Manganese Violet

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium manganese diphosphate, monoclinic and triclinic

Molecular formula:

MnKx (NH4)1-x P2O7 (0 =< x =< 0.5)

IUPAC Name:

Ammonium manganese diphosphate, monoclinic and triclinic

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: Fine violet powder
- Purity : > 99%
- Lot/batch No.: 124613
- Storage condition of test material: at room temperature, protected from humidity. Do not store near strong alkalis or flammable materials.

Method

Target gene:

TK Locus (Trifluorothymidine Resistance)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes.
- Periodically checked for Mycoplasma contamination: yes. Contamination by mycoplasma is checked using Mycoalert mycoplasma detection kit for each batch of the cells. Only the batches, which contain no mycoplasma, are used in the mutagenicity test.
- Periodically "cleansed" against high spontaneous background: yes. In order to maintain a low rate of spontaneous mutation, the L5178Y cells are purged of pre-existing TFT resistant cells according to the following procedure: 10E05 cells per mL are plated in 225 cm2 culture flasks in the RPMI 10 containing THMG (Cole et al., 1986):
9 μg/mL THYMIDINE
15 μg/mL HYPOXANTHINE
0.3 μg/mL METHOTREXATE
22.5 μg/mL GLYCINE
After 24 hours incubation at 37°C, the culture is centrifuged in order to eliminate methotrexate, the pellet is resuspended in RPMI 10 medium containing THG (Thymidine, Hypoxanthine, Glycine) at the same concentrations as those above mentioned. The cells are then incubated at 37°C, humidity near 95% and 5% of CO2 for 48 hours and subcultured 24 hours later at 3.105 cells per mL. After 48 hours of growing in THG medium, the cells are frozen and stored in liquid nitrogen at -196°C.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without S9 mix (3-hour treatment):
0.15 – 0.12 – 0.1 – 0.08 – 0.06 – 0.05 – 0.04 mM (assay 1)
0.17 – 0.15 – 0.13 – 0.11 – 0.09 – 0.08 mM (assay 2)
0.17 – 0.15 – 0.13 – 0.11 – 0.09 – 0.08 – 0.04 mM (assay 3)

Without S9 mix (24-hour treatment):
0.05 – 0.036 – 0.026 – 0.018 – 0.013 – 0.009 – 0.007 mM (assay 4)
0.040 – 0.036 – 0.033 – 0.030 – 0.027 – 0.025 – 0.012 mM (assay 5)

With S9 mix (3-hour treatment):
0.3 – 0.24 – 0.19 – 0.15 – 0.1 – 0.067 – 0.045 mM (assay 1)
0.6 – 0.5 – 0.41 – 0.34 – 0.29 – 0.24 – 0.2 mM (assay 2)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sodium monobasic phosphate buffer, pH 4
- Justification for choice of solvent/vehicle: Previous solubility assays were performed using 3 different solvents. When it was dissolved at 50 mg/mL in distilled water, the test item induced a homogeneous suspension which rapidly sedimented. In ethanol at 200 mg/mL, the test item induced a non homogeneous pasty suspension. Finally, at 100 mg/mL in DMSO, the test item induced a homogeneous suspension with very small grains that slowly sedimented in the tube. Concurrently, another solvent, i.e. a sodium monobasic phosphate buffer (pH 4) was assayed. It is noteworthy that the test item remains stable for pH < 7 in solution or culture medium.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
For a final volume of 20 mL of RPMI medium with 5% inactivated horse serum containing a suspension of 1 x 10E07 cells in 50 mL sterile centrifugation tubes, an appropriate volume (depending on the solvent used) of the test item in solution at each different concentration was added as well as 1 mL of 150 mM KCl. The treatment was performed under protection from light with stirring by means of a roller (20 rpm) for 3 hours at 37°C. At the end of the treatment period, the cells were washed twice with RPMI 10 medium and counted using a hemocytometer. The cells were then plated in RPMI 20 medium at a mean of 1.6 cells per well (2 plates of 96 wells per dose) for plating efficiency. The remaining cells were diluted at 2x10E05 cells per mL in RPMI 10 and cultured at 37°C.

