Sodium [[α,α'-(ethylenediimino)bis[2-hydroxybenzene-1-acetato]](4-)]ferrate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The source substance Fe(Na)EDDHA was examined in Bacterial Reverse Mutation Assays, in in vitro Mammalian Chromosome Aberration Test and in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay). The tests were performed with and without metabolic activation. The substance did not induce gene mutations by frameshift or base-pair substitution in the examined strains in the Ames test. Fe(Na)EDDHA tested up to cytotoxic concentrations did not induce structural chromosome aberrations in Chinese Hamster ovary cells and was therefore not considered as clastogenic in the tested system. Finally, Fe(Na)EDDHA showed no mutagenic effect in a Mouse Lymphoma assay. Therefore, the target substance o-o-EDDHA (CAS 16455-61-1) considered as non genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993-11-18 to 1994-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100, TA 102) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat-liver post mitochondrial supernatant

Test concentrations with justification for top dose:

Range-finding test: 20.6-5000 µg/plate;
Original experiment: 312.5 - 5000 µg/plate;
Confirmatory experiment: 312.5 - 5000 µg/plate.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA 100, TA 102, TA 98, TA 1537, WP2 uvrA with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

TA 1534 with metabolic activation

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without metabolic activation

Positive control substance:

other: 4-Nitroquinoline (4-NQO)

Remarks:

WP2 uvrA without metabolic activation

Positive control substance:

mitomycin C

Remarks:

TA 102 without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 plates per test substance concentration and control

Evaluation criteria:

Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positive response:
The test substance will be considered to be positive in the test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA 98, TA 1535, TA 1537, E.coli WP2 uvrA
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strains TA 100 or TA 102.
Generally a concentration-related effect should be demonstrable.

Statistics:

Not performed

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

Species / strain:

S. typhimurium TA 102

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:

E. coli WP2 uvr A

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:
Six concentrations of CGA 65047 SG 100, (A-5787 A) ranging from 20.6 - 5000 µg/plate were tested with strain Salmonella typhimurium TA100 and strain Escherichia coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. Although the test substance was applied as a fine homogeneous suspension, no precipitates were detectable on the plates. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 mg/plate with and 5000 µg/plate without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Analytical control:

To confirm that the cells were actually exposed to the intened test concentrations and to confirm the stability of the test substance in the vehicle used, determination of the concentration of the test substance in solution was performed by UV/VIS-spectroscopy. The values found by analysis of the different samples were in agreement with the intended concentrations (between 90.2 and 96.1 %), thus demonstrating a sufficient stability of the test substance in the vehicle.

There were no known or occurrences in this study that were considered to have affected the quiality or integrity of the test data.

