Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Reverse Mutation Assay (e.g. Ames test)
Negative (TA98, TA100, TA1535, TA1537, WP2uvrA), MnCl2, OECD 471, Thompson & Bowles (2009)

- In Vitro Mammalian Chromosome Aberration Test
Negative (human lymphocyte cells), MnCl2, OECD 473, Morris & Durward (2009)

- In Vitro Mammalian Cell Gene Mutation
Negative (L5178Y cells), MnCl2, OECD 476, Flanders (2009)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 2009 to 4 August 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.
Reason / purpose for cross-reference:
other: Target
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
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:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
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
Remarks on result:
other: all strains/cell types tested

Table 2: 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 3: 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 4: 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 5: Test Results, Experiment 1 – Without Metabolic Activation

Test Period

From 19 July 2009

To 22 July 2009

Test Material (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 6: Test Results, Experiment 1 – With Metabolic Activation

Test Period

From 19 July 2009

To 22 July 2009

Test Material (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 7: Test Results, Experiment 2 – Without Metabolic Activation

Test Period

From 19 July 2009

To 22 July 2009

Test Material (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

* p≤0.05

 

Table 8: Test Results, Experiment 2 – With Metabolic Activation

Test Period

From 19 July 2009

To 22 July 2009

Test Material (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 with and without metabolic activation

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

The mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.13/14.

During the study test strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and a tester strain of Escherichia coli (WP2uvrA) were exposed to the test material both in the presence and the absence of metabolic activation.Vehicle and positive controls were run concurrently. Two separate experiments were conducted, in the first five concentrations of test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. In the second experiment the test material and vehicle control were dosed using a pre-incubation method.

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 any of the bacterial strains, with any dose of the test substance, 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 the increase was within the range specified by the Standard Test Method, and 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.

Therefore, under the conditions of the study the test material was concluded to be non-mutagenic.

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material.
Reason / purpose for cross-reference:
read-across source
Species / strain:
other: S. Typhimurium TA 1535, TA 1537, TA 98 and 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
Endpoint:
in vitro gene mutation study in bacteria
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Documentation contains an insufficient level of information to enable a complete assessment on the reliability of the reported results. The study was conducted with silicomanganese slag, rather than with the registered substance itself. Use of data generated with silicomanganese slag, to address information requirements of ferromanganese slag, is considered to be justified on the basis of the similar compositions of the two substances. Both substances are UVCB substances, containing metallic oxides; each substance is obtained as a by-product in the manufacture of SiMn and FeMn alloy, respectively.
Reason / purpose for cross-reference:
other: Target
Qualifier:
no guideline followed
Principles of method if other than guideline:
The method was a standard Ames.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Conclusions:
Interpretation of results: negative with and without metabolic activation

Under the conditions of the study the test material was concluded to be non-mutagenic to S. typhimurium TA 98 and S. typhimurium TA 100 with and without metabolic activation.
Executive summary:

An Ames test was conducted with the test material SiMn slag using the Salmonella typhimurium tester strains TA98 and TA100. The bacterial tester strains were exposed to the test material both in the presence and absence of metabolic activation.

Under the conditions of the study the test material was concluded to be non-mutagenic.

Use of data generated with silicomanganese slag, to address information requirements of ferromanganese slag, is considered to be justified on the basis of the similar compositions of the two substances. Both substances are UVCB substances, containing metallic oxides; each substance is obtained as a by-product in the manufacture of SiMn and FeMn alloy, respectively.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Reason / purpose for cross-reference:
read-across source
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
3 July 2009 to 24 September 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.
Reason / purpose for cross-reference:
other: Targat
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
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 S9, 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.
Remarks on result:
other: all strains/cell types tested

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.
Executive summary:

The potential of the test material to induce structural chromosomal aberrations in human lymphocyte cells in vitro was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 473.

During the study duplicate cultures of human lymphocyte cells, treated with test material, were evaluated for chromosome aberrations over at least three dose levels. Vehicle and positive controls were run concurrently. Three treatment conditions were used for the study, as follows: firstly, cultures were exposed for 4 hours with a 20 hour expression time, both in the presence and absence of metabolic activation (S9 mix), secondly cultures were continuously exposed for 24 hours in the absence of metabolic activation. The frequencies of chromosome aberrations in both vehicle and positive controls were within the expected range and verified the sensitivity of the assay and the efficacy of the S9-mix.

