Reaction mass of iron chelate of sodium salt N-[1,2 dicarboxyethyl] D,L aspartic acid and sodium chloride

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:

June 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

666828-40-6

Cas Number:

666828-40-6

IUPAC Name:

666828-40-6

Constituent 2

Reference substance name:

Fe(III)IDHA

IUPAC Name:

Fe(III)IDHA

Specific details on test material used for the study:

Fe(III)IDHA Lot no. 906/P/Fe
Manufacturer code: 2415 Z
Productiondate: 14.09.2006
Fe: 9.0 % +/- 0.3 w/w

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

The post-mitochondrial fraction (S9) fraction (batch MCH110406, protein content 69.2 mg/ml, Protocol 1/2006 and batch Aro 110406, protein content 76.5 mg/ml) were used. S9 was derived from livers of adult male rats.

Test concentrations with justification for top dose:

five concentration from range 0.01 - 5.0 mg/plate
The maximum dose 5 mg/plate was selecte d according OECD 471 guideline as the highest tested dose; this concentration was not toxic for all three strains TA 97, TA 1535 and TA 100. Because toxicity at the highest dose was not recognised (without reduced level of spontaneous mutations or reduction of bacterial lawn) in all next experiments the dose 5 mg/plate was the highest test dose.

Vehicle / solvent:

steriledistilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Details on test system and experimental conditions:

Test system
Salmonella typhimurium strains TA 100, TA 98, TA 97, TA 102 and TA 1535 from Czech
collection of microorganism (CCM) were used. From lyophilised cultures were prepared cultures long term maintained in liquid nitrogen; dimethylsulphoxide (DMSO) was used as cryoprotective agent. Suspension for test was prepared as ovemight culture from stock culture in liquid nitrogen. The tester strains were inoculated in nutrient broth (CASO broth, Merck) and incubated at 37°C for 18-20 hours to give the bacterial density of 108-109/ml.

Metabolic activation
The post-mitochondria] fraction (S9) fraction (batch MCHI 10406, protein content 69.2 mg/ml, Protocol 1/2006 and batch Aro 110406, protein content 76.5 mg/ml) were used. S9 was derived from livers of adult Sprague-Dawley małe rats (MAT CONSULTING, Slovakia), weighing approximately 200 g according to standard procedure (SOP 9). The animals were pre-treated with the agent 20-methylcholanthrene (admini stered i.p. at 80 mg/kg) or Aroclor 1254 (administere s i.p . at 500 mg/kg) 5 days prior to killing.
S9 fraction was stored in liquid nitrogen (-196°C) (SOP 1O).
S9 homogenate was diluted with cofactors (S9 MIX): 33 mM KCI, 8 mM MgC h , 5 mM glucose-6-phosphate, 4 mM NADP and 100 mM phosphate buffer (pH = 7,4). The S9 conc. in S9 MIX was 10%.

Positive control:
without activation - sodium azide: TA 1535, TA 100- 0.0015 mg/plate, 2-nitrofluoren e (2NF)-0.003 mg/plate: TA 98, 9-aminoacridine (9-AA)-0.075 mg/plate -TA 97 , mitomycin C: TA 102- 0.00025 mg/plate
with activation - 2-acetamidofluorene (2-AAF)- O. I 00 mg/plate
Safety manipulation with positive controls was performed according to standard procedure (SOP 10).

Negative control/solvent control: distilled water

Control of strains
Genotypes of each strain were confirmed based on the methods described by Maron and Ames (1983) prior to the mutagenicity study, which included requirement of histidine (his·), the sensitivity to crystal violet (rfa mutation) and UV light (uvrB mutation), and the resistance to ampicilin (R factor) and spontaneous revertant counts (protocols 1, 2, 3, 4, 5, 6/2007).

Chemicals
D-biotin (Serva), L-histidine (Serva), glucose (Merck), MgS04 (Mikrochem), NaCl (Mikrochem), KCl (Mikrochem), MgCh (Calbiochem), NaH2P0 4 . H20 (Mikrochem), Na2HP04 . 12H20 (Fluka), -nicotinamideadeninedinucleotid phosphate (NADP) (Sigma), glucoso-6- phosphate(G-6-P) (Sigma), ampicilin (Biotika), NaOH (Fluka), crystal violet (Serva), tetracycline (Biotika), sodium azide (Sigma), 9-aminoacr idine (Serva), 2- nitrofluorene (Aldrich), mitomycin C (Sigma), Aroclor 1254 (Analabs), 2 acetamidofluorene (Sigma), dimethylsulphoxide (Fluka), 20-methylcholanthrene (Sigma).

Instruments
Vortex minishaeker, water-bath, digital pH meter Sentron Titan (SOP 2), laboratory autoclave (CV-EL 10 I, CertoClav) ( SOP 3), semi aseptic box (Fatran)( SOP 4), biological incubator BT 102 (SOP 5), analytical weight HllO (Sartorius) and analytical weight OWA(SOP 6).

