Ammonium iron(3+) hexakis(cyano-C)ferrate(4-)

Genetic toxicity: in vitro

Administrative data

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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 iron (3+) hexakis (cyano-C) ferrate (4-)

IUPAC Name:

Ammonium iron (3+) hexakis (cyano-C) ferrate (4-)

Constituent 2

Reference substance name:

Ammonium iron(3+) hexakis(cyano-C)ferrate(4-)

EC Number:

247-304-1

EC Name:

Ammonium iron(3+) hexakis(cyano-C)ferrate(4-)

Cas Number:

25869-00-5

Molecular formula:

C6FeN6.Fe.H4N

IUPAC Name:

ammonium iron(3+) hexakis(cyano-C)ferrate(4-)

Test material form:

other: solid

Details on test material:

- Name of test material : Ammonium iron (3+) hexakis (cyano-C) ferrate (4-)
- Physical state: solid
- Analytical purity: 93.6% (At first, 92.7% corrected for purity; then after treatment in the main test, the purity of the test substance was based on 93.6% according to information from the sponsor.)
- Lot/batch No.: K73-4766
- Stability under test conditions: the test substance was used within the expiration date (March 25, 2015; 5 years from the date of sending by the sponsor)
- Storage condition of test material: room temperature (actual temperature: 20.2 °C to 21.9 °C, permissible range: 10 °C to 30 °C) in an air-tight container

Method

Target gene:

thymidine-kinase locus (TK-locus)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-10 medium: 10 mL penicillin/ streptomycin solution (penicillin: 10,000 U/mL, streptomycin: 10,000 U/mL, Life Technologies Corp., Lot No. 584851), 5 mL of 4 w/v% sodium pyruvate (Wako Pure Chemical Industries, Ltd., Lot No. KWM0450) solution, and 5 mL of 10 v/v% pluronic solution (Life Technologies Corp., Lot No. 687835) were added to 1000 mL of RPMI1640 liquid medium (Life Technologies Corp., Lot No. 730665, 759165), 10 v/v%. inactivated Horse serum (Life Technologies Corp., 718041)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix treated with phenobarbital (PB, 4 times at 24-hour intervals at i.p. doses of 30, 60, 60, and 60 mg/kg, respectively) and 5,6-benzoflavone (once i.p. dose of 80 mg/kg on the same day of the third PB injection).

Test concentrations with justification for top dose:

3 h, –S9 mix assay: 39.4, 78.9, 158, 316, 631, 1262, 2524, and 5049 μg/mL

3 h, +S9 mix assay: 39.4, 78.9, 158, 316, 631, 1262, 2524, and 5049 μg/mL

24 h, –S9 mix assay: 39.4, 78.9, 158, 316, 631, 1262, 2524, and 5049 μg/m

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: CMC -Na (Sodium carboxymethyl cellulose);
- Justification for choice of solvent/vehicle: in the preliminary test for solubility, the test substance was not dissolved nor suspended properly in physiological saline at 50 mg/mL. The test substance was not dissolved nor suspended properly in neither dimethyl sulfoxide (Abbr.: DMSO) nor acetone at 500 mg/mL. The test substance was suspended properly in CMC-Na at 50 mg/mL.
Based on these results, CMC-Na was selected as the vehicle for the test substance and was used as the negative control substance in this study.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: 3 h, –S9 mix assay: MMS at 10 μg/mL and 24 h, –S9 mix assay: MMS at 5 μg/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: 3h,+S9 mix assay: CP at 3 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: whole wells counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

Statistics:

Statistical analysis for comparison with the negative control value:

The following statistical analysis for comparison with the negative control value was conducted for MF in the test substance treatment groups and the positive control substance treatment groups.

