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Genetic toxicity in vitro

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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restriction (limited documentation)
Justification for type of information:
The substance trisodium hydrogen ethylenediaminetetraacetate (CAS 150-38-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
uninduced and arochlor induced liver S9 mix of male Fischer 344 rats, B6C3F1 mice, and Syrian hamsters
Test concentrations with justification for top dose:
10; 33; 100; 333; 1000; 3333; 10000 µg/plate
Vehicle / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: 2-Nitrofluorene; n-Methyl-N`-nitro-N-nitrosoguanidine; +S9: 2-aminoanthracene; 2-2(furyl)-3-(5-nitro-2-furyl)acrylamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

NUMBER OF REPLICATIONS: 3
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/ cell type: S. typhimurium TA 1535
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restriction (limited documentation)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
uninduced and arochlor induced liver S9 mix of male Fischer 344 rats, B6C3F1 mice, and Syrian hamsters
Test concentrations with justification for top dose:
10; 33; 100; 333; 1000; 3333; 10000 µg/plate
Vehicle / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: 2-Nitrofluorene; n-Methyl-N`-nitro-N-nitrosoguanidine; +S9: 2-aminoanthracene; 2-2(furyl)-3-(5-nitro-2-furyl)acrylamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

NUMBER OF REPLICATIONS: 3
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/ cell type: S. typhimurium TA 1535
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study without detailed documentation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
no
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
Purity of the test item was 100%. Stock solutions were prepared before use.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
250, 500, 1000, 1500, 2000 µg/ml
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: for test without metabolic activation: ethyl methane sulfonate (EMS); for test with metabolic activation: 3-methylcholanthrene (MCA)
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no increases in the number of small colonies, and the proportion of large and small colonies remained constant for all compounds.

Mutagenicity and Cytotoxicity of Na2EDTA with and without metabolic activation

 Concentration (ug/ml)  S9  Relative growth (% of control) Mutant frequency per 10e6 survivors
 0  -  100.0  24
 250  -  95.5  27
 500  -  79.5  27
 1000  -  87.0 20 
 1500  -  83.5  30
 2000  -  65.5  29
 0.25 ug/ml EMS  -  42.0  693
       
 0  +  100.0  48
 250  +  78.5  55
 500  +  89.5  56
 1000  +  80.5  47
 1500  +  107.0  40
 2000  +  87.5  42
 5.0 ug/ml MCA  +  22.0  542
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study without detailed documentation
Justification for type of information:
The substance Ethylenediaminetetraacetic acid disodium salt dihydrate (CAS 139-33-3) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
no
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
Purity of the test item was 100%. Stock solutions were prepared before use.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
250, 500, 1000, 1500, 2000 µg/ml
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: for test without metabolic activation: ethyl methane sulfonate (EMS); for test with metabolic activation: 3-methylcholanthrene (MCA)
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no increases in the number of small colonies, and the proportion of large and small colonies remained constant for all compounds.

Mutagenicity and Cytotoxicity of Na2EDTA with and without metabolic activation

 Concentration (ug/ml)  S9  Relative growth (% of control) Mutant frequency per 10e6 survivors
 0  -  100.0  24
 250  -  95.5  27
 500  -  79.5  27
 1000  -  87.0 20 
 1500  -  83.5  30
 2000  -  65.5  29
 0.25 ug/ml EMS  -  42.0  693
       