In order to maintain exponential growth during all the expression time, on day 1 after treatment, the cells were counted and diluted in RPMI 10 medium to provide a cell density of 2x10E05 cells per mL. On day 2 (48 hours after the end of treatment), the cultures were counted again and part of the cells were plated in RPMI 20 at 1.6 cells per well (2 x 96-well microtiter plates per dose) to determine both the relative total growth and the plating efficiency at day 2, while the remaining cells were incubated in selective medium containing TFT (3 μg/mL) at 2 x10E03 cells per well (4*96-well microtiter plates per dose) to determine mutation frequency.

DURATION
- Exposure duration:
Without S9 mix: 3-hour treatment and 24-hour treatment
With S9 mix: 3-hour treatment
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): The cells were incubated at 37°C in a 5% CO2 incubator, with a humidity level close to 95% for 10-14 days, depending on the endpoint to be assessed. After the incubation time, plates containing cells in non-selective medium were scored for negative wells (i.e. wells without any viable colony) for assessing survival rate (RS0) and relative total growth. Plates containing TFT-selective medium were scored independently for "large" and "small" TFT resistant colonies using a binocular magnifying glass for determining mutation frequency

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicates

DETERMINATION OF CYTOTOXICITY
- Method: platting efficiency.
Short treatment (3 hours) and continuous treatment (24 hours)
Prior to the mutagenesis assay, a preliminary test was carried out to determine the cytotoxicity of the test item to L5178Y cells.
Short treatment: For a final volume of 10 mL of RPMI medium with 5% inactivated horse serum containing a suspension of 5 x 10E06 cells in 50 mL sterile centrifugation tubes, an appropriate volume of the test item in solution at each different concentration was added as well as 0.5 mL of 150 mM KCl.
Continuous treatment: For a final volume of 20 mL of RPMI 10 medium containing a suspension of 2.5 x 10E06 cells in 75 cm2 flasks, an appropriate volume of the test item in solution at each different concentration was added (KCl solution is not used in the continuous treatment).

The treatment was performed under the same conditions as in the mutation assay. After the 3-hour or 24-hour treatment, the surviving cells were counted by means of a hemocytometer. Part of the surviving cells were then re-plated in RPMI 20 medium (mean of 1.6 cells per well, 2 x 96-well microtiter plates per dose) and incubated at 37°C, humidity near 95% and 5% CO2. In parallel, the remaining cells were incubated at a density of 2x10E05 cells/mL in RPMI 10 medium for 48 hours. After 24-hour incubation (day 1), cell counting was performed and if necessary, the cell density was adjusted
at 2x10E05 cells/mL. Cell counting was repeated 24 hours later (day 2). The purpose of these cell countings was to detect a possible toxicity of the test item on cell growth (Turner et al., 1984). New plating efficiencies (2 x 96-well microtiter plates per dose at 1.6 cells per well) were concurrently performed in order to calculate the relative total growth (RTG) as described above. After 10 to 14 days, the number of empty wells containing no cell colonies was counted in order to calculate plating efficiencies

Evaluation criteria:

Each well of the mutation plates (in selective medium containing TFT) was scored as containing either a small colony, a large colony or no colony according to the following criteria:
SMALL COLONIES: colonies having a diameter less than 25% of the diameter of the well. A small colony should also have a dense colonal morphology and a clear contour.
LARGE COLONIES: colonies having a diameter greater than 25% of the diameter of the well. A large colony should show less densely packed cells, and blurred contour.
Any well containing one or more small colonies was scored as positive for small colony.
Any well containing one or more large colonies was scored as positive for large colony.
Any well containing a combination of large and small colonies was scored at the same time as large colony and small colony.
An empty well was one which contains no cell growth.

