Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1997-01-23 - 1997-07-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented GLP-guideline 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 magnessium (Mg) cation instead two Na+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Cross-referenceopen allclose all
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
June 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent as in a target substance (MgNa2IDHA). The only difference between the target and the source substance is presence of magnessium (Mg) cation instead Fe3+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 102
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
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.
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
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
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
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
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).




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.


Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: other: mutagenicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent as in a target substance (MgNa2IDHA). The only difference between the target and the source substance is presence of magnessium (Mg) cation instead Zn2+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
G 026
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Species / strain:
other: TA 98, TA 100, TA 97, TA 102, TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified

RESULTS AND DISCUSSION

 

Zn (II) IDHA was tested in range-fmdimg experiment performed using strainsSalmonella typhimuriumTA 97, TA 98, TA 1535, TA 102 and TA 100 up to a maximum dose 5.0 mg/plate (Table1).

Zn (II) IDHA at doses of 5 and 2.5 mg/plate reduced the number of spontaneous revertants most of tested strains.

The test article in concentration range of 1 - 0.0001 mg/plate was evaluated for mutagenicity in absence of S9 mix in standard plate assay without any modification and with preincubation, in presence of S9, with S9 fraction prepared from Sprague-Dawley rat after induction with 20-methylcholanthrene and with S9 fraction prepared from Sprague-Dawley rat after induction with Aroclor 1254. The raw data are attached in Tables 1-11. The results of mutagenicity assays with TA 100, TA 1535, TA 97, TA 102 and TA 98 are summarized in Tables12-16and are expressed as the average revertant number ± SD per plate from triplicates.

 

The concurrent strain-specific positive controls (TA 100, TA 1535: sodium azide - 0.0015 mg/plate, TA 98: 2-nitrofluorene (2NF) - 0.003 mg/plate, TA 97: 9-aminoacridine (9AA) -0.075 mg/plate, TA 102: mitomycin C (MMC) - 0.0005 mg/plate, 2-acetaminofluorene (2AAF) - 0.1 mg/plate) and negative controls were included in each assay.

 

Salmonella typhimuriumTA 100 (Tables 2, 3, 12. Annex 4):

Five independent experiments were performed, three without metabolic activation, two with metabolic activation. The mean levels of spontaneous mutations were within range 157-229 revertants / plate.

Zn (II) IDHA in experiments withot and with activation (20-metylcholanthere S9, Aroclor S9) was tested in concentration range from 1 to 0.0001 mg/plate. 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 second experiment without metabolic activation Zn (II) IDHA at concentrations of 1 mg/plate and 0.01 mg/plate statistical significant reduced the level of spontaneous mutations (Mf= 0.87 and 0.91) and in experiment with preincubation at concentration of 0.01 mg/plate statistical significant (p<0.01) reduced the level of spontaneous mutations (MF=0.97), but without biological relevance.

In experiment with metabolic activation (20-methylcholanthere S9) test article did not induce statistic significant increase of revertant frequency. In experiment with Aroclor S9 metabolic activation Zn (II) IDHA at concentrations of 0.01 and 0.0001 mg/plate statistical significant (p<0.05) increased the level of spontaneous mutations (MF= 1.23, 1.23) without biological significancy.

Salmonella typhimuriumTA 1535 (Tables 4, 5, 13. Annex 41:

Mutagenicity was evaluatedinfive independent experiments; three without metabolic activation, two with metabolic activation. The mean level of spontaneous mutation was within range 23-26 revertants / plate.

 

In experiments without metabolic activation Zn (II) IDHA was tested in concentration range from 1 to 0.0001 mg/plate.

In experiments without metabolic activation the level of revertant frequency was not increased in comparison with negative (solvent) control. Positive control (sodium azide) induced statistic significant increase of revertant frequency (with statistical significance p<0.01, p<0.001).

In experiments with metabolic activation Zn (II) IDHA was tested in concentration range from 1 to 0.0001 mg/plate (20-methylcholanthere S9, Aroclor S9). Test article did not induce statistic significant increase of revertant frequency.