Media
Minimal agar containing Vogel-Bonner minimal medium and glucose ( SOPI) was used. Top agar contains 0.6% Bacto agar and 0.5% NaCI in distilled water, which was autoclaved and stored at room temperature. Before plating, 10 ml of sterile 0.5 mM histidine/0.5 mM
biotin solution was added to the meltened top agar, kept at 45°C and used as an overlay on the minimal agar plate.
CASO broth (Merck) was used for growing tester strains. The growth medium was stored at 2-8°C. Media were prepared according to standar d procedure (SOP 7).

Test performance Type of Salmonella assay
Tests were performed in compliance with OECD 471 (OECD, 1997) (3) according to standard procedure (SOP 1). This procedure evaluates the mutagenie potentia! of test chemicals by their effect on five histidine requiring strains of the bacterium, Salmonella typhimurium in the absence and presence of a rat liver metabolising system.

Range-finding assay
A preliminary range-finding assay was perfonned using three tested strains to determine the optima} non toxic test doses of Fe(III) IDHA. Fe(III) IDHA was freshly prepared in sterile distilled water and five concentrations from range O.Ol - 5.0 mg/plate were tested without metabolic activation. An aliquot of the culture was added to 2 ml of melten top agar, along with 0.1 ml of the test substance. The contents were then mixed and poured onto the surface of minimum agar plate. The plates were incubated at 37°C for 48 hours. After the incubation period, the number of revertant colonies per plate was counted.
Cultures were set up in triplicate; negative control and positive control were also included.

Protocol
Fresh cultures of bacteria at the late exponential or early stationary phase of growth (app. 109 cells per ml) at 37°C were exposed to the test substance in the presence and absence of metabolic activation system. 0.1 ml of the culture was added to 2 ml of melted top agar; along with O. I ml of the test agent. For the assay with metabolie activation, 0.5 ml of metabolic activation mixture containing 1O % of postmitochondrial fraction (S9) together with the bacteria and the test substance were used. The contents were then mixed and pored onto the surface of a minimum agar plate. The triplicate plates were used at each dose level.
All plates were incubated at 37°C for 48-72 hours. After the incubation period, the number of revertant colonies per plate was counted.

Treatment of results
Data were presented as the number of revertant colonies per plate. The mean numbers of revertants per plate and the standard deviations were presented for the test substance and positive and negative controls.

Evaluation criteria:

Positive results: concentration -related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation , Mp> 2. Student's t-test was used for evaluation of statistical significanceof mutation frequency increasing against solvent control value.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 97

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

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

Additional information on results:

Salmonella typhimurium TA 100 (Tables 2, 3. 12):
Four independent experiments were performed, two without metabolic activation, two with metabolie activation. The mean levels of spontaneous mutations were within range 151-231 revertants/plate.
In all experiments Fe(IJI) IDHA was tested in concentration range from O.Ol - 5.0 mg/plate (5 concentrations). The rate of number revertant colonies per plate and the level of revertant colonies in solvent control plates higher than 2 (MF > 2) was not observed.

In experiments without activation the level of revertant frequency was not significantly increased in comparison with negative (solvent) control. Positive control (sodium azide) induced statistic significant (p<0.01, 0.05) increase in revertant frequency under experimental conditions.
In experiments with metabolic activation the mutagenie potentia) ofFe(III) IDHA was confirmed neither in experiment s with S 9 fraction isolated from rat liver after induction with 20-methylcholanthrene nor in experiments with S 9 fraction isolated from rat liver after induction with Aroclor 1254. Positive response on the treatment with 2AAF showed ability of both S9 systems in activating of promutagens (statistic significant p<0.001).

Salmonella typhimurium TA 1535 (Tables 4,5,13):
Four independent experiments were realis e d, two without metabolic activation , two with metabolic activation. The mean level of spontaneous mutation was within range 6-29 revertants /plate. In all experiments, test article was tested in concentration range from O.Ol -
5.0 mg/plate (5 concentrations).
In experiments without metabolic activation the level of revertant frequency was not increased in comparison with negative (solvent) control. In the Exp #2 at the dose 0.5
mg/plate was observed statistically significant decrease of revertant number in comparison with negative (solvent) control but without biologica! sign ificance, MF < 2 (MF = 0.62). Po s itive control (sodium azide) induced statistic significant increase in revertant frequency (with statistical significance).
In experiment with S 9 prepared from rat liver induced with 20-methylcholanthrene was not detected mutagenie effect ofFe(III) IDHA; when the S9 was prepared from Aroclor 1254 induced rat liver the results were negative, too. In Exp #4 at the dose 1.0 mg/plate was observed statistically significant increase of revertant number in comparison with negative (solvent) control but without biologica! significance, Mp< 2 (MF= 1.43).
The ability of S9 systems to activate of promutagens was evaluated in each experiment with use of other Salmonella typhimurium strains (TA 100 and TA 98 with positive control 2AAF).

