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

Genetic toxicity: in vitro

Administrative data

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

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

EC Number:

235-627-0

EC Name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

Cas Number:

12389-75-2

Molecular formula:

C14-H18-Fe-N3-O10.H.Na

IUPAC Name:

Iron(3+) ion sodium 5-[bis(carboxylatomethyl)amino]-3-{[bis(carboxylatomethyl)amino]methoxy}pentanoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description: yellow-green powder
Batch: CFC-10333 (429H0352)
Purity/Composition: ~97%
Test substance storage: at room temperature protected from light
Stability under storage conditions: stable
Expiry date: 1 December 2013
Test substance handling: use amber-coloured glassware or wrap container in tin-foil.
Stability in vehicle: Water: 1 year
Solubility in vehicle: Water: 110 g/L

No correction was made for the purity/composition of the test compound.

The test substance was dissolved in Milli-Q water (Millipore Corp., Bedford, MA., USA). The stock solution was treated with ultrasonic waves until the test substance had completely dissolved. The stock solution was filter (0.22 µm)-sterilized. Test substance concentrations were used within 1.5 hours after preparation

Method

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

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

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)

Controls

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

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.