V = [Hs{1 – (ys / ns)}] / [ys {ln(ys / ns)}2] + [Hm {1 – (ym / nm)}] / [ym {ln(ym / nm)}2]

Hs (Hm): heterogeneity factor calculated from the data for PE2 (MF)
The value calculated by the following formula was used in the above calculation.
Hs (Hm)= Hsta (19/20) + Hexp (1/20)
Hsta: standard value
Hexp: mean value of H in the treatment group (except for omitted data)
ys (ym): number of empty wells in PE2 (MF) plates
ns (nm): total number of wells in PE2 (MF) plates
var(Dt) = Vt + Vc
Vt (Vc): V for the treatment group (for the negative control)
Dt = ln(MFt) – ln(MFc)
MFt (MFc): MF for the treatment group (for the negative control)

Dt2 / var(Dt) was calculated by the above calculated values.

When Dt2 / var(Dt) was more than the standard value (–S9 mix assay: 4.67, +S9 mix assay: 4.97, 24-hour assay: 5.48, positive control group in each treatment condition: 2.71), it was judged that the MF increase significantly (p<0.05) compared to the negative control.

Statistical analysis for dose-dependency:

The dose-dependency was examined for each treatment condition by the following formulas.
W = 1 / V (MF)2
SW = Σ [W]
SWM = Σ [W × MF]
SWD = Σ [W × D]
SWDD = Σ [W × D2]
SWDM = Σ [W × D × MF]
D: concentration of the test substance (g/mL)
S1 = SWDM – SWD × SWM / SW
S2 = SWDD – (SWD)2 / SW
b = S1 / S2
var(b) = 1 / S2

b2 / var(b) was calculated by the above calculated values.

When b2 / var(b) was more than the standard value (2.71), it was judged that there was significant dose-dependency (p<0.05).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5049 μg/mL in the 24-hour assay

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment group under each treatment condition.

RANGE-FINDING/SCREENING STUDIES:

The cell growth inhibition test was conducted at 39.4, 78.9, 158, 316, 631, 1262, 2524, and 5049 μg/mL for 3 hours in the absence and presence of rat liver S9 mix in the short-term treatment assay (–S9 mix assay and +S9 mix assay) and for 24 hours in the absence of S9 mix in the long-term treatment assay (24-hour assay).
As a result, the relative survival rate (%RS) was less than 20% at 1262 μg/mL and above in the 24-hour assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results of the main test [ PE0, % RS0] (-S9 mix assay):

Concentration (µg/ mL)*	Culture	EW	TW	PE0 (%)	% RS0 (%)
Negative control [ CMC-Na]	1	33	192	102.9	100.0
	2	41	192
316 (313)	1	39	192	108.2	105.1
	2	29	192
631 (625)	1	22	192	120.3	116.9
	2	34	192
1262 (1250)	1	31	192	106.4	103.4
	2	39	192
2524 (2500)	1	41	192	95.0	92.3
	2	43	192
5049 (5000)	1	51	192	91.4	88.8
	2	38	192
Positive control [MMS 10 µg/ mL]	1	152	192	41.1	39.9
	2	47	192

CMC- Na: 1% w/v sodium carboxymethylcellulose solution

MMS: Methyl methanesulfonate

EW: Number of empty wells (without colonies)

TW: Total number of wells

PE0: Plating efficiency at day 0.

% RS0: Relative survival rate at day 0.

Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment groups.

*. The parenthesis showed the concentration based on correction for purity at 92.7 %.

Table 2. Results of the main test [PE2, % RS2] (-S9 mix assay):

Concentration (µg/ mL)*	Culture	EW	TW	HGF^(1)	PE0 (%)	% RS0 (%)
Negative control [ CMC-Na]	1	19	192	18.817	105.5	100.0
	2	52	192
316 (313)	1	34	192	2.493	119.2	113.0
	2	23	192
631 (625)	1	33	192	0.075	112.0	106.1
	2	31	192
1262 (1250)	1	43	192	0.015	92.8	88.0
	2	44	192
2524 (2500)	1	30	192	3.281	102.9	97.5
	2	44	192
5049 (5000)	1	66	192	(2)	70.1	66.5
	2	59	192
Positive control [MMS 10 µg/ mL]	1	28	192	3.888	105.5	100.0
	2	43	192

CMC- Na: 1% w/v sodium carboxymethylcellulose solution

MMS: Methyl methanesulfonate

EW: Number of empty wells (without colonies)

TW: Total number of wells

PE2: Plating efficiency at day 2.