The ability of S9 systems to activate of promutagens was evaluated in each experiment with use of otherSalmonella typhimuriumstrains (TA 100 and TA 98 with positive control 2AAF).

 

Salmonella typhimuriumTA 97 (Tables 6, 7. 14, Annex 41:

Five independent experiments were performed; three without metabolic activation, two with metabolic activation. The mean level of spontaneous mutation was within range 158-184 revertants/plate.

In experiments without activation Zn (II) IDHA was tested in concentration range from 1 to 0.0001 mg/plate. In first experiment without metabolic activation Zn (II) IDHA at concentrations of 1 mg/plate, 0.1 mg/plate and 0.0005 mg/plate statistical significant reduced the level of spontaneous mutations (Mf= 0.82, 0.86, 0.96). In experiment with preincubation Zn (II) IDHA at concentration of 0.01 mg/plate statistical significant (p<0.05) reduced the level of spontaneous mutations (Mf= 0.85).

Positive control (9-aminoanthracene) induced statistic significant increase of revertant frequency (p<0.01, p<0.05).

The experiments with metabolic activation were performed with S9 prepared from rat liver induced with 20-methylcholanthrene and with S9 prepared from rat liver induced with Aroclor 1254. In experiments with metabolic activation Zn (II) IDHA was tested in concentration range from 1 to 0.0001 mg/plate. Test article did not induce statistic significant increase of revertant frequency.

The efficacy of metabolic activation was confirmed in activation of promutagen 2AAF in experiment withSalmonella typhimuriumTA 100, 98 realised under the some conditions.

 

Salmonella typhimuriumTA 102 (Tables 8, 9, 15. Annex 41:

Five independent experiments were realised; three without metabolic activation, two with metabolic activation. The mean levels of spontaneous mutations were within range 170-323 revertants/plate. In experiments without activation Zn (II) IDHA was tested in concentration range from 1 to 0.0001 mg/plate. In these experiments Zn (II) IDHA did not induce statistic significant increase of revertant frequency. In first experiment without metabolic activation

In these experiments the mutagenic potential of Zn (II) IDHA was confirmed neither in experiment without metabolic activation nor in experiments with metabolic activation (S9 fraction isolated from rat liver after induction with 20-methylcholanthrene or Aroclor 1254). Positive control (sodium azide) induced statistic significant increase (p<0.05, p<0.01) of revertant frequency under experimental conditions without activation and positive response on

the treatment with 2AAF showed ability of both S9 systems in activating of promutagens (statistic significant p<0.05).

Zn (II) IDHA at concentration of 1 mg/plate statistical significant reduced the level of spontaneous mutations(MF=0.87).

Positive control (mitomycin C) induced statistic significant increase of revertant frequency (with statistical significance p<0.01).

In experiments with metabolic activation Zn (II) IDHA was tested in concentration range from 1 to 0.0001 mg/plate (20-methylcholanthere S9, Aroclor S9). Test article did not induce statistic significant increase of revertant frequency.

Positive response demonstrated the ability of the S9 systems to activate of promutagen was confirmed with positive control 2AAF on otherSalmonellastrains: TA 100 and/or TA 98.

 

Salmonella typhimuriumTA 98 (Tables 10,If16. Annex 4):

Five independent experiments were realised, three without metabolic activation, two with metabolic activation. The mean level of spontaneous mutation was within range 25 - 40 revertants/plate.

Zn (II) IDHA was tested in concentration range from 1 to 0.0001 mg/plate (at least 6 concentrations in each test) without metabolic activation and in concentration range from 1 to 0.0001 mg/plate with metabolic activation.

In experiments without metabolic activation and with preincubation the level of revertant frequency was not increased in comparison with negative (solvent) control. Positive control (2-nitrofluorene) induced statistic significant increase of revertant frequency (p<0.01 , MF=19.68, 13.91, 12.60).