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.

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2 in line with the criteria of Klimisch (1997). Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
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
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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:
3 July 2009 to 20 October 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.
Reason / purpose for cross-reference:
other: Target
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

NUMBER OF REPLICATIONS:Each dose level was performed in duplicate

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

Plate scoring: Microtitre plates were scored using a magnifying mirror box after 10-14 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. 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.

% 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.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
EVALUATION OF RESULTS
For a test substance to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency over the concurrent vehicle mutant frequency value.
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 substance 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 substance 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 substance induces modest resproducible increases in the mutation frequencies that do not exceed the GEF value, then scientific judgement is 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.
Statistics:
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.
Remarks on result:
other: all strains/cell types tested

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.
Executive summary:

The mutagenic potential of the test material was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 476.

Based on the results from the preliminary toxicity test, the doses selected for treatment of the initial mutagenesis assay ranged from 2.5 to 120 µg/mL and 20 to 160 µg/mL for the S9 non-activated and activated cultures, respectively. 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. Toxicity in the cloned cultures was observed at doses at 120 and 160 µg/mL without and with S9 activation, respectively.

Based on the results of the preliminary toxicity test, the doses chosen for treatment of the extended treatment assay ranged from 0.31 to 15 µg/mL for non-activated cultures with a 24-hour exposure. Toxicity in the cloned cultures was observed at doses of 10 and 15 µg/mL.

Overall, 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.

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2.

Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
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
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In Vivo Micronucleus Assay

Negative (mouse erythrocytes), MnCl2, OECD 474, Streicker (2009)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15 April 2009 to 4 September 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.
Reason / purpose for cross-reference:
other: Target
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 animal 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 range 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:
- Validity criteria
For a negative result, the highest test substance concentration evaluated for MN induction must, unless precluded 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.

- Evaluation of results
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 substance:
A test substance is considered to induce a positive response if a dose-related increase in micronucleated polychromatic erythrocytes is observed and one or more doses are statistically elevated relative to the vehicle control.
A test substance is judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response was observed at any sampling time.
Statistics:
Statistical analysis was conducted on the frequency of MN-RET, MN-NCE and RET. Using individual animal data, the analysis involved 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

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 material 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 the 1-hour post-treatment observation period, male and female mice in the 500 mg/kg treatment groups exhibited lethargy, unco-ordinated 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 treatment levels of 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:

The potential of the test material (manganese chloride) to induce chromosomal damage in vivo was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 476.

The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess the toxicity of the test material and set dose levels for the definitive study consisted of a toxicity study followed supplemental toxicity study. The second phase was the definitive micronucleus study.

In the range finding phase, male and female mice were treated with Mn2 + at 1000, 500, 250 and 125 mg/kg bw. However, based on the high levels of toxicity observed on the first day of treatment, the study was discontinued and restarted using doses of 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. Therefore, based on the range-finding study, the selected doses for the definitive study were 25, 50, 100 and 200 mg/kg manganese. Female mice only were dosed in the definitive test.

In the definitive micronucleus study there were no significant increases in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in the female mice at any treatment level.

The results of the assay indicate that under the conditions of the study, administration of the test material at doses up to 200 mg/kg bw did not induce a significant increase in micronucleated polychromatic erythrocytes in female mice. Therefore, the test material was considered to be negative in this mouse micronucleus assay.

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2.

Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
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:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; the compound therefore represents a more bioavailable form of manganese. The below data are therefore considered to represent the worst case scenario in terms of exposure to manganese.

 

IN VITRO

- Bacterial Reverse Mutation Assay (e.g. Ames test)

The mutagenic potential of the test material, manganese dichloride, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.13/14.

During the study test strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and a tester strain of Escherichia coli (WP2uvrA) were exposed to the test material both in the presence and the absence of metabolic activation. Vehicle and positive controls were run concurrently. Two separate experiments were conducted, in the first five concentrations of test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. In the second experiment the test materials and vehicle control were dosed using a pre-incubation method.

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 the increase was within the range specified by the Standard Test Method, and 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.

Therefore, under the conditions of the study the test material was concluded to be non-mutagenic.

