   7.5

19P

18P  (18)

16P   1.5

22

24P   (23)

22P    1.2

22P

23P   (25)

26P    3.2

13P

15P   (12)

9P      3.1

5000

88P

117P  (105)

111P   15.3

18P

21P  (19)

18P   1.7

26P

21P    (24)

24P     2.5

25P

23P    (25)

26P     1.5

9P

12P   (12)

16P    3.5

Name

Concentration

No. colonies per plate

ENNG

ENG

ENNG

4NQO

9AA

3

5

2

0.2

80

475

526  (492)

476   29.2

99

150  (115)

95     30.7

145

145   (145)

145    0.0

123

118   (119)

115    4.0

345

462   (400)

394    58.8

ENNG – N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO – 4-Nitroquinoline-1-oxide

9AA – 9-Aminoacridine

P – Precipitate

# - Standard deviation

 

Table 5: Test Results, Experiment 1 – With Metabolic Activation

 

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

97

96     (98)

100   8.3#

12

12   (11)

9     1.7

23

29    (28)

32     4.6

25

24   (23)

20    2.6

14

9     (12)

14    2.9

50

117

101   (106)

100    9.5

11

9     (10)

10    1.0

31

25     (25)

19      6.0

20

21    (20)

18     1.5

12

12    (12)

12     0.0

150

97

102   (101)

103    3.2

10

9    (9)

9    0.6

25

23    (25)

27     2.0

26

22    (24)

24     2.0

15

10    (12)

11     12.6

500

115

74     (94)

92      20.6

13

8   (10)

9    2.6

29

19     (23)

21      5.3

31

20     (24)

20      6.4

16

15    (15)

13     1.5

1500

90 P

82 P  (88)

91P    4.9

12P

9P    (11)

13P   2.1

26P

24P   (23)

20P    3.1

19P

18P   (24)

20P    6.4

11P

15P   (12)

9P      3.1

5000

92P

104P   (95)

90P      7.6

12P

9P     (11)

11P   1.5

24P

24P    (23)

20P     2.3

20P

24P    (22)

22P     2.0

12P

9P     (11)

13P    2.1

Name

Concentration

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2248

2526 (2506)

2743  248.1

209

151  (179)

177    29.1

172

225   (184)

154    36.9

206

184   (197)

202    11.7

278

217   (247)

247    30.5

2AA – 2-Aminoanthracene

BP – Benzo(a)pyrene

P – Precipitate

# - Standard deviation

 

Table 6: Test Results, Experiment 2 – Without Metabolic Activation

 

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

101

95      (104)

115    10.3#

20

16   (19)

20    2.3

21

24    (23)

25     2.1

26

26   (25)

23    1.7

11

16    (14)

15    2.6

50

106

97    (104)

106   5.2

21

21   (21)

21    0.0

18

26     (24)

29      5.7

20

26    (25)

2 9    4.6

8

15    (12)

13     3.6

150

113

107   (111)

114    3.8

24

20   (21)

20    2.3

22

29    (25)

23     3.8

20

19    (22)

27     4.4

10

8      (9)

10    1.2

500

96

104    (102)

106     5.3

16

22   (17)

14    4.2

23

24    (24)

25     1.0

23

25     (26)

30      3.6

15

8      (9)

10    1.2

1500

118P  (118)

119P   1.0

117       *

24P

20P  (23)

26P   3.1

21P

24P   (22)

22P    1.5

29P

21P   (25)

26P    4.0

10P

11P   (10)

9P      1.0

5000

101P

104P  (105)

110P   4.6

22P

23P  (22)

21P   1.0

27P

24P    (24)

21P     3.0

26P

25P    (25)

24P     1.0

12P

13P   (11)

9P      2.1

Name

Concentration

No. colonies per plate

ENNG

ENG

ENNG

4NQO

9AA

3

5

2

0.2

80

295

312  (309)

320   12.8

209

222    (212)

206     8.5

416

492   (462)

477    40.3

246

217   (228)

220    15.9

1078

2042 (1426)