Salmonella typhimurium TA 97 (Tables 6,7,14):
Four independent experiments were performed, two without metabolic activation, two with metabolic activation. The mean level of spontaneous mutation was within range 139-245 revertants/plate. In all experiments Fe(III) IDHA was tested in concentration range from O.Ol to 5 mg/plate (5 concentrations).
In experiments without activation the level of revertant frequency was in one experiment at conc. 5.0 mg/plate statistically significant increased in comparison with negative (solvent) control but without biological significance, MF<2 (MF=1.13).
The result confirmed not mutagenie effect ofFe(III) IDHA under condition without activation. Positive control (9-aminoanthracene) induced statistic significant increase of revertant frequency.
The experiments with metabolic activation were performed with S 9 prepared from rat liver induced with 20-methylcholanthrene and with S 9 prepared from rat liver induced with Aroclor 1254. In experiment with metabolic activation the level ofrevertant frequency was not increased in comparison with negative (solvent) control up to a maxima! dose 5 mg/plate. The efficacy ofmetabolic activation was confirmed in activation of promutagens 2-AAF in experiment with Salmonella typhimurium TA 100, 98 realised under the some conditions.

Salmonella typhimurium TA 102 (Tables 8,9,15):
Four independent experiments were realised, three without metabolic activation, three with metabolic activation. The mean levels of spontaneous mutations were within range 252-364 revertants/plate. In all experiments Fe(III) IDHA was tested in concentration range from O.Ol to 5 mg/plate (5 concentrations).
In experiments without metabolic activation the level of revertant frequency was not increased in comparison with negative (solvent) control. Positive control (mitomycin C) induced statistic significant increase in revertant frequency (p<0.05 and p<0.001).
In experiments with metabolic activation the mutagenie potentia! offe(III) IDHA was confirmed neither in experiment with S 9 fraction isolated from rat liver after induction with 20-methylcholanthrene nor in experiment with S 9 fraction isolated from rat liver after induction with Aroclor 1254.
Positive response demonstrated the ability of the S9 systems to activate of promutagens was confirmed with positive control 2AAF on other Salmonella strains: TA 100 and TA 98.

Salmonella typhimurium TA 98 (Tables 10.11. 16):
Four independent experiments were realised, two without metabolic activation, two with metabolie activation. The mean level of spontaneous mutation was within range 15 53 revertants/plate.
In experiments without activation the level of revertant frequenc y was in one experiment at
conc. 5.0 mg/plate statistically significant increased in comparison with negative (solvent) control but without biologica! significance, MF< 2 (MF=1.43). It was not confirmed in the second experiment.
The result confirmed not mutagenie effect of Fe(III) IDHA under condition without activation. Positive control (2-nitrofluorene) induced statistic significant increase of revertant frequency (p<0.05 and p<0.001).
In experiments with metabolic activation the mutagenie potentia! ofFe(III) IDHA was confirmed neither in experiment with S 9 fraction isolated from rat liver after induction with 20-methylcholanthrene nor in exper iment with S 9 fraction isolated from rat liver after induction with Aroclor 1254. Positive response on the treatment with 2AAF showed ability of both S9 systems in activating of promutagens (statistic significant p<0.05 and p<0.01, MF
=15.5 and MF=40.8).

Applicant's summary and conclusion

Conclusions:

In the Ames test, with use offive strains of Salmonella typhimurium (TA 97, TA 98, TA 100, TA 102 and TA 1535) was tested Fe(III) TDHA with and without the addition of activation system (S9) for mutagenicit y in concentration range O.Ol - 5.0 mg/plate.

The Fe(III) IDHA produce neither a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according these results is considerednon mutagenie in this system.

Executive summary:

Fe(III) IDHA was tested for mutagenie activity withSalmonella typhimuriumstrains TA 100, TA 98, TA97,TA1535 and TA102in bacterial reversemutation assay (Ames test) accordingtoOECD guideline 471 in compliance with GLP rules.

SubstanceFe(III)IDHAwasnottoxicuptoamaximumdose5.0mg/plateselectedaccording OECD 471 guideline as thehighesttested dose. Concentrations oftestcompound rangedbetweenO.Oland5.0mg/plate.

 

The test substance Fe(III) IDHA was evaluated formutagenicitywith use of Salmonella typhimurium strains TAlOO, TA98, TA 97, TA 1535 and TA102 in Ames standard plate assay without any modification in the absence of external metabolic activation, in standard plate assay without any modification. In experiments with the presence of extemal metabolic activation S9 fraction prepared from Sprague-Dawle y rat after induction with 20-methylcholanthrene and S9 fraction prepared from Sprague-Dawleyrat after induction with Aroclor1254 were used.

 

Adequate positive and negative controIs were performed and showed the reliability of the test system.

 

The test substance Fe(III)IDHA did not produce a significant increase in mutation frequency. at any tested concentration. It is concluded that does not exert mutagenie activity under the conditions of the test performed.