Statistics:

Statistical evaluation of data for the total number of mutants and for small colony mutants was performed using the method proposed by Robinson et al. (1990).
Briefly, the statistical analysis procedure includes the following steps:
• Test for consistency of duplicate cultures at each dose level for a single experiment. The limit is 10.8 times the current heterogeneity factor (H) (10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinite degrees of freedom. If the current heterogeneity factor is higher than 10.8 for survival heterogeneity factor (Hs) or mutation heterogeneity factor (Hm), this dose level is excluded from further consideration.
In case of heterogeneity in the negative control, the statistical evaluation of data is nevertheless performed.
• The new heterogeneity factor (Hexp) is calculated for doses not excluded. If it exceeds the value for that number of degrees of freedom in the experiment, the experiment may be discarded.
• For each dose level, mutant frequency (MF), log mutant frequency (LMF), the variance (V) of LMF and the weight (W) of MF are calculated for non-excluded dose levels (the value of MF could be slightly different in statistically evaluation compared to recapitulative tables because MF is calculated on pooled independent cultures, both series A and B).
• Each treatment is compared with the control by means of one-sided Dunnett's test for multiple comparisons with the same control.
• Test for linear trend of mutant frequency with dose is performed. The slope b and its variance var (b) are calculated to form the test statistic b2/var (b) that should be compared with tabulated critical values of chi2 with 1 degree of freedom.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at the highest concentrations)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at the highest concentrations)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS WITH METABOLIC ACTIVATION (assays 1 and 2)
During two independent assays performed with metabolic activation, no significant increase in the mutation frequency of total induced mutants (small and large colonies) or in the mean number of small colonies and in the mutation frequency of small colony mutants was noted at any concentrations tested in the presence of the test item.

RESULTS WITHOUT METABOLIC ACTIVATION (assays 1 and 2)
In return, in both independent assays performed without metabolic activation using a short-term treatment of 3 hours, statistically and biologically significant increases in the mutation frequency of total induced mutants were noted at 1 or 3 non toxic concentrations tested, with a dose-effect relationship associated with a statistically significant linear trend. The test item was thus considered as mutagenic in the 3-hour treatment without metabolic activation.

EFFECTS OF pH (assays 3, 4 and 5)
3-hour treatment (assay 3)
In order to check the possible implication of a potential degradation of the test item, a 3rd assay under the same experimental conditions but at lower pH-values (in the acceptable range of pH for this test system) was performed. A statistically and biologically significant increase in the mutation frequency of total induced mutants was noted at the highest non-toxic concentration tested of 0.17 mM, associated with a statistically significant linear trend. The mutagenic effect previously observed in two independent assays was confirmed when using experimental conditions ensuring the maintenance of pH<7 all along the treatment period.

24-hour treatment (assay 4)
In order to confirm, or not, these effects when using an extended time of exposure, i.e. 24-hour treatment, a complementary assay under this condition was carried out. A statistically and biologically significant increase in the mutation frequency of total induced mutants was noted at the highest concentration tested of 0.036 mM. However this concentration induced a very strong toxicity, and no concentration inducing an adjusted RTG comprised between 10 and 20% was available. It was thus decided to reiterate this assay but with a narrower range of concentrations in order to definitively conclude.

24-hour treatment (assays 5)
Statistically and biologically significant increases in the mutation frequency of total induced mutants were noted at both two highest concentration tested of 0.030 and 0.027 mM (that induced adjusted RTG acceptable or very close to the lowest accepted threshold for the mutagenicity assessment) with IMFs of +250.1 and +145.9 x10-6 mutants (i.e. clearly higher than +126 x10-6 mutants).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without metabolic activation after a 3-hour treatment, the test item revealed a high level of cytotoxicity in L5178Y cells at the 5 highest concentrations tested from 10 to 0.625 mM with no cell growth. The two inferior concentrations of 0.313 and 0.156 mM induced a very strong level of cytotoxicity with adjusted RTGs of 0.05 and 2.8%, respectively. The concentration of 0.15 was retained as the maximum concentration to be tested for the first mutagenicity test without S9-mix following a 3-hour treatment, and a large and narrowed range of concentrations was tested.

With metabolic activation, a very strong cytotoxicity in L5178Y cells at the 4 highest concentrations tested from 10 to 1.25 mM with no cell growth. The two inferior concentrations of 0.625 and 0.313 mM induced a very strong level of cytotoxicity with adjusted RTGs of 0.05 and 0.5%, respectively. The concentration of 0.156 mM induced a moderate cytotoxicity with an adjusted RTG of 30.9%. The concentration of 0.3 mM was choosen as the maximum concentration to be tested in the first assay with S9-mix.