1157  535.2

ENNG – N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO – 4-Nitroquinoline-1-oxide

9AA – 9-Aminoacridine

P – Precipitate

# - Standard deviation

* p0.05

 

Table 7: Test Results, Experiment 2 – With Metabolic Activation

 

Test Period

From 19 July 2009

To 22 July 2009

Test Substance (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

108

106     (108)

109      1.5#

10

9     (11)

13    2.1

25

27    (26)

26     1.0

22

21   (22)

22    0.6

16

16   (15)

12    2.3

50

93

91   (106)

107    8.7

15

9     (12)

13    3.1

22

22     (25)

31      5.2

21

26    (22)

22     0.6

13

15    (15)

16     1.5

150

98

89     (99)

110   10.5

13

8     (10)

13    3.1

22

23    (22)

31     0.6

19

24    (22)

22     2.5

14

14    (12)

7       4.0

500

91

106    (102)

110   10.0

12

14   (12)

9     2.5

30

29     (30)

30      0.6

26

21     (23)

22      2.6

16

14    (14)

12     2.0

1500

81P

100P  (96)

110P  10.0

9P

13P    (10)

9P       2.3

25P

31P   (27)

25P    3.5

25P

25P   (24)

22P    1.7

9P

16P     (14)

16P      4.0

5000

90P

84P     (92)

102P    9.2

11P

10P    (10)

8P     1.5

26P

23P    (25)

27P     2.1

24P

21P    (22)

22P     1.5

12P

15P    (12)

10P     2.5

Name

Concentration

No. colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2474

2427 (2510)

2629  105.7

374

332  (332)

291   41.5

342

264   (313)

333    42.7

261

620   (457)

490    181.8

497

494   (498)

504    5.1

2AA – 2-Aminoanthracene

BP – Benzo(a)pyrene

P – Precipitate

# - Standard deviation

 

The test material was found to cause no visible reduction in growth of the bacterial background lawn at any dose and was therefore tested up to the maximum dose level of 5000 µg/plate A particulate precipitate was at 1500 µg/plate and above. This was considered not to prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for and of the bacterial strains, with any dose of the test material, with or without metabolic activation. In the TA100 revertant colony, a small but statistically significant increase was observed on the 1500 µg/plate in Experiment 2 (increase of less than 1.5 times). However these were within the range specified by the Standard Test Method, this increase proved non-reproducible over two separate experiments. This was concluded to have no biological or toxicological relevance. All of the positive control substances induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

Conclusions:
Interpretation of results: negative

The test material has been found to be non-mutagenic under the test conditions reported.
Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: all forms of genotoxicity in vitro.
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Review of related compounds
Adequacy of study:
supporting study
Justification for type of information:
Please see the read across justification.
Reason / purpose for cross-reference:
read-across source
Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: other: all forms of genotoxicity in vitro
Type of information:
other: review of related compounds
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The document does no contain any data for permanganate; however a number of studies with other forms of manganese are reported.
Qualifier:
no guideline available
Principles of method if other than guideline:
Literature review and critical assessment
GLP compliance:
no
Remarks:
: not relevant
Type of assay:
other: literature review
Target gene:
Various
Test concentrations with justification for top dose:
Various

 