An Ames test, has also been conducted with silicomanganese slag using the Salmonella typhimurium tester strains TA98 and TA100. The bacterial tester strains were exposed to the test material both in the presence and absence of metabolic activation.

Under the conditions of the study the test material was concluded to be non-mutagenic.

Use of data generated with silicomanganese slag, to address information requirements of ferromanganese slag, is considered to be justified on the basis of the similar compositions of the two substances. Both substances are UVCB substances, containing metallic oxides; each substance is obtained as a by-product in the manufacture of SiMn and FeMn alloy, respectively. The finding from this study supports findings from the study with manganese chloride and further substantiates the argument for not repeating the studies with the registered substance itself.

 

- In Vitro Mammalian Chromosome Aberration Test

The potential of the test material, manganese dichloride, to induce structural chromosomal aberrations in human lymphocyte cells in vitro was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 473.

During the study duplicate cultures of human lymphocyte cells, treated with test material, were evaluated for chromosome aberrations over at least three dose levels. Vehicle and positive controls were run concurrently. Three treatment conditions were used for the study, as follows: firstly, cultures were exposed for 4 hours with a 20 hour expression time, both in the presence and absence of metabolic activation (S9 mix), secondly cultures were continuously exposed for 24 hours in the absence of metabolic activation. The frequencies of chromosome aberrations in both vehicle and positive controls were within the expected range and verified the sensitivity of the assay and the efficacy of the S9-mix.

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.

 

- In Vitro Mammalian Cell Gene Mutation

The mutagenic potential of the test material, manganese dichloride, was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 476.

Based on the results from the preliminary toxicity test, the doses selected for treatment of the initial mutagenesis assay ranged from 2.5 to 120 µg/mL and 20 to 160 µg/mL for the S9 non-activated and activated cultures, respectively. 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. Toxicity in the cloned cultures was observed at doses at 120 and 160 µg/mL without and with S9 activation, respectively.

Based on the results of the preliminary toxicity test, the doses chosen for treatment of the extended treatment assay ranged from 0.31 to 15 µg/mL for non-activated cultures with a 24-hour exposure. Toxicity in the cloned cultures was observed at doses of 10 and 15 µg/mL.

Overall, 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.

 

IN VIVO

- In Vivo Micronucleus Assay

The potential of the test material, manganese dichloride, to induce chromosomal damage in vivo was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 476.

The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess the toxicity of the test material and set dose levels for the definitive study consisted of a toxicity study followed supplemental toxicity study. The second phase was the definitive micronucleus study.

In the range finding phase, male and female mice were treated with Mn2 +at 1000, 500, 250 and 125 mg/kg bw. However, based on the high levels of toxicity observed on the first day of treatment, the study was discontinued and restarted using doses of 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. Therefore, based on the range-finding study, the selected doses for the definitive study were 25, 50, 100 and 200 mg/kg manganese. Female mice only were dosed in the definitive test.

In the definitive micronucleus study there were no significant increases in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in the female mice at any treatment level.

The results of the assay indicate that under the conditions of the study, a administration of the test material at doses up to 200 mg/kg bw did not induce a significant increase in micronucleated polychromatic erythrocytes in female mice. Therefore, the test material was considered to be negative in this mouse micronucleus assay.

 

All studies with manganese dichloride were conducted under GLP conditions and in accordance with standardised guidelines. Since the studies were conducted on a more bioavailable form of manganese, rather than on the registration substance itself, they have been assigned a reliability score of 2 in line with the criteria of Klimisch (1997). Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from these studies are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Justification for selection of genetic toxicity endpoint
Multiple studies have been provided to address the different endpoint of genetic toxicity, each addressing different types of genetic toxicity. Since all the studies showed negative results, a single study could not be selected as key over the others.

In accordance with Annex XI of REACH, Section 1.1 (use of existing data), a further study is not required. A comprehensive literature review, which is attached to this endpoint summary (Jenkinson P (2009), Manganese and its inorganic compounds: 2. Genotoxicity Aspects), showed no evidence of genetic toxicity for inorganic managnese. To support this, in-vitro and in-vivo genetic toxicity studies conducted on a more bioavailable substance (manganese chloride) were negative; these studies are therefore representative of a worst case following exposure to inorganic manganese.

Justification for classification or non-classification

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