Potassium; 2-[bis[2-[bis(carboxylatomethyl)amino]ethyl]amino]acetate; iron(3+)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

DTPA-FeHNa tested in the Salmonella Typhimurium Reverse Mutation Assay and the Eschericha Coli reverse mutation assay did not result in an increased number of revertant colonies in strains S. typhimurium strains TA 98, 100, 1535, 1537. An in vitro micronucleus test in human lympocytes did not result in an increased number of micronuclei following exposure for 4 h (with and without S9 mix), but it did following exposure for 20 h (without S9 mix).

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:

key study

Study period:

July-August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The characteristics of the different Salmonella typhimurium strains are as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver (S9-mix)

Test concentrations with justification for top dose:

Dose range finding test: 0, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Main test 1: 0, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Repeat test: 0, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.

Vehicle / solvent:

Milli-Q water (Millipore Corp., Bedford, MA., USA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Remarks:

see below

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale: recommended test system in international guidelines (e.g. OECD, EC).
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates: Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Acros Organics).

Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions: All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.7 – 39.2°C) in the dark. Temporary deviations of maximally 2 hours (in the range of 35.7 – 36.0°C and 38.0 – 38.7°C) occurred due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. In the first experiment DTPA-FeHNa was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

No statistical analysis doen; see at evaluation criteria

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DRF test: In tester strain WP2uvrA , no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain TA100, extreme reductions in the number of revertant colonies were observed at the test substance concentration of 1000 μg/plate in the absence of S9-mix and at 3330 μg/plate in the presence of S9-mix. No revertant colonies were observed at test substance concentrations of 3330 and 5000 μg/plate in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix. A slight reduction of the bacterial background lawn was only observed at 5000 µg/plate in the absence and presence of S9-mix.

Main test 1: There was no reduction of the bacterial background lawn in any of the tester strains. The reduction in the number of revertants is presented in Table 1 (see below).

Main test 2: In tester strain WP2uvrA , no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. There was no reduction of the bacterial background lawn in any of the tester strains. The reduction in the number of revertants in the tester strains TA1535, TA1537, TA98 and TA100 is presented in Table 2 (see below).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1            Experiment 1: Mutagenic response of DTPA-FeHNa in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay

 

Dose                   Mean number of revertant colonies/3 replicate plates (± S.D.) with

(µg/plate)             different strains ofSalmonella typhimuriumand oneEscherichia colistrain

                                        TA1535              TA1537       TA98              TA100        WP₂uvrA

                                               Without S9-mix

positive control              812 ± 13       749 ± 44       1030 ± 12       738 ± 26       932 ± 66

 solvent control                 11 ±  3       5 ±  1              39 ±  4         127 ±  7        34 ±  6

                    3                                                                                          124 ±  8         29 ±  6

                 10                      11 ±  1           5 ±  1               40 ±  1           131 ± 10        36 ±  4

                 33                       9 ±  2          5 ±  1               36 ±  3            127 ±  3         29 ±  4

                100                       9 ±  3          3 ±  2               37 ±  8           106 ± 10         28 ±  1

                333                       7 ±  2          2 ±  1               31 ±  0           102 ± 10         30 ±  4

               1000                     2 ±  1          1 ±  1               32 ±  3              26 ±  2         31 ±  2

               3330                     0 ±  1          0 ±  0               27 ±  4                0 ±  0         35 ±  4

               5000                                                                                         0 ±  0 s       31 ±  4

------------------------------------------------------------------------------------------------------------

                                               With S9-mix¹

 positive control            342 ± 28     326 ± 57              798 ± 12          1036 ± 19       344 ± 17

  solvent control                8 ±  3          5 ±  1                34 ±  2               112 ± 16         29 ±  3

                  3                                                                                              105 ±  4         31 ±  3

                 10                                                                                              106 ± 11        34 ±  5

                 33                       6 ±  1          4 ±  2               35 ±  3             117 ±  4         32 ±  1

                100                       7 ±  2          5 ±  1               32 ±  4             119 ±  2         35 ±  7

                333                       6 ±  2          4 ±  1               29 ±  4             117 ± 17         32 ±  4

               1000                       2 ±  1          2 ±  2               28 ±  3             92 ±  5         39 ±  3

               3330                       0 ±  1          2 ±  0               32 ±  6                32 ±  2         39 ±  6

               5000                       0 ±  0          0 ±  0               29 ±  3                0 ±  0 s       39 ±  3

 

Solvent control: 0.1 ml Milli-Q water

1    The S9-mix contained 5% (v/v) S9 fraction

s     Bacterial background lawn slightly reduced

Table 2            Experiment 2: Mutagenic response of DTPA-FeHNa in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay

 

Dose                   Mean number of revertant colonies/3 replicate plates (± S.D.) with

(µg/plate)             different strains ofSalmonella typhimuriumand oneEscherichia colistrain

TA1535              TA1537       TA98              TA100       WP₂uvrA

                                               Without S9-mix

positive control                 759 ± 23        676 ± 29       923 ± 101       808 ± 46       981 ± 62

 solvent control                        5 ±  2          4 ±  2            25 ±  1          104 ±  6          30 ±  3

                 33                            3 ±  1          5 ±  2          26 ±  2              99 ±  8       30 ±  9

                100                              4 ±  1       2 ±  2       18 ±  4              92 ±  5       34 ±  7

                333                            1 ±  1       2 ±  2       21 ±  4              83 ±  4       36 ±  3

               1000                            1 ±  1       4 ±  3       12 ±  3              16 ±  2       31 ±  9

               3330                            1 ±  1       1 ±  1       7 ±  5               0 ±  0       36 ±  5

               5000                              1 ±  1       2 ±  1       0 ±  0               0 ±  1       34 ±  3

------------------------------------------------------------------------------------------------------------

                                               With S9-mix¹

positive control                    224 ± 28       344 ± 25      460 ± 66       1149 ± 18       396 ± 21

 solvent control                     6 ±  1              4 ±  1       31 ±  4              81 ±  8       32 ±  3

                 33                              6 ±  3              3 ±  2       25 ±  3            96 ± 20       38 ±  2

                100                            5 ±  1              4 ±  1       23 ±  1            81 ±  9       32 ±  4

                333                            6 ±  3              3 ±  2       30 ± 10           74 ±  9       40 ± 10

               1000                            1 ±  1              4 ±  1       30 ±  9           72 ±  9       39 ±  4

               3330                            1 ±  1              3 ±  1       22 ±  2        39 ±  6       40 ±  9

               5000                            0 ±  1              1 ±  1       19 ±  5           29 ±  7       43 ±  6

Solvent control: 0.1 ml Milli-Q water

1    The S9-mix contained 10% (v/v) S9 fraction

 

 

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that DTPA-FeHNa is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

DTPA-FeHNa was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium(TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9 -mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch CFC-10333 (429H0352) of DTPA-FeHNa was a yellow-green powder with a purity of ~97%. The test substance was dissolved in Milli-Q water.

In the dose range finding test, DTPA-FeHNa was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix in the strains TA100 and WP₂uvrA. DTPA-FeHNa did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 1000 μg/plate and above in the absence of S9-mix and at 3330 and 5000 μg/plate in the presence of S9-mix. In tester strain WP₂uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

Based on the results of the dose range finding test, DTPA-FeHNa was tested in the first mutation assay at a concentration range of 10 to 3330 µg/plate in the absence of S9-mix at 33 to 5000 µg/plate in the presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in the tester strains TA1535 and TA1537.

In an independent repeat of the assay with additional parameters, DTPA-FeHNa was tested at a concentration range of 33 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. Toxicity was observed in all tester strains, except in the tester strains TA98 in the presence of S9-mix and in tester strain WP₂uvrA in the absence and presence of S9-mix.

DTPA-FeHNa did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that DTPA-FeHNa is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well perfomed and reported GLP study

Justification for type of information:

The underlying hypothesis for the read-across is that chelates have the same mode of action based on their ability to chelate, remove or add metal cations to body causing perturbation of body’s micronutrients balance.
The source substance is a chelating agent in a target substance. The only difference between the target and the main source substance is presence of iron (Fe) and potassium (K) cations instead H+ cations. As iron and potassium are an essential macro- and microelements required by all forms of life, is considered not to influence the toxicological activity.

Qualifier:

according to guideline

Guideline:

other: OECD 487

Principles of method if other than guideline:

OECD guideline 487 for the testing of chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit); adopted 22 July 2010.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Chromosome aberration

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Blood samples were obtained by venapuncture from young healthy, non-smoking individuals (29 and 33 years old) with no known recent exposures to genotoxic chemicals or radiation.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver (S9 mix)

Test concentrations with justification for top dose:

first test: 4682, 2341, 1171, 585, 293, 146, 73.0, 36.6, 18.3 and 9.1 µg/ml
second test: 4682, 4000, 3500, 3000, 2530, 2000, 1500, 1000, 500, 250 and 125 µg/ml

Vehicle / solvent:

The test substance could be dissolved in culture medium up to the maximum required stock concentration of 46.82 mg/ml (100 mmol/l), based on a purity of 97%.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

and Vinblastine sulphate

Details on test system and experimental conditions:

In the presence of phytoheamagglutinine (PHA-L), aliquots of 0.5 ml of whole blood in 4.5 ml culture medium, were incubated for 48 hours at 37ºC in humidified air containing 5% CO2. The incubation was carried out in sterile screw-capped (loose) centrifuge tubes. After the 48-hour incubation period, the cells were exposed to different concentrations of the test substance, in both the presence and absence of the S9-mix. Culture medium was used as a solvent for the test substance. In the presence of S9-mix, a clastogenic compound (Cyclophosphamide), which requires metabolic activation, was used as positive control. In the absence of S9-mix, a direct acting clastogenic compound (Mitomycin C) and an aneugenic compound (Vinblastine sulphate) were used as positive control substances. Culture medium was used as a solvent for all positive control substances. In all instances duplicate cultures were used.
See further at remarks.

Evaluation criteria:

The frequencies of binucleated cells with micronuclei were used for the evaluation of micronuclei induction. The CBPI was calculated for treated (selected doses) and control cultures as a measure of cell cycle delay. The test concentrations selected covered the appropriate range of cytotoxicity from maximum to little or no cytotoxicity. The standard maximum cytotoxicity level for the selection of the test substance concentration was 55 ± 5%.

The study was considered valid if the selected clastogenic and aneugenic positive controls gave a statistically significant increase in the number of binucleated cells containing micronuclei and the negative controls were within the historical data performed at the test facility.

A response was considered positive if a statistically significant concentration-related or a reproducible statistically significant increase in the number of binucleated cells containing micronuclei was induced, at any of the test points.

A response was considered equivocal if the percentage of binucleated cells containing micronuclei was statistically marginal higher than that of the negative control (0.05
A test substance was considered negative if it produces neither a statistically significant concentration-related nor a reproducible statistically significant increase in the number of binucleated cells containing micronuclei, at any of the test points.

See also at remarks.

Statistics:

The frequencies of micronuclei found in the cultures treated with the test substance and positive control cultures were compared with those of the concurrent solvent control using Chi-square test (one-sided). The results were considered statistically significant when the p-value of the Chi-square test was less than 0.05 (P<0.05).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity observed in the first in vitro micronucleus test:
In the pulse treatment groups with and without metabolic activation (S9-mix), none of the dose levels analyzed (4682 to 9.1 µg/ml) showed cytotoxicity, when compared to the concurrent negative control. The positive control substances Cyclophosphamide (20 µg/ml), Mitomycin C (0.4 µg/ml) and Vinblastine sulphate (0.025 µg/ml) showed 63%, 60% and 43% cytotoxicity, respectively.

Cytotoxicity observed in the second in vitro micronucleus test:
In the continuous treatment group without metabolic activation, the highest two dose levels (4682 and 4000 µg/ml) showed 20% and 22% cytotoxicity, respectively. At the lowest dose levels (3500, 3000, 2530, 2000, 1500, 1000, 500, 250 and 125 µg/ml) the cytotoxicity fluctuated (between 3% and 10%).
The positive control substances Mitomycin C (0.4 µg/ml) and Vinblastine sulphate (0.025 µg/ml) showed 31% and 19% cytotoxicity, respectively.

Remarks on result:

other: other: 4 h treatment

Remarks:

Migrated from field 'Test system'.

See tables attached.

Conclusions:

Interpretation of results (migrated information):
other: positive (aneugenic activity), negative (no clastogenic activity)

From the results obtained in two independent in vitro micronucleus tests it is concluded that the test substance DTPA-FeNaH induced a statistically significant increase in the number of binucleated cells containing micronuclei in the continuous treatment when compared to the negative control under the conditions used in this study.
The proportion of the large micronuclei and small micronuclei induced by the test substance was not statistically different from the response of Vinblastine sulphate at acceptable toxicity levels. The observed similar proportion of large micronuclei is considered to be an indication for aneugenic effects under the conditions used in this study.

Executive summary:

The test substance DTPA-FeNaH was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix) according to the OECD guideline 487. Two independent in vitro micronucleus tests were conducted for which blood was obtained from two different donors. Culture medium was used as solvent for the test substance. Dose levels ranging from 9.1 to 4682 µg/ml (4682 µg/ml = 10 mM) were tested as final concentrations in the culture medium. Duplicate cultures were used. Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment/recovery time was 4/20 hours (pulse treatment), respectively. In the second test,in thecontinuous treatment groups, the treatment/recovery times were 20/28 hours, respectively.