While manganese sulphate was shown to not be mutagenic toSalmonella typhimuriumstrains TA97, TA98, TA100, TA1535, or TA1537 either in the presence or absence of S9 from Aroclor 1254-induced liver from rats or Syrian hamsters, it was shown to be mutagenic to strain TA97 elsewhere. In yeast (Saccharomyces cerevisiaestrain D7), a fungal gene conversion/reverse mutation assay indicated that manganese sulphate was mutagenic (Singh 1984). Manganese chloride was reportedly not mutagenic inS. typhimuriumstrains TA98, TA100, and TA1535, but it was mutagenic in strain TA1537, and conflicting results were obtained for TA102.In vitroassays in mammalian cells also gave conflicting results concerning manganese mutagenicity. Manganese chloride produced gene mutations in cultured mouse lymphoma cells. Manganese chloride caused DNA damagein vitrousing human lymphocytes at a concentration of 25 μm without metabolic activation, but not at the lower tested concentrations of 15 and 20 μm. The compound also caused DNA damage in human lymphocytes using the single-cell gel assay technique in the absence of metabolic activation, but caused no DNA damage when S9 was present. Manganese sulphate induced sister chromatic exchange in Chinese hamster ovary (CHO) cells in both the presence and absence of S9 from Aroclor 1254-induced rat liver. In a separate assay, manganese sulphate also induced chromosomal aberrations in CHO cells in the absence of S9 but not in its presence. Manganese chloride caused chromosome aberrations in human lymphocytes without metabolic activation, but only when treated in the G2 phase of the cell cycle; treatment in the G1, G1/S, and S1 phases of the cell cycle did not result in chromosome aberrations. The compound was also found to be clastogenic in root tip cells ofVicia faba,but not in cultured FM3A cells in the absence of metabolic activation. Manganese chloride caused cell transformation in Syrian hamster embryo cells.

Conclusions:
Interpretation of results: Ambiguous

The ATSDR document concludes that the results of in vitro studies with manganese compounds are conflicting.
Executive summary:

The ATSDR document represents a comprehensive and up-to-date review of the genotoxicity of various manganese compounds. The review concludes that mutagenicity studies in both bacteria and mammalian strains are conflicting. While manganese sulphate was shown to not be mutagenic to Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537 either in the presence or absence of S9 from Aroclor 1254-induced liver from rats or Syrian hamsters, it was shown to be mutagenic to strain TA97 elsewhere. In yeast (Saccharomyces cerevisiae strain D7), a fungal gene conversion/reverse mutation assay indicated that manganese sulphate was mutagenic (Singh 1984). Manganese chloride was reportedly not mutagenic in S. typhimurium strains TA98, TA100, and TA1535, but it was mutagenic in strain TA1537, and conflicting results were obtained for TA102. In vitro assays in mammalian cells also gave conflicting results concerning manganese mutagenicity. Manganese chloride produced gene mutations in cultured mouse lymphoma cells. Manganese chloride caused DNA damage in vitro using human lymphocytes at a concentration of 25 μm without metabolic activation, but not at the lower tested concentrations of 15 and 20 μm. The compound also caused DNA damage in human lymphocytes using the single-cell gel assay technique in the absence of metabolic activation, but caused no DNA damage when S9 was present. Manganese sulphate induced sister chromatic exchange in Chinese hamster ovary (CHO) cells in both the presence and absence of S9 from Aroclor 1254-induced rat liver. In a separate assay, manganese sulphate also induced chromosomal aberrations in CHO cells in the absence of S9 but not in its presence. Manganese chloride caused chromosome aberrations in human lymphocytes without metabolic activation, but only when treated in the G2 phase of the cell cycle; treatment in the G1, G1/S, and S1 phases of the cell cycle did not result in chromosome aberrations. The compound was also found to be clastogenic in root tip cells of Vicia faba, but not in cultured FM3A cells in the absence of metabolic activation. Manganese chloride caused cell transformation in Syrian hamster embryo cells.

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
other justification
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Justification for type of information:
In accordance with column 2 of REACH Annex VIII, In vitro cytogenicity study in mammalian cells or in vitro micronucleus study (required in section 8.4.2. does not usually need to be conducted as adequate data from an in vivo cytogenicity test are available.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key Study: Erythrocyte Micronucleus Assay: Read-Across Source MnCl2 – Streicker (2009)


Manganese administered twice at 24 ± 0.5 hour intervals to female B6C3F1 mice by oral gavage at 25, 50, 100 or 200 mg/kg BW did not induce chromosomal damage.