Without metabolic activation after a 24-hour treatment, all the concentrations tested ranging from 0.156 to 10 mM induced a potent cytotoxicity in L5178Y cells, with no cell growth for concentrations ranging from 10 to 0.625 mM. So, no toxicity data were available at these concentrations. The assay was thus reiterated with lower concentrations (i.e. from 0.15 to 0.04 mM). In the new assay, a potent cytotoxicity was noticed on the whole range of concentrations with 0.1 to 7.4% of adjusted RTG.

Any other information on results incl. tables

Table 1: Assay 1 without S9

 

ASSAY 1 WITHOUT S9 3-Hour treatment	Concentrations in mM	0	0.04	0.05	0.06	0.08	0.10	0.12	0.15	MMS 10 μg/mL
	Adjusted RTG ( Relative total growth )	100	135.2	83.6	98.4	50.0	45.6	30.8	14.3	83.6
	MUTATION FREQUENCY x 10-6 cells	128.0	107.5	180.4	164.3	227.7	263.1	307.7	307.8	591.3
	Mean Induction Ratio	-	0.8	1.4	1.3	1.8	2.1	2.4	2.4	4.6
	Induced Mutation Frequency x 10-6 cells	-	-20.5	52.4	36.3	99.7	135.1	179.7	179.8	463.3
	Statistical significance (Dunnett's test)	-	N.S.	N.S.	N.S.	<0.05	*	*	<0.05	<0.05

 

Table 2: Assay 2 without S9

 

ASSAY 2 WITHOUT S9 3-Hour treatment	Concentrations in mM	0	0.08	0.09	0.11	0.13	0.15	0.17	MMS 10 μg/mL
	Adjusted RTG ( Relative total growth )	100	54.7	47.4	30.0	25.0	14.6	6.1	94.9
	MUTATION FREQUENCY x 10-6 cells	114.8	168.0	187.7	216.4	221.0	302.2	**	540.0
	Mean Induction Ratio	-	1.5	1.6	1.9	1.9	2.6	**	4.7
	Induced Mutation Frequency x 10-6 cells	-	53.3	73.0	101.6	106.3	187.4	**	425.3
	Statistical significance (Dunnett's test)	-	N.S.	<0.05	<0.05	<0.05	*	**	<0.05

 

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

**: Adjusted RTG being inferior to the limits accepted for mutagenicity analysis, this concentration was not taken into account in the interpretation of the results

 

Table 3: Assay 1 with S9

 

ASSAY 1 WITH S9 3-Hour treatment	Concentrations in mM	0	0.045	0.067	0.10	0.15	0.19	0.24	0.30	CPA 2 μg/mL
	Adjusted RTG ( Relative total growth )	100	110.8	109.6	142.0	118.1	95.8	77.6	27.0	92.9
	MUTATION FREQUENCY x 10-6 cells	122.1	117.4	117.1	79.3	92.9	95.3	78.1	146.0	428.6
	Mean Induction Ratio	-	1.0	1.0	0.6	0.8	0.8	0.6	1.2	3.5
	Induced Mutation Frequency x 10-6 cells	-	-4.8	-5.1	-42.8	-29.2	-26.9	-44.0	23.9	306.5
	Statistical significance (Dunnett's test)	-	N.S.	N.S.	N.S.	*	*	*	N.S.	<0.05

Table 4: Assay 2 with S9

ASSAY 2 WITH S9 3-Hour treatment	Concentrations in mM	0	0.20	0.24	0.29	0.34	0.41	0.50	0.60	CPA 2 μg/mL
	Adjusted RTG ( Relative total growth )	100	46.8	29.4	28.0	2.4	1.8	0.8	0.5	95.8
	MUTATION FREQUENCY x 10-6 cells	154.2	219.1	230.9	172.8	185.7	**	**	**	690.9
	Mean Induction Ratio	-	1.4	1.5	1.1	1.2	**	**	**	4.5
	Induced Mutation Frequency x 10-6 cells	-	64.9	76.7	18.6	31.5	**	**	**	536.7
	Statistical significance (Dunnett's test)	-	N.S.	N.S.	N.S.	*	**	**	**	<0.05

 N.S.: Not statistically significant (with a = 0.05 critical value); CPA: cyclophosphamide