In experiments with metabolic activation the mutagenic potential of Zn (II) IDHA was confirmed neither in experiment with S9 fraction isolated from rat liver after induction with 20-methylcholanthrene nor in experiment with S9 fraction isolated from rat liver after induction with Aroclor 1254. In experiment with 20-methylchoianthrene rat S9 Zn (II) IDHA at concentration of 0.1 mg/plate statistical significant (pO.Ol) reduced the level of spontaneous mutations (Mp = 0.78). Zn (II) IDHA (concentration range from 1 to 0.0001 mg/plate) didn't increase the level of revertants above the level of frequency of spontaneous mutations.

Positive response on the treatment with 2AAF showed ability of both S9 systems in activating of promutagens (statistic significant p,0.0l, p<0.05,MF=28.41 and 53.72).

 

The presented results are acceptable because the presence of the uvr B mutation, rfa mutation, the pKM 101 plasmid (TA 98, TA 100, TA 102, TA 97) and pAQl plasmid (TA 102) were demonstrated. The spontaneous revertant numbers forSalmonella strainswere within the normal range (2) and the laboratory's data range.

The positive mutagens yielded a significant increase in the number of revertants as compared to solvent controls.

The positive control adequately demonstrated the sensitivity of the assay - sensitivity of each strain to detect mutagens and positive response with 2AAF showed ability of S9 system to activate mutagens.

 

The results indicate that substance Zn (II) IDHA neither produce 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 considered non mutagenic in this system.

Conclusions:
CONCLUSIONS

Zn (IT) IDHA was tested in the Ames test, with use of five strains of Salmonella typhimurium (TA 97, TA 98, TA 100, TA 102 and TA 1535) with and without the addition of activation system (S9) for mutagenicity in concentration range from 1 to 0.0001 mg/plate.
The Zn (II) 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 considered non mutagenic in this system.
Executive summary:

Five strains of Salmonella typhimuriumTA 100, TA 98, TA 97, TA 1535 and TA 102 were used for evaluation of mutagenic activity of Zn (II) IDHA (product of Przedsiębiorstwo Produkcyjno-Consultingowe,Poznań,Poland) in bacterial reverse mutation assay (Ames test). The tests were performed according to OECD guideline 471 in compliance with GLP rules (3,4). Substance Zn (II) IDHA was tested in range finding assay up to a maximum dose 5.0 mg/plate selected according to OECD 471 guideline as the highest tested dose. Because the highest doses 5 and 2.5 mg/plate reduced the number of spontaneous revertants most of tested strains, the concentrations of test compound in following experiments ranged between 0.0001 and 1 mg/plate.

The test substance Zn (II) IDHA was evaluated for mutagenicity with use ofSalmonella typhimuriumstrains TA 100, TA 98, TA 97, TA 1535 and TA 102 in Ames standard plate assay without any modification and with preincubation in the absence of external metabolic activation, in standard plate assay without any modification in the presence of external metabolic activation with S9 fraction prepared from Sprague-Dawley rat after induction with 20-methylcholanthrene and with S9 fraction prepared from Sprague-Dawley rat after induction with Aroclor 1254. Adequate positive and negative controls were performed and showed the reliability of the test system.The test substance Zn (II) IDHA did not produce a significant increase of mutation frequency in strainsSalmonella typhimuriumTA 100, TA 98, TA 97, TA 1535, TA 102 up to the dose 1 mg/plate. It is concluded that Zn (II) IDHA does not exert mutagenic activity under the conditions of the test performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): IDS, Na-Salz = Iminodisuccinate, Na-salt or Sodium-iminodisuccinate
- Molecular formula (if other than submission substance): C8H7NO8Na4
- Molecular weight (if other than submission substance): 337.1 [g/mol]
- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: chelate
- Physical state: white powder
- Analytical purity: 67,2 %
- Composition of test material, percentage of components: 67.2% IDS, Na-Salz, 10.1% DL-Asparic acid, Na-salt, 8.3% H20, 7.8% Fumaric acid, Na-salt, 4.2% DL-Malic acid, Na-salt, 1.1% Sodium hydroxide, 0.9% Maleinic acid, Na-salt (analytical result dated February 27, 1997)
- Purity test date: February 27, 1997
- Lot/batch No.: SAV B 0004
- Expiration date of the lot/batch: January 14, 1998
- Storage condition of test material: at room temperature