 


Supporting Study: Various In Vivo Endpoints: Read-Across Source: Mn Substances – ATSDR (2008)


The results of in vitro studies show that at least some chemical forms of manganese have mutagenic potential. However, as the results of in vivo studies in mammals are inconsistent, no overall conclusion can be made about the possible genotoxic hazard to humans from exposure to manganese compounds.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Animal number 23 which showed hunched posture at the 1 hour post dose observation on Day 1 of the study. All other animals were found to be normal throughout the study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-04-15 to 2009-09-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to current accepted guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC USA
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: 18.9-22.2 g
- Assigned to test groups randomly: yes under following basis: Animals were stratified by body weight and assigned to a dose group such that mean animal weights across dose groups were approximately equal.
- Housing: Animals were housed individually housed in polycarbonate cages with absorbent hardwood bedding (Betachip, Northeastern products Corp., Warrensbury, NY USA). Animals were transferred to clean cages weekly.
- Diet : Purina Certified Rodent Chow 5002 (Ralston Purina, St. Louis, MO, USA) available ad libitum.
- Water : Reverse-osmosis water was provided ad libitum using plastic water bottles with stainless steel sipper tubes. Fresh water was supplied weekly.
- Acclimation period: All animals were examined by a veterinarian or other appropriate persons during the acclimation period to assess the health status. All animals were re-examined at the end of the acclimation period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: physiol. saline
- Amount of vehicle : 10 mL/kg BW
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were made from manganese chloride tetrahydrate and concentrations calculated as manganese (Mn2+). The manganese chloride dosing solutions were freshly prepared in 0.9 % saline on each day of treatment.

DOSE ADMINISTRATION
The animals were dosed on days 1 and 2 via oral gavage in a single dose using a stainless steel or disposable gavage needle. Each dose was administered 24 ± 0.5 hours after the first dose.

JUSTIFICATION OF DOSING ROUTE
An appropriate route of human exposure is oral.
Duration of treatment / exposure:
Single oral exposures
Frequency of treatment:
Animals were dosed twice within 24 hours.
Post exposure period:
46 hours
Remarks:
Doses / Concentrations:
25 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals were included in each dosing group
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control : cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 25 mg/kg/day in 0.9 % saline
Tissues and cell types examined:
Blood was collected from each mouse for analysis. 350 µL of MicroFlowPLUS kit Solution B (anticoagulant) was aliquoted into an appropriately labelled sterile microcentrifuge tube for each animal. One day prior to collection of blood, 2 mL of MicroFlowPLUS Solution A (fixative) was aliquoted into two appropriately labelled 15 mL polypropylene conical tubes for each study animal. Immediately prior to use, each tube containing Solution B was shaken immediately before bleeding each animals to coat the inside of the tube. Following anaesthesia by isofluorane, blood was collected from the retro-orbital sinus by heparinized hemocrit. 120 (± 20) µL of blood was dispensed into the corresponding microcentrifuge tube containing Solution B and mixed by inverting several times. The stabilised blood samples were maintained at room temperature and fixed within 5 hours of collection.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Results from a renge finding study.


METHOD OF ANALYSIS: Blood samples were fixed by 180 µL of each blood-Solution B sample was withdrawn from the tube. All tubes of fixed cells were quickly transferred back to the freezer and stored at -75 ± 10 °C for at least 24 hours prior to processing for flow cytometry analysis of reticulocytes. Flow cytometry of MN was performed using MicroFlowPLUS (Mouse) kit reagents (Litron Laboratories, Rochester NY) according to the kit’s instructional manual with minimal modification, 1) 1 mL of fixed cells for each experimental sample was transferred to a tube containing 12 mL of Solution C and washed in preparation for labelling and 2) volumes of labelling solution I, labelling solution II and DNA stain were adjusted as appropriate each day to minimise the use of excess reagents. The MicroFlowPLUS method utilises CD71 and CD61 antibodies as well as a fixed malaria biostandard that contains DNA in the same size range as MN to properly configure the flow cytometer to exclude platelets (CD61+) and only count micronucleus events in erythrocytes. For each peripheral blood sample, 20,000 immature (CD71+) reticulocytes were analysed to determine the frequency of both MN-RET and micronucleated mature (CD41-) normochromatic erythrocytes (NCE). A BD FACSCalibur™, a four-colour, dual laser benchtop system, was used for MN-RET and MN-NCE analysis.
Evaluation criteria:
Positive control:
The reference control (positive control) cyclophosphamide, is expected to induce an increase in the frequency of MN-RET at p < 0.05.
Test item:
For negative studies, the highest test item concentration evaluated for MN induction must, unless precleded by the use of the limit dose of 2000 mg/kg, induce bone marrow or animal toxicity or be a dose only slightly lower than that which would be expected to induce mortality.

Criteria for a positive response:
A decision by an experienced scientific investigator to classify a test item as negative, equivocal, or positive for genotoxicity in this assay is based on the biological relevance of the equivocal, or positive for genotoxicity based on the biological relevance of the results, taking into consideration the appropriateness of the concurrent control data, the results of the statistical analysis of the experimental data and the extent of cytotoxicity.
Statistics:
Statistical analysis was conducted on the frequency of MN-RET, MN-NCE and RET. Using individual animal data, the analysis involveed the use of the Shapiro-Wilk test with a confidence level of 95 % to determine the normality of the MN-RET data in the vehicle control group.

Normally distributed MN-RET frequency data were then analysed for linearity and variance between treatment groups using linear regression and one-way ANOVA analyses, respectively. The Dunnett multiple comparison test was used to determine if a treatment groups was significantly different (p < 0.05) from vehicle controls. A one-tailed independent t-test was used to verify a positive response to the control compound, cyclophosphamide.
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Animal number 23 which showed hunched posture at the 1 hour post dose observation on Day 1 of the study. All other animals were found to be normal throughout the study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 125-1000 mg/kg bw
- Clinical signs of toxicity in test animals: Please refer to Range finding study under section: Remarks on results including tables and figures for full details.

RESULTS OF DEFINITIVE STUDY
- Micronucleus Assay Results (incl. statistical analysis): The percent of micronucleated cells among 20,000 RET and ≥900,000 NCE from the peripheral blood of B6C3F1 mice treated with the vehicle, reference chemical, and test item as analysed by flow cytometry. Based on a one-way ANOVA (p = 0.415) test and Dunnett pair wise comparison of each dose group against the concurrent control, the group %MN-RET means comparison of each dose group against the concurrent control, the group %MN-RET means were not significantly increased by treatment with manganese at dose levels 25, 50, 100 or 200 mg/kg BW. No dose response was indicated by linear regression analysis (p = 0.509). There was also no impact on treatment with manganese on %MN-NCE (p = 0.3939) or %RET (p = 0.7375). Treatment with 25 mg/kg/day of the reference control, cyclophosphamide, induced a statistically significant increase in MN-RET (p < 0.0001) as well as MN-NCE (p = 0.0012) and resulted in a 40.6% decrease in %RET.
- Clinical signs of toxicity in test animals: Cage side observations were conducted prior to dosing and at 1 and 4 hours post-dose each day. All animals in the definitive study were considered normal throughout with the exception of animal number 23 which showed hunched posture at the 1 hour post dose observation on Day 1 of the study.

Range finding study:

Male and female mice were treated with Mn2 +at 1000, 500, 250 and 125 mg/kg BW. A few minutes after dosing, the fist animal (male) in the 1000 mg/kg treatment group exhibited seizures and convulsions. This animal was euthanised and no further mice were treated in this dose group. At 1 -hour post-treatment observation period, male and female mice in the 500 mg/kg treatment groups exhibited lethargy, uncoordinated movement, and abnormal breathing. These mice were euthanised after the 1 -hour observation period. Also at the 1 -hour post-treatment observation period, the mice in the 250 mg/kg treatment group exhibited hunched posture, decreased movement and piloerection. At the 4 -hour post-treatment observation, the mice in the 250 mg/kg group continued to exhibit a hunched posture, decreased movement and lethargy, with one animal found dead. These mice were immediately euthanised. The control mice and the mice in the 125 mg/kg treatment group were normal throughout both days of treatment.