Table: Original experiment

With(+) or without(-) S9 Mix	Test substance concentration (Mg/plate)	Number of revertants (number of colonies/plate)
		Base-pair substitution type				Frameshift type
		TA 100	TA 1535	UP2 uvrA	TA 102	TA 98	TA 1537
S9 Mix (+)	Solvent control	173 183 185  (180)	33 16 21  (23)	36 33 36  (35)	357 360 282  (333)	53 40 51  (48)	8 16 7 (10)
	312.50	185 197 187  (190)	19 18 25  (21)	40 41 43  (41)	330 331 321  (327)	48 41 52  (47)	7 8 8 (8)
	625.00	181 195 157  (178)	21 20 19  (20)	36 40 49  (42)	344 357 337  (346)	45 68 60  (58)	8 16 13  (12)
	1250.00	192 196 180  (189)	28 25 15  (23)	25 28 30  (28)	342 328 331  (334)	51 52 50  (51)	13 7 12  (11)
	2500.00	171 182 189  (181)	25 19 18  (21)	44 43 32  (40)	379 365 360  (368)	57 48 51  (52)	9 14 6  (10)
	5000.00	177 180 173  (177)	27 13 17  (19)	39 48 40 (42)	336 376 354  (355)	52 44 52  (49)	16 7 6  (10)
S9 Mix (-)	Solvent control	161 127 168  (152)	24 17 24  (22)	41 41 24  (35)	222 255 206  (228)	44 27 36  (36)	18 13 8  (13)
	312.50	134 149 157  (147)	25 19 27  (24)	27 32 41  (33)	128 194 218  (180)	29 26 27  (27)	8 19 16  (14)
	625.00	151 161 174  (162)	27 25 24  (25)	29 31 49  (36)	293 351 314  (319)	36 44 41  (40)	12 15 17  (15)
	1250.00	137 140 162  (146)	19 24 18  (20)	26 25 37  (29)	276 308 368  (317)	44 33 38  (38)	14 12 16  (14)
	2500.00	157 151 168  (159)	17 19 20  (19)	33 40 28  (34)	234 256 236  (242)	30 41 30  (34)	17 15 13  (15)
	5000.00	149 159 164  (157)	21 24 15  (20)	30 30 36  (32)	72 141 240  (151)	32 21 38  (30)	16 16 14  (15)
Positive control requiring S9 Mix	Name	2-Amino-anthracene	Cyclophosphamide	2-Amino-anthracene	2-Amino-anthracene	2-Amino-anthracene	2-Amino-anthracene
	Concentration (µg/plate)	2.50	400.00	50.00	20.00	2.50	2.50
	Number of colonies/plate	1284 916 1466 (1222)	243 344 410  (332)	737 847 894  (826)	789 1028 1500 (1106)	595 925 740  (753)	139 167 147  (151)
Positive control not requiring S9 Mix	Name	Sodium azide	Sodium azide	4-N00	Hitomycin-C	2-Nitro-fluorene	9-Amino-acridine
	Concentration (µg/plate)	5.00	5.00	2.00	2.00	20.00	150.00
	Number of colonies/plate	834 831 676  (780)	953 928 900  (927)	552 620 488  (553)	1497 1364 571 (1144)	1805 1808 1686 (1766)	1704 1357 1707 (1589)
Notes: 1. When inhibition is found against growth of the bacteria, mark the applicable value with an asterix 2. Fill the average number of colonies in each concentration in the () 3. "Number of revertants"-Fi11 in the observed value and average value in order beginning with low concentration of the test substance

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of these experiment and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of S.thyphimurium and E.coli used.

Executive summary:

FeNaEDDHA was tested for mutagenic effects in vitro in histidine-required strains of Salmonella typhimurium and in a tryptophan-required strain of Escherichia coli. The following strains were used: S.thyphimurium TA 98, TA100, TA 102, TA 1535, TA 1537 and E.coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in bidistilled water and testen at 5 concentrations in the range of 312.5 - 5000 µg/plate in the presence and absence of metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 312.5 - 5000 µg/plate. The concentration of 5000 µg/plate represents the test limit dose. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as a positive control.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of the test substance FeNaEDDHA led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

read-across source

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line CCL 61 (Chinese hamster ovary cells, CHO) was maintained in culture medium consisting of Nutrient Mixture F-12 supplemented with 10 % fetal calf serum + Penicillin/Streptomycin 100 units/mL ug/mL in 75 cm2 tissueculture (plastic) flaks. The cultures were incubated at 37 degree C in humidified atmosphere containing 5% CO2. The cell culture were periodically checked for mycoplysma contamination.

Genome stability of the cell line:
The cell line CHO CCL 61 has been used for cytogenetic studies for several years. The stability of the genome of these cells can be assessed on the basis of the regular (cytogenetic) analysis of control cultures in the course of the cytogenetic studies. It is judged to be adequate for the particular purpose of cytogenetic studies.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver post mitochondrial fraction S9

Test concentrations with justification for top dose:

Experiments without metabolic activation:
- 18 hours treatment time:
original experiment: 7.81, 15.63 and 31.25 µg/mL
confirmatory experiment: 7.81, 15.63 and 31.25 µg/mL
- 42 hours treatment time: 7.81, 15.63 and 31.25 µg/mL

Experiments with metabolic activation:
- 3 hours treatment followed by 15 hours recovery period:
original experiment: 31.25, 62.5 and 125 µg/mL
confirmatory experiment: 31.25, 62.5 and 125 µg/mL
- 3 hours treatment followed by 39 hours recovery, period: 31.25, 62.5 and 125 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- The final concentration of the vehicle was 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24 hours before treatment
- Exposure duration: without metabolic activation: 18 hours and 42 hours; with metabolic activation: 3 hours (followed by 15 and 39 hours recovery period)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemide 0.4 µg/mL for 2 hours (2 hours before cell harvest)


NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
The cytotoxicity test was performed as an integral part of the mutagenicity test.
- Method: mitotic index

Evaluation criteria:

Criteria for a positive response
The test substance is generally considered to be active in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0 (based on historical negative control range) and differs statistically significant from the respective value ofthe negative control.
- A concentration-related response should be demonstrable.