Method

Target gene:
his-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: normal nutrient broth
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

TA 1535 and TA 100 bear the base-pair substitution, his G 46, and TA 100 additionally contains the plasmid pKM 101.
This R factor, also contained in TA 98 and TA 102, codes for an ampicillin resistance and should raise the sensitivity of the strains.
TA 102 carries the ochre Mutation his G 428 on the multicopy plasmid pAQl, which codes in addition for tetracycline resistance.
TA 1537 and TA 98 bear frameshift markers.
TA 1537 exhibits the +1 mutant, his C 3076, while TA 98 bears the +2 type, his D 3052.
With the exception of TA 102, all strains have reduced capability to repair DNA-damage which increases the likelihood that such damage results in mutations.
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: normal nutrient broth
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

TA 1535 and TA 100 bear the base-pair substitution, his G 46, and TA 100 additionally contains the plasmid pKM 101.
This R factor, also contained in TA 98 and TA 102, codes for an ampicillin resistance and should raise the sensitivity of the strains.
TA 102 carries the ochre Mutation his G 428 on the multicopy plasmid pAQl, which codes in addition for tetracycline resistance.
TA 1537 and TA 98 bear frameshift markers.
TA 1537 exhibits the +1 mutant, his C 3076, while TA 98 bears the +2 type, his D 3052.
With the exception of TA 102, all strains have reduced capability to repair DNA-damage which increases the likelihood that such damage results in mutations.
Metabolic activation:
with and without
Metabolic activation system:
the 9000 g fraction of homogenized mammalian Livers together with co-factors
Test concentrations with justification for top dose:
0, 40, 158, 500, 1581 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water (formed a clear colourless solution
- the positive controls were dissolved in DMSO
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether, and DMF according to information given by the internal sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
sodium azide (10 µg/plate, TA1535), nitrofurantoin (0.2 µg, TA100), 4-nitro-1,2-phenylene diamine (10 µg, TA1537), 4-nitro-1,2-phenylene diamine (0.5 µg/plate TA98), Cumene hydroperoxide (50 µg/plate) and 2-aminoanthracene (3 µg/plate, TA102)
Positive control substance:
sodium azide
cumene hydroperoxide
other: nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2 aminoantracene
Remarks:
No "untreated" negative control was set up for the used solvent, since sufficient evidence was available ia the literature and from our own experience , indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation)

For the mutant count, three plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained three plates per strain.
The amount of solvent for the test substance and for the controls was 0.1 mL/plate.
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 µg or 5 µL per plate were used as the highest dose. At least four, additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
The first repeat was performed as preincubation in a water bath at 37°C for 20 minutes. At the end of the preincubation period 2 mL of molten soft agar were added to the tubes, the content mixed and plated.
For the mutant count, three plates were used for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive-control also contained three plates per strain. In experiments without S9 mix buffer was used as replacement.
The dose of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as µg/tube for better separation of plate incorporation and preincubation trials, despite the fact that µg/plate and µg/tube could be used synonymously.
The toxicity of the substance was assessed in two ways. The first method was a gross appraisal of background growth on the plates for mutant determination. A reduction in background growth was indicated by the letter "b" after the mutant count and only a single "b", without any other values, is noted for a concentration, this "b" represents three plates with reduced background growth. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37°C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure by determining the titers of the negative controls with S9 mix.
The dilution of bacterial suspensions used for the determination of titers was 1:1, 000, 000 . Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased fivefold to permit the complete growth of bacteria.
The tests were performed both with and without S9 mix.
The count was made after the plates had been incubated for 48 hours at 37°C. If no immediate count was possible, plates were temporarily stored in a refrigerator.
Evaluation criteria:
The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data and/or the laboratories' own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

Results and discussion

Test resultsopen allclose all
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:
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:
S. typhimurium TA 102
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
Additional information on results:
other: Due to the results of the first trial, doses ranging from 1000 µg to 5000 µg per tube were chosen for the repeat tests.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The colony number of each plate and mean values of the plate incorporation assay are listed for each dose in the tables 4 to 8, 13 and 14. As may be seen, there was no indication of a bacteriotoxic effect of IDS, Na-Salz at doses of up to and including 1581 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. 5000 µg/plate had only a weak, strain-specific bacteriotoxic effect. Therefore this dose could nevertheless be used for assessment purposes.