% RS2: Relative survival rate at day 2.

Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment groups.

*. The parenthesis showed the concentration based on correction for purity at 92.7 %.

 (1) Heterogeneity factor, standard value 1.8

(2) HGF was not calculated because data was not accepted.

Table 3. Results of the main test [RTG] (-S9 mix assay):

 

Concentration (µg/ mL)*	Culture	Cell concentration at the beginning of the incubation ( x10^4cells/ mL)	Cell concentration at about 24 h after cell treatment ( x10^4cells/ mL)	DCG-1	Cell concentration adjusted about 24 h after cell treatment ( x10^4cells/ mL)	Cell concentration at about 48 h after cell treatment ( x10^4cells/ mL)	DCG-2	RSG (%)	RTG (%)
Negative control [ CMC-Na]	1	20.0	52.0	2.60	17.3	78.1	4.51	100.0	100.0
	2	20.0	55.8	2.79	13.9	79.1	5.69
316 (313)	1	20.0	55.5	2.78	13.9	65.3	4.70	92.0	104.0
	2	20.0	56.4	2.82	14.0	61.3	4.35
631 (625)	1	20.0	53.8	2.69	13.4	77.4	5.78	114.5	121.5
	2	20.0	54.5	2.73	13.6	79.6	5.85
1262 (1250)	1	20.0	67.0	3.35	13.4	66.6	4.97	126.8	111.6
	2	20.0	70.4	3.52	14.1	73.1	5.18
2524 (2500)	1	20.0	58.5	2.93	14.6	47.5	3.25	75.2	73.3
	2	20.0	55.5	2.78	13.9	55.6	4.00
5049 (5000)	1	20.0	33.5	1.68	11.2	7.5	0.67	12.1	8.0
	2	20.0	35.9	1.80	12.0	14.9	1.24
Positive control [MMS 10 µg/ mL]	1	20.0	8.9	0.45	4.5	20.5	4.56	62.5	62.5
	2	20.0	63.8	3.19	15.9	78.0	4.91

DCG-1: Daily cell growth at day-1      RSG: Relative suspension growth

DCG-2: Daily cell growth at day-2      RTG: Relative total growth

 

Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment groups.

*. The parenthesis showed the concentration based on correction for purity at 92.7 %.

Table 4. Results of the main test [ PE0, % RS0] (+ S9 mix assay):

Concentration (µg/ mL)*	Culture	EW	TW	PE0 (%)	% RS0 (%)
Negative control [ CMC-Na]	1	43	192	88.0	100.0
	2	51	192
316 (313)	1	36	192	96.5	109.7
	2	46	192
631 (625)	1	50	192	85.4	97.0
	2	48	192
1262 (1250)	1	58	192	72.2	82.1
	2	63	192
2524 (2500)	1	57	192	73.2	83.2
	2	62	192
5049 (5000)	1	49	192	81.0	92.1
	2	56	192
Positive control [CP 3 µg/ mL]	1	91	192	45.3	51.5
	2	95	192

CMC- Na: 1% w/v sodium carboxymethylcellulose solution

CP: Cyclophosphamide monohydrate

EW: Number of empty wells (without colonies)

TW: Total number of wells

PE0: Plating efficiency at day 0.

% RS0: Relative survival rate at day 0.

Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment groups.

*. The parenthesis showed the concentration based on correction for purity at 92.7 %.

Table 5. Results of the main test [PE2, % RS2] (+ S9 mix assay):

Concentration (µg/ mL)*	Culture	EW	TW	HGF^(1)	PE0 (%)	% RS0 (%)
Negative control [ CMC-Na]	1	33	192	1.342	102.1	100.0
	2	42	192
316 (313)	1	39	192	0.016	98.8	96.8
	2	40	192
631 (625)	1	31	192	0.019	113.0	110.7
	2	32	192
1262 (1250)	1	33	192	0.711	166.0	113.7
	2	27	192
2524 (2500)	1	47	192	0.340	84.7	83.0
	2	52	192
5049 (5000)	1	59	192	6.083	86.0	84.2
	2	38	192
Positive control [MMS 10 µg/ mL]	1	88	192	0.168	50.2	49.2
	2	84	192

CMC- Na: 1% w/v sodium carboxymethylcellulose solution

CP: Cyclophosphamide monohydrate

EW: Number of empty wells (without colonies)

TW: Total number of wells

PE2: Plating efficiency at day 2.