Based on the results of the first day of treatment, the study was discontinued and restarted using 175, 200 and 225 mg/kg BW. At the 1 -hour post-treatment observation, the female mice in the 225 mg/kg group exhibited a hunched posture, decreased movement and piloerection. These mice were immediately euthanised. All other animals were considered normal throughout the study.

Based on the range-finding study, the selected doses for the definitive study were 25, 50, 100 and 200 mg/kg manganese. Female mice were dosed chosen for the definitive test.

Definitive test:

Table 2: Frequency of RET, MN-RET and MN-NCE in peripheral blood reticulocytes of female B6C3F1 mice administered manganese and cyclophosphamide by oral gavage

 

Dose (mg/kg)

% MN-RET

SEM

% MN-NCE

SEM

% RET

SEM

Vehicle

0

0.202

0.006

0.118

0.001

1.725

0.172

Manganese

25

50

100

200

0.173

0.202

0.197

0.180

0.012

0.017

0.019

0.008

0.120

0.113

0.122

0.112

0.007

0.005

0.003

0.004

1.527

1.565

1.697

1.675

0.087

0.191

0.067

0.105

Cyclophosphamide

25

1.057

0.020

0.144

0.004

1.025

0.077

Conclusions:
Interpretation of results: Negative
Manganese administered twice at 24 ± 0.5 hour intervals to female B6C3F1 mice by oral gavage at 25, 50, 100 or 200 mg/kg BW did not induce chromosomal damage.
Executive summary:

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality for classification and labelling and for risk assessment.

 

The reference control, cyclophosphamide, administered at 25 mg/kg/day under the same dosing regimen, induced a significant increase in the frequency of MN-RET (p<0.0001) and MN-NCE (p = 0.0012) and a decrease in % RET. Also the quality criteria of the study have been fulfilled as the study was conducted to OCED 474 (Mammalian Erythrocyte) guidelines and to GLP.

  

Lack of any observed effects in % MN-RET, %MN-NCE and % RET in the study supports the lack of classification and labelling of this substance.

Endpoint:
genetic toxicity in vivo, other
Remarks:
Genotoxicity in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Please see the read across justification.
Endpoint:
genetic toxicity in vivo, other
Remarks:
Genotoxicity in vivo
Type of information:
other: review
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published review of literature data on the genotoxicity in vivo of manganese compounds
Qualifier:
no guideline available
Principles of method if other than guideline:
Review of published literature genotoxiity studies.
GLP compliance:
no
Remarks:
: not relevant
Type of assay:
other: review of various study types
Species:
other: various

Manganese chloride did not produce somati mutations inDrosophila melanogasterfruit flies in one study, and manganese sulfate did not induce sex-linked recessive lethal mutations in germ cells of maleD. melanogaster. In vivoassays in mice showed that oral doses of manganese sulphate caused micronuclei and chromosomal aberrations in bone marrow. In contrast, oral doses of manganese chloride did not cause chromosomal aberrations in the bone marrow or spermatogonia of rats.

Conclusions:
Interpretation of results: Ambiguous
The results of in vitro studies show that at least some chemical forms of manganese have mutagenic potential. However, as the results of in vivo studies in mammals are inconsistent, no overall conclusion can be made about the possible genotoxic hazard to humans from exposure to manganese compounds.
Executive summary:

The ATSDR document represents a comprehensive and up to date review of the genotoxicity of manganese compounds. Manganese chloride did not produce somatic mutations in Drosophila melanogaster fruit flies in one study, and manganese sulfate did not induce sex-linked recessive lethal mutations in germ cells of male D. melanogaster. In vivo assays in mice showed that oral doses of manganese sulphate caused micronuclei and chromosomal aberrations in bone marrow. In contrast, oral doses of manganese chloride did not cause chromosomal aberrations in the bone marrow or spermatogonia of rats.