Salmonella typhimurium TA 102 revealed without S9 mix an in crease in mutant counts of about 80 as compared to the respective negative control (Table 5). This increase could not be confirmed (Tables 11 and 12), and is therefore to be regarded as a random result.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

The colony number of each plate and mean values of the preincubation assay are listed for each dose in Tables 6 to 10. As may be seen, there was no indication of a bacteriotoxic effect of IDS, Na-Salz at doses of up to and including 5000 µg per tube. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.

None of the five strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls (Tables 6 to 10) and thus confirmed the results of the plate incorporation method.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Table 2: Tabulated Summary of Data
Summary of Mean Values Without S9 Mix From Tables 1-12
Table and group Strain
TA 1535 TA 100 TA 1537 TA 98 TA 102
1-5
µ/plate  
0 6 97 9 22 151
50 7 92 8 20 160
158 6 90 9 17 149
500 5 87 6 17 165
1581 4 88 7 22 191
5000 7 92 6 24 231
Na-azide 924        
NF   209      
4-NPDA     64 129  
Cumene         242
6-10
µg/tube  
0 7 127 8 21 315
1000 7 125 9 28 325
2000 6 128 7 29 327
3000 6 98 6 31 304
4000 7 136 4 32 255
5000 6 122 5 18 265
Na-azide 824        
NF   216      
4-NPDA     69 171  
Cumene A..       599
11-12
µg/plate  
0         145 113
1000         142 120
2000         156 141
3000         123 160
4000         133 165
5000         163 159
Cumene         223 234

Table 3: Summary of Mean Values With S9 Mix From Tables 1-12 
Table and group Strain
TA 1535 TA 100 TA 1537 TA 98 TA 102
1-5
µg/plate  
0 11 105 9 30 314
50 13 96 14 26 271
158 12 111 10 26 243
500 9 104 8 33 295
1581 7 122 8 25 265
5000 12 119 8 25 332
2-AA 74 1675 91 2083 623
6-10
µg/tube  
0 6 104 6 26 353
1000 5 105 5 24 357
2000 7 98 6 26 384
3000 8 108 5 29 380
4000 5 108 6 22 381
5000 6 95 6 21 404
2-AA 66 1227 26 1625 719
11-12
µg/plate  
0         196 159
1000         174 181
2000         161 183
3000         168 151
4000         188 154
5000         178 156
2-AA         361 337

