Dimanganese trioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available for the target substance. Thus, to assess the genotoxic potential of dimanganese trioxide, data from manganese dichloride (source substance) was used in read-across approach. The source substance was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, OECD 473 and OECD 490, GLP).

In a bacterial reverse gene mutation test conducted according to OECD 471, the source substance did not induce mutagenicity. In a mammalian cell mutation assay conducted according to OECD 490, the source substance was also tested negative for inducing mutagenic effects. Moreover, the manganese dichloride was tested negative in an in vitro cytogenicity study in human lymphocytes conducted according to OECD 473. Based on the available data from the read-across partner, the target substance dimanganese trioxide can be considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For justification of read-across please refer to the read-across statement in IUCLID 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

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/10^6viable 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)	24-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:

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:

In a mammalian cell gene mutation assay conducted in accordance to OECD guideline 476 (nowadays OECD 490), L5178Y mouse lymphoma cells cultured in vitro were exposed to manganese dichloride (> 95% purity), solved in R0 medium. Test concentrations chosen for the main experiments were based on the results from a preliminary toxicity test. In the first main experiment, the test item was tested in the presence of metabolic activation up to 160 µg/mL and in the absence of metabolic activation up to 120 µg/mL. The incubation time was 4 hours. In the second main experiment, the test item was tested in the absence of metabolic activation up to 15 µg/mL. The incubation time was 24 hours. Cytotoxicity were observed in all main experiments. The positive controls did induce the approriate response. 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.

This information is used in a read-across approach in the assessment of the target substance. 

For justification of read-across please refer to the attached read-across statement (see IUCLID section 13).

Reference 2

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:

weight of evidence

Justification for type of information:

For justification of read-across please refer to the read-across statement in IUCLID 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

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.

TEST RESULTS:
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.

Table 2: Mitotic index – 4(20)-Hours

Dose Level µg/mL	4 Hours Treatment Without S9				4 Hours Treatment With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	5.70	7.10	6.40	100	5.00	3.80	4.40	100
9.84 (78.75)^a	-	-	-	-	-	-	-	-
19.69 (157.5)^a	5.25	5.95	5.60	88	4.10	3.00	3.55	81
39.38 (315)^a	5.70	5.35	5.53	86	3.50	2.85	3.18	72
78.75 (630)^a	3.30	3.40	3.35	52	1.90 P	3.10 P	2.50	57
157.5 (945)^a	2.25	1.80	2.03	32	1.20 P	2.40 P	1.80 P	41
315 (1260)^a	NM P	NM P	NM P	0	1.15 P	2.40 P	1.78 P	40
MMC 0.4	1.75	2.20	1.98	31	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	0.75	0.85	0.80	18

a= dose levels with S9

MMC= mitomycin C

CP= cyclophosphamide

P= precipitate

NA= not applicable

-= not assessed for mitotic index

NM= no scoreable metaphases

Table 3: Mitotic Index – 24 Hours

Dose Level µg/mL	24 Hours Treatment Without S9
	A	B	Mean	% of Control
0	3.45	2.65	3.05	100
1.23	3.85	3.70	3.78	124
2.46	3.35	3.85	3.60	118
4.92	2.90	2.65	2.78	91
9.84	1.85	1.80	1.83	60
19.69	NM	NM	NM	0
30.38	NM	NM	NM	0
MMC 0.2	1.25	0.95	1.10	36

 NM = No scorable metaphases

MMC = Mitomycin C

Table 4: Results of Chromosome Aberration Test – 4(20)-Hours Without Metabolic Activation (S9)