Criteria for a negative response
The test substance is generally considered to be inactive in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in all treatment groups is less than or equal to 6.0 (based on historical negative control range) and does not differ statistically significant from the respective value ofthe negative control.

Statistics:

In the preliminary tests the data were assessed for flask effects (dependence of cells within each culture) using a chi-squared test. The non significant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly a chi-square test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased, resulting in a rather save judgement ofthe observed effects.
The tests were performed based upon the presence of any specific aberration.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: cytotoxicity test was performed , results were not indicated

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 ug/ml (without metabolic activation) or 125 ug/ml (with metabolic activation) could not be scored.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxicity test / Selection of concentrations

The selection of the highest scorable concentrations could not primarily be based on cytotoxicity data. Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 µg/mL (without metabolic activation) or 125 µg/mL (with metabolic activation) could not be scored. An inhibition in mitotic activity by 62.3% could be observed in the original experiment performed with metabolic activation (3h/15h) at the highest scorable concentration of 125 µg/mL. In the respective confirmatory experiment cytotoxicity was noted at the non-scorable concentration of 250 µg/mL only. In the absence of metabolic activation toxicity appeared only after 42 hours treatment at the highest concentration of 1000 µg/mL (non-scorable).

Original mutagenicity study

In the experiment performed without metabolic activation (experiment 1; 18 hours treatment), 2.0% of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentrations of 7.81 µg/mL, 15.63 µg/mL and 31.25 µg/mL 1.0%, 2.0% and 1.5% of cells with specific chromosomal aberrations were found.

In the experiment performed with metabolic activation (experiment 2; 3 hours treatment/15 hours recovery), 1.0% of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 31.25 µg/mL, 62.5 µg/mL and 125 µg/mL the respective values were 2.5%, 2.0% and 5.0%. The value obtained with the highest concentration of the second experiment showed a statistically significant difference when compared with the respective negative control. The value however is below the critical limit required for a positive response and it is within the historical range for negative controls. Furthermore no increase at all was observed in the respective confirmatory experiment. The slight increase in the frequency of aberrant metaphases is therefore considered to be spontaneous in origin.

Flow cytometry

The influence of the test substance on the cell cycle of CHO cells was tested at the concentrations selected for chromosome analysis. The DNA distribution was determined by flow cytometry and compared with the profile of the respective control culture. In the absence and in the presence of metabolic activation a shift in the DNA distribution profile could not be detected. Therefore, no evidence of a cell cycle disturbance by the test substance was obtained in the CHO cells.

Analytical results

The test material in suspension was analysed by UV/VIS-spectroscopy to confirm the intended concentrations to be used in the mutagenicity tests and the stability of the test substance in the vehicle used. The concentration values found were 120.6 and 124.1% of the calculated concentrations, thus indicating a sufficient stability of the test substance in the vehicle.

TABLE1 MITOTIC INDEX VALUES / CYTOTOXICITY
Original study, experiment 1        18h treatment without metabolic activation
Cells scored			Mitosis	M.I. %	Frequency % of control
Solvent control		2000	263	13.15	100
CGA65047SG100 (A-5787A)
1000 µg/mL		2000	250	12.50	95.06
500 µg/mL		2000	234	11.70	88.97
250 µg/mL		2000	268	13.40	101.90
125 µg/mL		2000	273	13.65	103.80
62.5 µg/mL		2000	282	14.10	107.22
31.25 µg/mL		2000	290	14.50	110.27
15.63 µg/mL		2000	300	15.00	114.07
7.81 µg/mL		2000	310	15.50	117.87
Original study, experiment 2        3h treatment with metabolic activation/ 15h recovery
Cells scored			Mitosis	M.I. %	Frequency % of control
Solvent control		2000	220	11.00	100.00
CGA65047SG100 (A-5787A)
1000 µg/mL		2000	114	5.70	51.82
500 µg/mL		2000	70	3.50	31.82
250 µg/mL		2000	62	3.10	28.18
125 µg/mL		2000	83	4.15	37.73
62.5 µg/mL		2000	218	10.90	99.09
31.25 µg/mL		2000	224	11.20	101.82
15.63 µg/mL		2000	238	11.90	108.18
7.81 µg/mL		a)

