Trimanganese tetraoxide

Genetic toxicity: in vitro

Administrative data

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:

2009-07-10 to 2009-08-04

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.

Justification for type of information:

A comprehensive literature review (Jenkinson P (2009), Manganese and its inorganic compounds: 2. Genotoxicity Aspects) showed no evidence of genetic toxicity for the registered substance, trimanganese tetraoxide. To support this, in-vitro and in-vivo genetic toxicity studies conducted on a more bioavailable substance (manganese chloride) were negative; therefore, according to REACH Annex XI section 1.2, a further study is not necessary

Cross-reference

Reason / purpose for cross-reference:

other: Target substance

Reference

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: Read-across

Justification for type of information:

A comprehensive literature review (Jenkinson P (2009), Manganese and its inorganic compounds: 2. Genotoxicity Aspects) showed no evidence of genetic toxicity for the registered substance, trimanganese tetraoxide. To support this, in-vitro and in-vivo genetic toxicity studies conducted on a more bioavailable substance (manganese chloride) were negative; therefore, according to REACH Annex XI section 1.2, a further study is not necessary.

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:

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 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 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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

manganese chloride

IUPAC Name:

manganese chloride

Constituent 2

Reference substance name:

Manganese dichloride

EC Number:

231-869-6

EC Name:

Manganese dichloride

Cas Number:

7773-01-5

Molecular formula:

Cl2Mn

IUPAC Name:

manganese(2+) dichloride

Details on test material:

- Molecular formula of test material : MnCl2 (Eramet)
- Substance type: Light pink solid flakes
- Physical state: Solid
- Analytical purity: > 95 %
- Lot/batch No.: 104896-05
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Histidine synthesis in the Salmonella typhimurium strains and tryptophan synthesis in the E. coli strain used.

Species / strain

Species / strain 1

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 2

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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: plate incorporation (Experiment 1) and preincubation (Experiment 2)

DURATION
- Preincubation period: 10 hours at 36 °C
- Exposure duration: 48 hours

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.

Results and discussion

Test results

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:

RANGE-FINDING/SCREENING STUDIES: The test material 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

Any other information on results incl. tables

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 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 5: 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 6: 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 7: 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.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

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.

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