Treatment Group	Replicable	Mitotic Index (%)	Number of Cells Scored	Number of Aberrations						Total Number of Aberrations		Frequency of Aberrant Cells (%)
				Gaps	Chromatid		Chromosome		Others X	(+ Gaps)	(-Gaps)	(+ Gaps)	(-Gaps)
					Breaks	Exchanges	Breaks	Exchanges
Vehicle Group	A	5.70	100	0	0	0	0	0	0	0	0	0	0
	B	7.10	100	1	0	0	0	0	0	1	0	1	0
	Total	(100)	200	1	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)
19.59 µg/mL	A	5.25	100	0	0	0	0	0	0	0	0	0	0
	B	5.95	100	0	1	0	0	0	0	1	1	1	1
	Total	(88)	200	0	1	0	0	0	0	1	1	1 (0.5)	1 (0.5)
39.38 µg/mL	A	5.70	100	0	0	0	0	0	0	0	0	0	0
	B	5.35	100	0	0	0	0	0	0	0	0	0	0
	Total	(86)	200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)
78.75 µg/mL	A	3.30	100	0	0	0	0	0	0	0	0	0	0
	B	3.40	100	0	1	0	0	0	0	1	1	1	1
	Total	(52)	200	0	1	0	0	0	0	1	1	1 (0.5	1 (0.5)
Positive Control MMC 0.4 µg/mL	A	1.75	50^a	2	24	17	2	0	0	45	43	25	28
	B	2.20	50^a	7	18	15	2	1	0	13	36	26	25
	Total	(31)	100	9	42	32	4	1	0	88	79	54 (54.0)	53*** (53.0)

MMC = Mitomycin C

a = Slide evaluation terminated at 50 cells because approximately 30-50 % of cells with aberrations had been observed

*** = P < 0.001

Table 5: Results of Chromosome Aberration Test – 4(20)-Hours With Metabolic Activation (S9)

Treatment Group	Replicable	Mitotic Index (%)	Number of Cells Scored	Number of Aberrations						Total Number of Aberrations		Frequency of Aberrant Cells (%)
				Gaps	Chromatid		Chromosome		Others X	(+ Gaps)	(-Gaps)	(+ Gaps)	(-Gaps)
					Breaks	Exchanges	Breaks	Exchanges
Vehicle Group	A	5.00	100	0	0	0	0	0	0	0	0	0	0
	B	3.80	100	1	0	0	0	0	0	1	0	1	0
	Total	(100)	200	1	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)
157.5 µg/mL	A	4.10	100	0	1	0	0	0	0	1	1	1	1
	B	3.00	100	1	0	0	0	0	0	1	0	1	0
	Total	(81)	200	1	1	0	0	0	0	2	1	2 (1.0)	1 (0.5)
315 µg/mL	A	3.50	100	1	0	0	0	0	0	1	0	1	0
	B	2.85	100	0	0	0	0	0	0	0	0	0	0
	Total	(72)	200	1	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)
630 µg/mL	A	1.90	100	0	1	0	0	0	0	2	2	2	2
	B	3.10	100	1	0	0	0	0	0	1	0	1	0
	Total	(57)	200	1	1	0	0	0	0	3	2	3 (1.5)	2 (1.0)
945 µg/mL	A	1.20	100	3	5	0	0	0	0	8	5	7	4
	B	2.40	100	1	2	0	0	0	0	3	2	3	2
	Total	(41)	200	4	7	0	0	0	0	11	7	10 (5.0)	6* (3.0)
Positive Control CP 5 µg/mL	A	0.75	100	2	18	7	2	0	0	29	27	22	22
	B	0.85	100	6	30	2	11	0	0	49	43	31	30
	Total	(18)	200	8	48	9	13	0	0	78	70	53 (26.5)	52*** (26.0)

CP = Cyclophosphamide

* = P < 0.05

*** = P < 0.001

Table 6: Results of Chromosome Aberration Test – 24-Hours Without Metabolic Activation (S9)

Treatment Group	Replicable	Mitotic Index (%)	Number of Cells Scored	Number of Aberrations						Total Number of Aberrations		Frequency of Aberrant Cells (%)
				Gaps	Chromatid		Chromosome		Others X	(+ Gaps)	(-Gaps)	(+ Gaps)	(-Gaps)
					Breaks	Exchanges	Breaks	Exchanges
Vehicle Group	A	3.45	100	0	0	0	0	0	0	0	0	0	0
	B	2.65	100	2	1	0	0	0	0	3	1	3	1
	Total	(100)	200	2	1	0	0	0	0	3	1	3 (1.5)	1 (0.5)
19.59 µg/mL	A	3.35	100	1	0	0	1	0	0	2	1	2	0
	B	3.85	100	1	0	0	0	0	0	1	0	1	1
	Total	(118)	200	2	0	0	1	0	0	3	1	3 (1.5)	1 (0.5)
39.38 µg/mL	A	2.90	100	0	2	0	3	0	0	5	5	3	3
	B	2.65	100	1	2	0	1	0	0	4	3	3	3
	Total	(91)	200	1	4	0	4	0	0	9	8	6 (3.0)	6 (3.0)
78.75 µg/mL	A	1.85	100	1	8	0	0	0	0	9	8	7	7
	B	1.80	100	5	5	0	0	0	0	10	5	7	5
	Total	(60)	200	6	13	0	0	0	0	19	13	14 (7.0)	12** (6.0)
Positive Control MMC 0.4 µg/mL	A	1.25	50^a	0	21	32	2	0	1	56	56	34	34
	B	0.96	50^a	4	32	14	6	0	0	56	51	33	32
	Total	(36)	100	4	53	46	8	0	1	112	108	67 (67.0)	66*** (66.0)