a) When three subsequent concentrations with a frequency of 70% mitosis or more in relation to the solvent control are found, the evaluation of the lower concentrations is omitted.

TABLE 2 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 1
18 h treatment without metabolic activation
Treatment	total no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exc	cs del	cs exc	mab	pol	end
Solvent control	200	2.0	1	2		1		1	7
CGA 65047 SG 100 (CA-5787 A)
7.81 µg/mL	200	1.0	3			1	1		3
15.63 µg/mL	200	2.0	2	1		2	1		3
31.25 µg/mL	200	1.5	3			2	1		6
positive control (Mito-C, 0.2 µg/mL	50 )	24.0***	5	6	6				2
TABLE 3 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 2
3 h treatment with metabolic activation / 15 h recovery
Treatment	total no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exc	cs del	cs exc	mab	pol	end
Solvent control	200	1.0	6			2			8
CGA 65047 SG 100 (CA-5787 A}
31.25 µg/mL	200	2.5	6	2		2	1		3	1
62.5 µg/mL	200	2.0	13	1		3			9
125 µg/mL	200	5.0*	10	6	3		1		5	1
positive control (CPA, 20 µg/mL)	100	17.0***	2	6	9	3			3

	Legend to Tables2 -3
ctdel	Chromatid deletions (including deletions, breaks, fragments)
ct exc	Chromatid exchanges (including triradials, quadriradials, endfusions and acentric rings)
csdel	Chromosome deletions (including deletions, breaks, fragments)
cs exc	Chromosome exchanges (including dicentrics, polycentrics, centric and acentric rings)
mab	Multiple aberrations: metaphases containing more than10 aberrations of different types or more than 5 aberrations of one particular type (excluding gaps)
gaps	Chromatid and chromosome type gaps
pol	Polyploid metaphases (>30 centromers)
end	Endoreduplications
CPA	Cyclophosphamide
Mito-C	Mitomycin-C
*)	Statistical significance:0.05 =P> 0.01
	Statistical significance:0.01 =P> 0.001
	Statistical significance: P< 0.001
#)	%cells with aberrations excluding gaps and numerical alterations(pol,end)

Conclusions:

It was concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with FeNaEDDHA. The same result is expected for the target substance since it has the same constituents that act via the same mechanism as the source substance. No differences in the magnitude of the effects are expected.

Executive summary:

FeNaEDDHA was investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary cells in vitro with and without extrinsic metabolic activation (S9). The test compound FeNaEDDHA was dissolved in DMSO and tested at each of the following conditions:

Experiments without metabolic activation:

- 18 hours treatment time:

original experiment: 7.81, 15.63 and 31.25 µg/mL

confirmatory experiment: 7.81, 15.63 and 31.25 µg/mL

- 42 hours treatment time: 7.81, 15.63 and 31.25 µg/mL

Final concentrations higher than 31.25 ug/mL of culture medium could not be scored due to solubility limitations. Mitomycin C (0.2 ug/mL) was used as a positive control in the 18 hours experiments.

Experiments with metabolic activation:

- 3 hours treatment followed by 15 hours recovery period:

original experiment: 31.25, 62.5 and 125 µg/mL

confirmatory experiment: 31.25, 62.5 and 125 µg/mL

- 3 hours treatment followed by 39 hours recovery, period: 31.25, 62.5 and 125 µg/mL

Final concentrations higher than 125 ug/mL of culture medium could not be scored due to solubility limitations. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/15 hours experiments.

In addition, DNA distribution of cultures treated under the above described conditions (18 hours only) was determined by flow cytometry. These measurements allow to analyse the influence of the test substance on the cell cycle of CHO cells.

In both the experiments performed without and with metabolic activation no significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed.

Flow cytometry experiments did not reveal any evidence for cell cycle disturbing activities of FeNaEDDHA either in the absence or in the presence of metabolic activation.

Reference 3

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:

1993-11-09 to 1994-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk locus (5-trifluoro-thymidine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Four types of RPMI 1640 were prepared: RM0, RM5, RM10 and RM20
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 rat liver post-mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

The cell cultures were evaluated at the following concentrations:
Experiment I:
without S9 mix: 3.91; 15.63; 62.50; 250; and 1000 µg/mL
with S9 mix: 0.49; 1.95; 7.81; 31.25; and 125 µg/mL
Experiment II:
without S9 mix: 3.91; 15.63; 62.50; 250; and 1000 µg/mL
with S9 mix: 0.98; 3.91; 15.63; 62.50; and 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The highest concentration of the test substance was determined in a preliminary solubilisation test to be 100.0 mg/mL soluble in DMSO, which resulted in a homogeneous suspension after sonication during 10 to 20 minutes. Higher concentration produced non tolerable precipitates in the vehicle. The final concentration of DMSO in the culture medium was 1%.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with metabolic activation

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: overnight
- Exposure duration: 4h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 1-2 weeks: "At the end of the expression period cultures were plated for viability and 5-trifluorothymidine (TFT) restistance. A sample of the post-expression culture was diluted to give 100 mL at 1 x 10E4 cells/mL ('mutation cultures').TFT was added to the 'mutation cultures' to give a final concentration of 4 µg/mL. Each TFT treated culture was dispensed at 200 µL per well into four 96-well microtitre plates (2000 cells per well). These plates were incubated for 1-2 weeks to allow cell growth.
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) was added to the "mutation cultures" to give a final concentration of 4 µg/mL.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 5.0x10E6 L5178Y cells were placed in growth medium (RM10) for the determination of mutation rate and toxicity, respectively.

DETERMINATION OF CYTOTOXICITY
A range-finding cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiments. The concentrations applied ranged from 0.49 to 1000.0 mg/mL separated by 2-fold intervals. 1000.0 mg/mL represent the highest concentration which could be applied in the experiment.

Evaluation criteria:

The test substance was considered to be mutagentic if:
- the assay was valid
- the mutant frequency at one or more concentrations was statistically significant greater than that of the negative control
- there was a statistically significant dose-relationship as indicated by the linear trend analysis
- the effects observed were reproducible

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. These test required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxicity

In the cytoxicity range-finder experiment, 12 concentrations of the test substance were tested. In the part with metabolic activation, the concentrations ranged from 0.49 to 1000.0 µg/mL separated by 2 fold intervals. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. In the part with metabolic activation, down to the concentration of 31.25 µg/mL, a growth inhibiting effect greater than 50.0% in comparison to the negative control could be seen. No relevant cytotoxicity could be detected after treatment with the test chemical in the part without metabolic activation.

Accordingly, five concentrations were selected for the original mutagenicity experiment. In the part with metabolic activation the following concentrations were selected: 0.49, 1.95, 7.81, 31.25 and 125.0 µg/mL. At the top concentration of 125.0 µg/mL the mean zero hour survival value was 49.33%. The relative total growth at the end ofthe expression period revealed a mean value of 31.40%. In the part without metabolic activation the concentrations appplied were: 3.91, 15.63, 62.50, 250.0 and 1000.0 µg/mL. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. The highest concentration showed a mean relative survival of 62.01%. The mean relative total growth value was 80.31%. All concentrations were selected to determine viability and TFT-resistance two days after treatment.

In the confirmatory mutagenicity experiment, in the part with metabolic activation, the concentration range was increased. The concentrations applied were: 0.98, 3.91, 15.63, 62.50 and 250.0 µg/mL. In the part without metabolic activation the same concentration range as in the original experiment was selected. The mean relative survival value obtained in the part with metabolic activation was 47.45% at the highest concentration. The mean relative total growth value obtained after the two days expression period was 42.05%. The zero hour survival value in the part without metabolic activation was 81.90%. The relative total growth determined after the expression revealed a value of 75.74%. All concentrations were selected to determine viability and TFT-resistance 2 days after treatment.