 


The results of in vitro studies show that at least some chemical forms of manganese have mutagenic potential. However, as the results of in vivo studies in mammals are inconsistent, no overall conclusion can be made about the possible genotoxic hazard to humans from exposure to manganese compounds.

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

Additional information

In Vitro


Key Study: Ames Read-Across Source MnCl2 – Thompson & Bowles (2009)


The test material has been found to be non-mutagenic under the test conditions reported. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


 


Key Study: Chromosome Aberration Read-Across Source MnCl2 – Morris & Durwood (2009)


The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups in the absence or presence of a liver enzyme metabolising system or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.  The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


 


Key Study: Mouse Lymphoma Read-Across Source MnCl2 – Flanders (2009)


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 study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


 


Supporting Study: Various In Vitro Endpoints Read-Across Source Mn Substances – ATSDR (2008)


The ATSDR document represents a comprehensive and up-to-date review of the genotoxicity of various manganese compounds. The review concludes that mutagenicity studies in both bacteria and mammalian strains are conflicting. While manganese sulphate was shown to not be mutagenic to Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537 either in the presence or absence of S9 from Aroclor 1254-induced liver from rats or Syrian hamsters, it was shown to be mutagenic to strain TA97 elsewhere. In yeast (Saccharomyces cerevisiae strain D7), a fungal gene conversion/reverse mutation assay indicated that manganese sulphate was mutagenic (Singh 1984). Manganese chloride was reportedly not mutagenic in S. typhimurium strains TA98, TA100, and TA1535, but it was mutagenic in strain TA1537, and conflicting results were obtained for TA102. In vitro assays in mammalian cells also gave conflicting results concerning manganese mutagenicity. Manganese chloride produced gene mutations in cultured mouse lymphoma cells. Manganese chloride caused DNA damage in vitro using human lymphocytes at a concentration of 25 μm without metabolic activation, but not at the lower tested concentrations of 15 and 20 μm. The compound also caused DNA damage in human lymphocytes using the single-cell gel assay technique in the absence of metabolic activation, but caused no DNA damage when S9 was present. Manganese sulphate induced sister chromatic exchange in Chinese hamster ovary (CHO) cells in both the presence and absence of S9 from Aroclor 1254-induced rat liver. In a separate assay, manganese sulphate also induced chromosomal aberrations in CHO cells in the absence of S9 but not in its presence. Manganese chloride caused chromosome aberrations in human lymphocytes without metabolic activation, but only when treated in the G2 phase of the cell cycle; treatment in the G1, G1/S, and S1 phases of the cell cycle did not result in chromosome aberrations. The compound was also found to be clastogenic in root tip cells of Vicia faba, but not in cultured FM3A cells in the absence of metabolic activation. Manganese chloride caused cell transformation in Syrian hamster embryo cells. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).


 


In Vivo


Key Study: Erythrocyte Micronucleus Assay: Read-Across Source MnCl2 – Streicker (2009)


The reference control, cyclophosphamide, administered at 25 mg/kg/day under the same dosing regimen, induced a significant increase in the frequency of MN-RET (p<0.0001) and MN-NCE (p = 0.0012) and a decrease in % RET. Also, the quality criteria of the study have been fulfilled as the study was conducted to OCED 474 (Mammalian Erythrocyte) guidelines and to GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


Lack of any observed effects in % MN-RET, %MN-NCE and % RET in the study supports the lack of classification and labelling of this substance.


 


Supporting study: Various In Vivo Endpoints: Read Across Source: Mn Substances – ATSDR (2008)


The ATSDR document represents a comprehensive and up to date review of the genotoxicity of manganese compounds. Manganese chloride did not produce somatic mutations in Drosophila melanogaster fruit flies in one study, and manganese sulphate did not induce sex-linked recessive lethal mutations in germ cells of male D. melanogaster. In vivo assays in mice showed that oral doses of manganese sulphate caused micronuclei and chromosomal aberrations in bone marrow. In contrast, oral doses of manganese chloride did not cause chromosomal aberrations in the bone marrow or spermatogonia of rats. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the material does not require classification with respect to genetic toxicity.