In both the first and the second test, the negative controls were comparable to the historical data of the test facility. Treatment with the positive controls Cyclophosphamide, Mitomycin C and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the cultures treated with the solvent control (negative control). This demonstrates the validity of the study.

In the first test,in the pulse treatment groups with and without metabolic activation, the test substance was not cytotoxic to the cells when compared to the concurrent negative control. In both the pulse treatment groups, three dose levels (4682, 2341 and 1171 µg/ml) of the test substance, together with the negative controls and positive controls, were analyzed for micronucleus induction in binucleated lymphocytes.In the pulse treatment group, with metabolic activation (S9-mix), the test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analysed.In the absence of a metabolic activation system, at the two highest dose levels (4682 and 2341µg/ml) the test substance induced a slight but statistically non-significant higher percentage of binucleated cells containing micronuclei when compared to the concurrent vehicle control. These percentages were within the range of the historical data of the test facility.

In the second test (continuous treatment group without metabolic activation), at the two highest dose levels analysed, the test substance was slightly cytotoxic to the cells when compared to the concurrent negative control. Five dose levels of the test substance (4682, 4000, 3000, 2000 and 1000 µg/ml), together with the negative controls and positive controls, were analyzed for micronucleus induction in binucleated lymphocytes.At the four highest dose levels analysed, the test substance induced a statistically significant increase in the number of binucleated cells containing micronuclei, respectively. At the lowest dose level analysed, the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei, when compared to the number found in the concurrent negative control (culture medium).

To discriminate aneugens from clastogens, size-classified micronucleus counting was performed on the slides of three dose levels (4682, 4000 and 3000 µg/ml), together with those of the positive controls Mitomycin C and Vinblastine sulphate.

In the second test, with respect to the size-classified micronucleus counting, treatment with the positive controls Mitomycin C and Vinblastine sulphate resulted in a statistically significant different response in the number of small and large micronuclei. The small/large micronuclei proportion for Vinblastine sulphate and Mitomycin C were 49%/51% and 70%/30%, respectively. This demonstrates the validity of the size-classified micronucleus counting carried out in the second in vitro micronucleus test.

From the results obtained in the first and second in vitro micronucleus test it is concluded that, under the conditions used in this study, the test substance DTPA-FeNaH induced a statistically significant increase in the number of binucleated cells containing micronuclei in the continuous treatment, when compared to the negative control. Based on the results of the size-classified micronucleus counting, the proportion of  the large micronuclei and small micronuclei found at the three test substance dose levels analysed ( 4682, 4000 and 3000 µg/ml) was not statistically different from the response of Vinblastine sulphate at acceptable toxicity levels. The observed similar proportions of large and small micronuclei is considered to be an indication for an aneugenic effect, under the conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

DTPA-FeHNa was negative in the Ames test and in the in vitro micronucleus test using a treatment period of 4 h (with and without S9 -mix). In the in vitro micronucleus test using a treatment period of 20 h (continuous treatment without S9 -mix), DTPA-FeHNa was positive at levels >= 2000 µg/mL, inducing aneugenic but no clastogenic effects. This long treatment period together with the high concentrations of chelant may have resulted in exchange and substantial binding of essential elements such as zinc. Similar results were obtained with EDTA-FeNa and EDTA-CuNa2 (see robust summaries and read across document; section 13). Heimbach et al (2000; see also robust summary) concluded that the lack of effects by the Zn-EDTA salt in contrast to effects induced by Ca-, Na- and Mn-salts of EDTA, provided evidence that zinc is required for the initiation or continuation of DNA synthesis and maintaining cell function. As such, the significance of mutations produced by DTPA-FeHNa (and also EDTA-FeNa and EDTA-CuNa2) at non-physiological concentrations in an in vitro screening system is difficult to extrapolate for relevance to intact organisms.

Although no in vivo genotoxicity studies have been carried out with DTPA-FeHNa, several in vivo genotoxicity studies are available for other EDTA-compounds such as EDTA-Na2H2. No genotoxic activity was observed (see also read across document in section 13).

Therefore, the overall findings indicate that DTPA-FeHNa lacks significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function.

Justification for classification or non-classification

The test substance gave negative results in two in vitro mutagenicity studies, viz. the Ames test and the micronuclueus test following exposure for 4 h (with and without S9 mix) but gave positive results (aneugenicity but not clastogenicity ) following exposure for 20 h (without S9 -mix). The latter was most probably explained by induction of Zn deficiency. Overall, it was concluded that classification for genotoxicity is not warranted.