MMC = Mitomycin C

a = Slide evaluation terminated at 50 cells because approximately 30-50 % of cells with aberrations had been observed

** = P < 0.01

*** = P < 0.001

Table 7: Mean Frequency of Polyploid Cells (%)

Dose Level µg/mL	Harvest Time 24 Hours
	4 Hours Without S9	4 Hours With S9	24 Hours Without S9
0	1.0	0.5	0.5
2.46 (157.5)^a	NA	0.0	0.0
4.92 (315)^a	NA	0.0	0.0
9.84 (630)^a	-	0.0	0.0
19.69 (945)^a	0.0	0.0	-
39.38	0.0	NA	-
78.75	0.5	NA	NA
MMC 0.2	NA	NA	0.0
MMC 0.4	0.0	NA	NA
CP5	NA	0.0	NA

 a = Dose levels with S9

MMC = Mitomycin C

CP = Cyclophosphamide

NA – Not applicable

- = Not determined

Conclusions:

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:

In a mammalian cell cytogenetic assay conducted according to OECD guideline 473, human peripheral lymphocyte cultures were exposed to manganese dichloride (> 95% purity), solved in MEM cell culture medium. Concentrations chosen for the main experiments were based on the results from a preliminary toxicity test. In the first main experiment, cells were treated for 4 hours in the presence and absence of metabolic activation. In the main second experiment cells were treated for 24 hours with metabolic activation. Afterwards, slides were prepared, stained with 5% Giemsa and where possible the first 100 consecutive well-spread metaphases from each culture were counted. All 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. Based on the results, manganese dichloride is considered to be not clastogenic in human peripheral lymphocytes. This study is classified as acceptable and satisfies the requirement for the in vitro mammalian chromosomal aberration test according to OECD 473.

This information is used in a read-across approach in the assessment of the target substance. 

For justification of read-across please refer to the attached read-across statement (see IUCLID section 13).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For justification of read-across please refer to the read-across statement in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: S. typhimurium TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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.

RESULTS OF THE STUDY:
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.

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 * p≤0.05

 

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

 

Conclusions:

In conclusion, manganese chloride is not genotoxic in the bacterial reverse gene mutation assay (OECD 471) in the presence and absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria conducted according to OECD guideline 471, S. typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA were exposed to manganese dichloride (> 95% purity) at concentrations of 0, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

The test item was tested up to the limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation assay).

This information is used in a read-across approach in the assessment of the target substance. 

For justification of read-across please refer to the attached read-across statement (see IUCLID section 13).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data is available for the target substance. Thus, to assess the genotoxic potential of dimanganese trioxide, data from manganese dichloride (source substance) was used in read-across approach. The source substance was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, OECD 473 and OECD 490, GLP).

In a bacterial reverse gene mutation test conducted according to OECD 471, the source substance did not induce mutagenicity. In a mammalian cell mutation assay conducted according to OECD 490, the source substance was also tested negative for inducing mutagenic effects. Moreover, the manganese dichloride was tested negative in an in vitro cytogenicity study in human lymphocytes conducted according to OECD 473. Based on the available data from the read-across partner, the target substance dimanganese trioxide can be considered to be non-genotoxic.

Justification for classification or non-classification

Based on the lack of mutagenicity/cytogenicity in all conducted in vitro assays with the source substance, dimanganese trioxide is considered to be non-genotoxic and thus no classification is warranted in accordance with CLP Regulation 1272/2008.