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

**: Adjusted RTG being inferior to the limits accepted for mutagenicity analysis, this concentration was not taken into account in the interpretation of the results

Table 5: Assay 3 without S9 (3 -hour treatment, pH < 7)

ASSAY 3 WITHOUT S9 3-Hour treatment	Concentrations in mM	0	0.04	0.08	0.09	0.11	0.13	0.15	0.17	MMS 10 μg/mL
	Adjusted RTG (Relative total growth )	100	96.0	68.1	62.7	57.4	39.5	33.9	22.2	60.8
	MUTATION FREQUENCY x 10-6 cells	137.8	146.7	149.3	150.7	181.6	242.0	215.3	336.5	765.3
	Mean Induction Ratio	-	1.1	1.1	1.1	1.3	1.8	1.6	2.4	5.6
	Induced Mutation Frequency x 10-6 cells	-	8.9	11.5	13.0	43.8	104.2	77.5	198.7	627.5
	Statistical significance (Dunnett's test)	-	*	N.S.	N.S.	*	<0.05	N.S.	<0.05	<0.05

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

Table 6: Assay 4 without S9 (24 -hour treatment, pH < 7)

ASSAY 4 WITHOUT S9 24-Hour treatment	Concentrations in mM	0	0.007	0.009	0.013	0.018	0.026	0.036	MMS 2 μg/mL
	Adjusted RTG (Relative total growth )	100	118.0	120.7	117.3	90.6	57.4	7.5	127.1
	MUTATION FREQUENCY x 10-6 cells	185.9	189.5	152.7	160.8	206.2	261.4	513.6	656.3
	Mean Induction Ratio	-	1.0	0.8	0.9	1.1	1.4	2.8	3.5
	Induced Mutation Frequency x 10-6 cells	-	3.6	-33.2	-25.1	20.3	75.5	327.7	470.4
	Statistical significance (Dunnett's test)	-	N.S.	N.S.	N.S.	N.S.	N.S.	<0.05	<0.05

 

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

Table 7: Assay 5 without S9 (24 -hour treatment, pH < 7)

ASSAY 5 WITHOUT S9 24-Hour treatment	Concentrations in mM	0	0.012	0.025	0.027	0.030	0.033	MMS 2 μg/mL
	Adjusted RTG (Relative total growth )	100	66.8	27.6	22.2	9.8	6.2	184.5
	MUTATION FREQUENCY x 10-6 cells	127.2	215.1	218.8	273.2	377.3	**	365.2
	Mean Induction Ratio	-	1.7	1.7	2.1	3.0	**	2.9
	Induced Mutation Frequency x 10-6 cells	-	87.9	91.5	145.9	250.1	**	237.9
	Statistical significance (Dunnett's test)	-	<0.05	<0.05	*	<0.05	**	<0.05

Mean Induction ratio = Mutation frequency (treated) / Mutation frequency (control)

Induced Mutation Frequency = Mutation frequency (treated) - Mutation frequency (control)

*: Because of heterogeneity between the 2 cultures, statistical analysis could not be assessed

N.S.: Not statistically significant (with α = 0.05 critical value); MMS: methylmethanesulfonate

Applicant's summary and conclusion

Conclusions:

Under these experimental conditions, the test item induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture without metabolic activation, in 5 independent assays following either a short or a continuous treatment. Furthermore, the clear increase in the number of small colonies is rather in favour of a clastogenic activity. In return, neither biologically nor statistically significant mutagenic activity was observed in L5178Y mouse lymphoma cell culture after treatments with the test item in presence of metabolic activation, during two independent assays.

Executive summary:

The search for any mutagenic activity of the test item was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 476, in 2 independent assays with metabolic activation, 3 independent assays without metabolic activation following a short treatment and 2 independent assays without metabolic activation following a continuous treatment. The acceptance criteria for the assay were fulfilled. The current study was considered as valid. Under these experimental conditions, the test item induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture without metabolic activation, in 5 independent assays following either a short or a continuous treatment. Furthermore, the clear increase in the number of small colonies is rather in favour of a clastogenic activity. In return, neither biologically nor statistically significant mutagenic activity was observed in L5178Y mouse lymphoma cell culture after treatments with the test item in presence of metabolic activation, during two independent assays.