Bayer A.G. Table : 4 Study Number: T 5053854
Department of Toxicology Study Director: Dr.Herbold
Pharma Research Center Technician: Düver
Wuppertal Elberfeld Date: Feb. 13.1997
AMES Test with IDS Na-salt   Strain: S.typhimurium TA 1535
Dose/Plate (µg/Plate) Revertants per plate Titer Quotient
10% Dilution per mL
-S9 M SD +S9 M SD 10+6 10+8 -S9 +S9
Water 6 6 1 12 11 2 61 6.9 1.0 1.0
5 8 77
7 12
50 8 7 1 11 13 2 % / 1.2 1.3
7 14
7 15
158 8 6 2 10 12 2 % / 1.1 1.2
6 14
5 13
500 7 5 2 9 9 2 % / 0.9 0.8
4 10
5 7
1581 5 4 1 7 7 1 % / 0.7 0.7
4 7
4 8
5000 8 7 1 13 B 12 1 % / 1.2 1.2
6 11 B
7 13 B
Na-azide 10 918 924 23 % / / % / 154. 1* /
905
950
2-AA
3
% / / 77 74 5 % / / 6.9*
68
77
*: mutagenic effect B: Background lawn reduced
%: not tested SD: Standard-Deviation
M: Mean +S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G. Table : 5 Study Number: T 5053854
Department of Toxicology Study Director: Dr.Herbold
Pharma Research Center Technician: Düver
Wuppertal Elberfeld Date: Feb. 13.1997
AMES Test with IDS Na-salt   Strain: S.typhimurium TA 100
Dose/Plate (µg/Plate) Revertants per plate Titer Quotient
10% Dilution per mL
-S9 M SD +S9 M SD 10+6 10+8 -S9 +S9
Water 106 97 9 102 105 3 42 5.0 1.0 1.0
89 106 58
97 108
50 86 92 7 90 96 7 % 0.9 0.9
91 104
100 95
158 80 90 13 123 111 15 % / 0.9 1.1
105 94
86 116
500 81 87 11 106 104 15 % / 0.9 1.0
100 117
81 88
1581 99 88 10 126 122 4 % / 0.9 1.2
84 118
80 122
5000 93 92 4 114 119 6 % / 0.9 1.1
88 117
95 126
NF
0.2
208 209 20 % / / % / 2.1* /
190
229
2-AA
3
% / / 1785 1675 238 % / / 15.9*
1838
1402
*: mutagenic effect B: Background lawn reduced
%: not tested SD: Standard-Deviation
M: Mean +S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G. Table : 6 Study Number: T 5053854
Department of Toxicology Study Director: Dr.Herbold
Pharma Research Center Technician: Düver
Wuppertal Elberfeld Date: Feb. 13.1997
AMES Test with IDS Na-salt   Strain: S.typhimurium TA 1537
Dose/Plate (µg/Plate) Revertants per plate Titer Quotient
10% Dilution per mL
-S9 M SD +S9 M SD 10+6 10+8 -S9 +S9
Water 8 9 1 7 9 2 88 8.9 1.0 1.0
9 9 89
9 10
50 8 8 1 13 14 1 % / 0.9 1.6
7 14
9 14
158 8 9 1 12 10 2 % / 1.0 1.2
8 9
10 10
500 9 6 3 10 8 2 % / 0.7 0.9
6 7
4 6
1581 8 7 1 10 8 2 % / 0.8 0.9
7 7
6 6
5000 7 6 1 7 8 2 % / 0.7 0.9
6 10
6 7
4-NPDA
10
54 64 8 % / / % / 7.3* /
69
68
2-AA
3
% / / 105 91 12 % / / 10.5*
81
87
*: mutagenic effect B: Background lawn reduced
%: not tested SD: Standard-Deviation
M: Mean +S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G. Table : 7 Study Number: T 5053854
Department of Toxicology Study Director: Dr.Herbold
Pharma Research Center Technician: Düver
Wuppertal Elberfeld Date: Feb. 13.1997
AMES Test with IDS Na-salt   Strain: S.typhimurium TA 98
Dose/Plate (µg/Plate) Revertants per plate Titer Quotient
10% Dilution per mL
-S9 M SD +S9 M SD 10+6 10+8 -S9 +S9
Water 27 22 5 31 30 3 209 21.2 1.0 1.0
17 33 215
23 27
50 22 20 8 27 26 4 % / 0.9 0.9
12 22
27 29
158 24 17 6 26 26 1 % / 0.8 0.9
13 27
14 26
500 14 17 5 34 33 3 % / 0.8 1.1
14 29
23 35
1581 19 22 3 25 25 2 % / 1.0 0.8
24 27
22 23
5000 22 24 3 31 25 6 % / 1.1 0.8
23 19
28 24
4-NPDA
0.5
138 129 8 % / / % / 5.8* /
127
122
2-AA
3
% / / 1970 2083 103 % / / 68.7*
2108
2172
*: mutagenic effect B: Background lawn reduced
%: not tested SD: Standard-Deviation
M: Mean +S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G. Table : 8 Study Number: T 5053854
Department of Toxicology Study Director: Dr.Herbold
Pharma Research Center Technician: Düver
Wuppertal Elberfeld Date: Feb. 13.1997
AMES Test with IDS Na-salt   Strain: S.typhimurium TA 102
Dose/Plate (µg/Plate) Revertants per plate Titer Quotient
10% Dilution per mL
-S9 M SD +S9 M SD 10+6 10+8 -S9 +S9
Water 151 151 6 302 314 15 68 6.0 1.0 1.0
145 309 52
156 331
50 169 160 17 254 271 15 % / 1.1 0.9
140 278
171 282
158 168 149 17 247 243 6 % / 1.0 0.8
141 245
137 236
500 175 165 9 258 295 35 % / 1.1 0.9
164 301
157 327
1581 180 191 14 307 265 40 % / 1.3 0.8
185 261
207 228
5000 269 231 33 350 332 17 % / 1.5² 1.1
211 329
213 317
Cumene
50
249 242 17 % / / % / 1.6* /
222
254
2-AA
3
% / / 629 623 14 % / / 2.0*
607
634
² see Chapter 5.1 *: Mutagenic effect
%: not tested SD: Standard-Deviation
M: Mean +S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G. Table : 9 Study Number: T 5053854
Department of Toxicology Study Director: Dr.Herbold
Pharma Research Center Technician: Düver
Wuppertal Elberfeld Date: Feb. 20.1997
AMES Test with IDS Na-salt   Strain: S.typhimurium TA 1535
Dose/Plate (µg/Plate) Revertants per plate Titer Quotient
10% Dilution per mL
-S9 M SD +S9 M SD 10+6 10+8 -S9 +S9
Water 7 7 1 7 6 1 223 23.8 1.0 1.0
7 6 252
6 6
1000 7 7 2 5 5 1 % /  1.0 0.8
8 5
5 6
2000 5 6 1 5 7 2 % / 0.9 1.1
5 7
7 8
3000 6 6 2 7 8 1 % / 1.0 1.2
5 9
8 7
4000 8 7 2 4 5 2 % / 1.0 0.7
5 7
7 3
5000 6 6 0 5 6 2 % / 0.9 0.9
6 5
6 8
Na-azide 10 841 824 59 % / / % / 123.6* /
758
872
2-AA
3
% / / 73 66 7 % / / 10.4*
60
65
*: mutagenic effect B: Background lawn reduced
%: not tested SD: Standard-Deviation
M: Mean +S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G. Table : 10 Study Number: T 5053854
Department of Toxicology Study Director: Dr.Herbold
Pharma Research Center Technician: Düver
Wuppertal Elberfeld Date: Feb. 20.1997
AMES Test with : IDS Na-salt   Strain: S.typhimurium TA 100
Dose/Plate (µg/Plate) Revertants per plate Titer Quotient
10% Dilution per mL
-S9 M SD +S9 M SD 10+6 10+8 -S9 +S9
Water 135 127 7 105 104 2 197 19.7 1.0 1.0
124 102 196
121 105
1000 121 125 4 95 105 10 % / 1.0 1.0
126 107
128 114
2000 138 128 9 108 98 12 % / 1.0 0.9
125 101
122 85
3000 96 98 13 105 108 12 % / 0.8 1.0
86 121
111 97
4000 126 136 17 118 108 12 % / 1.1 1.0
126 110
155 95
5000 123 122 6 103 95 9 % / 1.0 0.9
127 85
115 97
NF
0.2
218 216 11 % / / % / 1.7* /
204
225
2-AA
3
% / / 1219 1227 18 % / / 11.8*
1248
      1214            
*: mutagenic effect B: Background lawn reduced
%: not tested SD: Standard-Deviation
M: Mean +S9: with S9 Mix
-S9: without S9 Mix

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Therefore, IDS, Na-Salz was considered to be non-mutagenic with and without S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.
Executive summary:

IDS, Na-Salz was investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants (Herbold, B, 1997a, OECD 471). These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. In a first experiment, doses up to and including 1581 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had only a weak, strain-specific bacteriotoxic effect. Due to the weakness of this effect this range could nevertheless be used for assessment purposes. Moreover, IDS, Na-Salz was investigated in an independent repeat using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per tube after preincubation for 20 minutes at 37°C on the same Salmonella typhimurium LT2 mutants. In this experiment, doses up to and including 5000 µg per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

In both experiment, evidence of mutagenic activity of IDS, Na-Salz was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls. Therefore, IDS, Na-Salz was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.