Mutagenicity

In the presence and absence of metabolic activation, in the original and confirmatory experiments, reproducible, statistically significant and dose-related relevant increases in mutant frequencies were not observed. In the original experiment, in the part with metabolic activation, the highest concentration of 125.0 µg/mL was excluded from statistical analysis due excessive heterogeneity in the duplicate survival plates. In the part without metabolic activation the concentration of 15.36 µg/mL was excluded from statistical anlysis due to heterogeneity in the survival plates. Nevertheless, the mutant frequency values found at these concentrations are lying within the normal range and do not influence the validity of the study in any respect.

Large and small colonies

For the negative and positive controls the number of wells containing small colonies and the number of wells containing large colonies were scored. With metabolic activation the mean proportion of small colonies in the negative controls ranged from 40.0 to 44.4%, whereas with the positive controls (DMN) an increased proportion of 55.4 to 59.5% was observed. Without metabolic activation the proportion of small colonies in the negative controls ranged from 67.3 to78.0%, whereas with the positive controls (EMS) a proportion of 42.0 to 53.3% was observed.

Small and large colony numbers are not reported for CGA 65 047 SG 100, (A-5787 A) as the data did not fulfil the criteria for a positive response.

Table1 : Summary of the mutagenicity test (Experiment with metabolic activation)

Treatment	Zero hour survival %	Relative total growth (RTG) %
Negative control DMN 2 µL/mL	100.00 78.35	100.00 48.88
CGA 65 047 SG 100  (A-5787 A):
125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL	49.33 103.76 93.79 85.79 75.22	31.40 86.83 64.80 63.13 60.61
Treatment	Mutant frequency X 10E-6	Significance (P)
Negative control DMN 2 µL/mL	24.58 387.77	#
CGA 65 047 SG 100.  (A-5787 A):
125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL	54.08 27.73 25.17 36.01 27.83	§ NS NS NS NS
Test for linear trend: NS

Table2 : Summary of the mutagenicity test (Experiment without metabolic activation)

Treatment	Zero hour survival %	Relative total growth (RTG) %
Negative control EMS 1 µL/mL	100.00 1.82	100.00 2.54
CGA 65 047 SG 100 (A-5787 A):
1000.00 µg/mL 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL 3.91 µg/mL	62.01 74.51 100.13 134.44 144.04	80.31 109.86 96.75 92.87 115.83
Treatment	Mutant frequency x 10E-6	Significance (P)
Negative control EMS 1 µL/mL	33.76 841.91	#
CGA 65 047 SG 100 (A-5787 A):
1000.00 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL  3.91 µg/mL	38.61 22.40 35.37 40.38 39.89	NS NS NS § NS
Test for linear trend		NS

 § excluded from experiment;

NS not significant.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the result of two indipendently performed experiments and under the given experimental conditions, it is concluded that FeNaEDDHA and its metabolites did not show any mutagenic activity at the tk locus of mouse lymphoma L5178Y cells in the presence and absence of metabolic activation.

Executive summary:

FeNaEDDHA was tested for its ability to induce mutations at the tk locus (5-trifluoro-thymidine resistance) in L5178Y mouse lymphoma cells. The study consisted of a preliminary cytotoxicity range-finder and two independent experiments, each performed with and without metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial supernatant (S9 fraction).

In a first range-finder experiment the concentrations applied ranged from 0.49 to 1000.0 µg/mL separated by 2-fold intervals. Based on the results of the range finding study, 5 concentrations were chosen for the first mutagenicity experiment, separated by 4-fold intervals and ranging from 0.49 to 125.0 µg/mL in the part with and from 3.91 to 1000.0 µg/mL in the part without metabolic activation. At the highest concentrations the zero hour survival was 49.33% and 62.01% in the presence and absence of metabolic activation. In the confirmatory experiment, in the part with metabolic activation, the concentration range was increased ranging from 0.98 to 250.0 µg/mL. Without metabolic activation the same concentration range was used. With metabolic activation the relative survival at the highest concentration was 47.45%, without metabolic activation the value found was 81.90%. Negative (vehicle) and positive control treatments were included in each experiment with and without metabolic activation. The mutant frequencies of the negative controls were within normal ranges and the positive controls N-Nitrosodimethylamine (DMN, with metabolic activation) and Ethylmethansulfonate (EMS, without metabolic activation) produced statistically significant increases of mutant frequency. In the part performed with and without metabolic activation, no relevant increases in mutant frequency were observed after treatment with FeNaEDDHA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Salmonella and E.Coli tests in vitro (AmesTests):

Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to induce mutagenic effect in histidine-requiring strains of Salmonella mutagenic (TA 98, TA 100, TA 102, TA 1535, TA 1537) and in a tryptophan-requiring strain of Escherichia coli (WP2 uvr A) (OECD Guideline 471) (CIBA-GEIGY, 1994b; Report No. 931146a). The test compound was dissolved in bidistilled water and tested at five concentrations ranging from 312.5 to 5000 µg/plate with and without metabolic activation. Suitable positive controls were used for each strain. All experiments were repeated in order to confirm the results. The results revealed no increased incidence of mutants by the test item with and without metabolic activation. Therefore, it was concluded that the test compound did not show mutagenic activity in S. typhimurium and E. coli. The positive controls induced mutagenic activity. In conclusion, Fe(Na)EDDHA provoked no mutagenic activity in this test system.

In-vitro Mammalian Chromosome Aberation Test

The test compound Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke clastogenic effects in Chinese hamster ovary cells (CCL61) in vitro (OECD TG 473) (CIBA-GEIGY, 1994c; Report No. 931147). The compound was dissolved in DMSO and tested without metabolic activation at concentrations of 0, 7.81,15.63 and 31.25 µg/mL for 18 and 42 hours. With metabolic activation (liver S9 fraction from Aroclor 1254 induced rat liver) concentrations of 0, 31.25, 62.5 and 125 µg/mL were applied for 3 hours followed by 15 hours recovery or 3 hours followed by 39 hours recovery. Higher concentrations could not be reached due to solubility limitations.

Three independent experiments of each with and without metabolic activation were performed. Two replicate culture per concentration and 200 cells per concentration were evaluated.

The results showed in both experiments with and without metabolic activation no increased number of metaphases with chromosomal aberrations. In contrast, the positive controls (Mitomycin 0.2 µg/mL and Cyclophosphamide 20 µg/mL) induced clastogenic effects. In conclusion, the test substance provoked no clastogenic activity in this test in vitro.

In-vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay):

Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke mutations at the tk locus in L5178Y mouse lymphoma cells in vitro (OECD Guideline 476) (CIBA-GEIGY, 1994a; Report No. 931146b). The test compound was dissolved in DMSO. The range finding experiments showed that 1000 µg/mL was the highest concentration which could be used. Higher concentrations (greater than 100 mg/mL) produced precipitates in the vehicle.

In the presence of metabolic activation (liver S9 -fraction from Aroclor 1254 treated rats) the two highest concentrations revealed cytotoxicity. In absence of metabolic activation no toxicity was noted.

For the mutagenicity experiment, concentrations ranging from 0 to 125 µg/mL with metabolic activation and from 0 to 1000 µg/mL without metabolic activation were used. In the confirmatory experiment with metabolic activation concentrations ranging from 0 to 250 µg/mL were applied. The same concentrations (0 to 1000 µg/mL) were used in the confirmatory experiment without metabolic activation.

Corresponding positive controls (N-Nitrosodimethylamine, with metabolic activation and Ethylmethansulfonate without metabolic activation) were included. The mouse lymphoma cells were treated for 4 hours. After two days expression time, mutations at the tk locus were selected by resistance to 5-trifluorothymidine. Two types of colonies were selected, large colonies (base-pair substitutions and deletions) and small colonies (chromosome aberrations). The results showed no increase incidence of mutations at the tk locus of mouse lymphoma L5178Y cells in presence or absence of metabolic activation. Positive controls showed mutagenic activity. In conclusion, Fe(Na)EDDHA was not mutagenic in this test system in vitro.

Justification for classification or non-classification

Based on results of the three different in vitro genetic toxicity studies, performed with the source substance Fe(Na)EDDHA, the target substance o,o-Fe(Na)EDDHA does not need to be classified and labelled as genotoxic according to Regulation 1272/2008/EC (CLP).