% RS2: Relative survival rate at day 2.

Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment groups.

*. The parenthesis showed the concentration based on correction for purity at 92.7 %.

(1) Heterogeneity factor, standard value 1.8

 

Table 6. Results of the main test [RTG] (-S9 mix assay):

  

Concentration (µg/ mL)*	Culture	Cell concentration at the beginning of the incubation ( x10^4cells/ mL)	Cell concentration at about 24 h after cell treatment ( x10^4cells/ mL)	DCG-1	Cell concentration adjusted about 24 h after cell treatment ( x10^4cells/ mL)	Cell concentration at about 48 h after cell treatment ( x10^4cells/ mL)	DCG-2	RSG (%)	RTG (%)
Negative control [ CMC-Na]	1	20.0	60.3	3.02	15.1	76.3	5.05	100.0	100.0
	2	20.0	54.0	2.70	13.5	68.8	5.10
316 (313)	1	20.0	76.4	3.82	15.3	68.8	4.50	107.0	103.6
	2	20.0	61.4	3.07	15.3	69.0	4.51
631 (625)	1	20.0	83.4	4.17	13.9	63.5	4.57	109.0	120.6
	2	20.0	53.9	2.70	13.5	62.6	4.64
1262 (1250)	1	20.0	50.1	2.51	16.7	61.1	3.66	65.2	74.1
	2	20.0	40.8	2.04	13.6	63.4	4.66
2524 (2500)	1	20.0	35.6	1.78	17.8	72.5	4.07	58.4	48.5
	2	20.0	47.6	2.38	15.9	64.8	4.08
5049 (5000)	1	20.0	28.1	1.41	14.1	27.0	1.91	20.2	17.0
	2	20.0	34.4	1.72	17.2	31.4	1.83
Positive control [CP 3 µg/ mL]	1	20.0	40.8	2.04	13.6	76.5	5.63	76.8	37.8
	2	20.0	43.9	2.20	14.6	71.4	4.89

DCG-1: Daily cell growth at day-1      RSG: Relative suspension growth

DCG-2: Daily cell growth at day-2      RTG: Relative total growth

 

Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment groups.

*. The parenthesis showed the concentration based on correction for purity at 92.7 %.

Table 7. Results of the main test [PE0, % RS0] (24 - hour assay):

Concentration (µg/ mL)*	Culture	EW	TW	PE0 (%)	% RS0 (%)
Negative control [ CMC-Na]	1	25	192	116.0	100.0
	2	35	192
63.1 (62.5)	1	25	192	103.8	89.4
	2	48	192
126  (125)	1	34	192	98.0	84.5
	2	46	192
252 (250)	1	39	192	104.6	90.2
	2	33	192
505 (500)	1	29	192	114.0	98.2
	2	33	192
757 (750)	1	54	192	89.3	77.0
	2	38	192
1010 (1000)	1	88	192	60.0	51.7
	2	59	192
1262 (1250)	1	73	192	63.1	54.4
	2	67	192
2524 (2500)	1	134	192	23.4	20.2
	2	130	192
5049 (5000)	1	159	192	14.4	12.4
	2	146	192
Positive control [ MMS 5µg/ mL]	1	68	192	79.3	68.3
	2	40	192

CMC- Na: 1% w/v sodium carboxymethylcellulose solution

MMS: Methyl methanesulfonate

EW: Number of empty wells (without colonies)

TW: Total number of wells

PE0: Plating efficiency at day 0.

% RS0: Relative survival rate at day 0.

Precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment groups.

*. The parenthesis showed the concentration based on correction for purity at 92.7 %.

 

 

 

  

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative