Reaction mass of disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]zincate(2-) and sodium nitrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other: mutagenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

G 026

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]zincate(2-)

Cas Number:

666748-59-0

Molecular formula:

C8H7NNa2O8Zn

IUPAC Name:

disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]zincate(2-)

Constituent 2

Reference substance name:

Zinc (II) chelate of sodium salt 1,2 dicarboxyethyl D,L aspartic acid

IUPAC Name:

Zinc (II) chelate of sodium salt 1,2 dicarboxyethyl D,L aspartic acid

Details on test material:

TEST ARTICLE

Identification:
Zn (II) IDHA mikrogranulat

Lot Number:
906/P/Zn

Purity:
Certificate of analysis No. ZN-006/906/P (Annex 2) Sponsor:
Przedsiebiorstwo Produkcyjno-Consultingowe, Ul. Warszawska 43, 61 028 Poznan, Poland

Stability:
Life time of the test article is 36 months from manufacturing date (14.09.2006).
Storage Conditions:
Product is stable at temperature from - 10°C to + 30°C. The test article has been stored approximately at 20°C, protected against sunlight in the laboratory No. 412A (SOP13, 14). The storage conditions were monitored (SOP 12). The records from monitoring of temperature in laboratory No. 412A will be archivised according to Principle of GLP-Department of Biology 2008, chapter 10 (4).
The test substance has been maintained in the Department of Microbiology during the study and until one year after finishing of study and remaining test article will be returned to the customer.

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

Results and discussion

Test results

Species / strain:

other: TA 98, TA 100, TA 97, TA 102, TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Any other information on results incl. tables

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 (M_F> 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 (M_F=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 (M_F= 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(M_F=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 , M_F=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,M_F=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.

Applicant's summary and conclusion

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.