 0  +  100.0  48
 250  +  78.5  55
 500  +  89.5  56
 1000  +  80.5  47
 1500  +  107.0  40
 2000  +  87.5  42
 5.0 ug/ml MCA  +  22.0  542
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
BALB/c-3T3 transformation assay was evaluated because chemical-induced morphologically transformed cells are easily recognized and induced at relatively high frequencies in this assay.
BALB/c-3T3 transformation assay: BALB/c-3T3 cells were grown in soft agar. Non-malignant cells grow at low frequencies in soft agar, transformed cells readily grew in soft agar and were tumorigenic in vivo.
GLP compliance:
no
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
Supplied by Radian.
Species / strain / cell type:
other: BALB/c-3T3
Metabolic activation:
without
Test concentrations with justification for top dose:
First Experiment: 250; 361; 500; 750 µg/ml (0.907; 1.36; 1.81; 2.72 mM)
Second Experiment: 231; 461; 643; 770 µg/ml (0.837; 1.67; 2.33; 2.79 mM)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
Each transformation assay contained: a standard clonal survival assay, a co-culture clonal survival assay, a transformation assay. In each experiment, chemical-induced transformation was detected in 18-20 vessels/dose seeded with 3.2 x 10E4 cells/vessel. Chemical doses were applied to cell cultures for 48 hr, days 2-4, using standard procedures. The chemical was tested at four treatment doses in two independent trials.
Evaluation criteria:
The number of type I-III transformed foci of BALB/c-3T3 cells were identified microscopically using published criteria, and type III foci had three phenotypic properties: piling and overlapping cells, disorientation of cells at the periphery of the focus, and invasion of transformed cells into a contact-inhibited monolayer of WT cells. Type I and II foci also appeared in many different sizes, but they lacked one or more of the three phenotypic properties of the type III transformed focus.
Statistics:
rank t-statistics
Key result
Species / strain:
other: BALB/c-3T3
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Na3EDTA was moderately cytotoxic to the BALB/c-3T3 cells and had an average LD50 of 1.89 mM. The statistical sensitivities of transformation assay trials 1 and 2 were 80 and 67/110, respectively; the detection sensitivities for BaP of trials 1 and 2 were 89 and 78/110, respectively. The test chemical had a limited activity transformation response in the first experiment, and a sufficient negative response in the second experiment. Ethylenediamine tetraacetic acid, trisodium salt, was evaluated as inactive in the transformation assay, and its actual and estimated rank t-statistics were 1.41 and 2.01, respectively.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The substance Trisodium hydrogen ethylenediaminetetraacetate (CAS 150-38-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
BALB/c-3T3 transformation assay was evaluated because chemical-induced morphologically transformed cells are easily recognized and induced at relatively high frequencies in this assay.
BALB/c-3T3 transformation assay: BALB/c-3T3 cells were grown in soft agar. Non-malignant cells grow at low frequencies in soft agar, transformed cells readily grew in soft agar and were tumorigenic in vivo.
GLP compliance:
no
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
Supplied by Radian.
Species / strain / cell type:
other: BALB/c-3T3
Metabolic activation:
without
Test concentrations with justification for top dose:
First Experiment: 250; 361; 500; 750 µg/ml (0.907; 1.36; 1.81; 2.72 mM)
Second Experiment: 231; 461; 643; 770 µg/ml (0.837; 1.67; 2.33; 2.79 mM)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
Each transformation assay contained: a standard clonal survival assay, a co-culture clonal survival assay, a transformation assay. In each experiment, chemical-induced transformation was detected in 18-20 vessels/dose seeded with 3.2 x 10E4 cells/vessel. Chemical doses were applied to cell cultures for 48 hr, days 2-4, using standard procedures. The chemical was tested at four treatment doses in two independent trials.
Evaluation criteria:
The number of type I-III transformed foci of BALB/c-3T3 cells were identified microscopically using published criteria, and type III foci had three phenotypic properties: piling and overlapping cells, disorientation of cells at the periphery of the focus, and invasion of transformed cells into a contact-inhibited monolayer of WT cells. Type I and II foci also appeared in many different sizes, but they lacked one or more of the three phenotypic properties of the type III transformed focus.
Statistics:
rank t-statistics
Key result
Species / strain:
other: BALB/c-3T3
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Na3EDTA was moderately cytotoxic to the BALB/c-3T3 cells and had an average LD50 of 1.89 mM. The statistical sensitivities of transformation assay trials 1 and 2 were 80 and 67/110, respectively; the detection sensitivities for BaP of trials 1 and 2 were 89 and 78/110, respectively. The test chemical had a limited activity transformation response in the first experiment, and a sufficient negative response in the second experiment. Ethylenediamine tetraacetic acid, trisodium salt, was evaluated as inactive in the transformation assay, and its actual and estimated rank t-statistics were 1.41 and 2.01, respectively.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
other: Chromosomal aberration Assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix
Test concentrations with justification for top dose:
50; 75; 100 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Results of the Chromosome Aberrations Test for Na3EDTA.
Study Result: Negative
Activation Trial Trial Call
No Activation 1 Negative
Induced Rat Liver S9 2 Negative
Trial #:1   Activation: No Activation   Date: 10/17/1984   Harvest Time: 13.5 hrs   Trial Call: Negative  
Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
Abs. Per With Abs. Per With Abs. Per With Abs. Per With
Cell Abs. Cell Abs. Cell Abs. Cell Abs.
Abs: Aberrations
Vehicle Control: Dimethylsulfoxide 10          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
Positive Control: Mitomycin C 0.25       100 29 0.29 26 24 0.24 22 5 0.05 5 0 0 0
1          50 31 0.62 46 24 0.48 44 7 0.14 14 0 0 0
Test Chemical: Ethylenediamine tetraacetate, trisodium salt (EDTA)  25          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
50          100 2 0.02 2 1 0.01 1 1 0.01 1 0 0 0
75          100 5 0.05 5 3 0.03 3 2 0.02 2 0 0 0
100          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
Trend: 1.106 1.331 0.333
Probability: 0.134 0.092 0.37
Trial #:2   Activation: Induced Rat Liver S9   Date: 10/31/1984   Harvest Time: 14.0 hrs   Trial Call: Negative  
Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
Abs. Per With Abs. Per With Abs. Per With Abs. Per With
Cell Abs. Cell Abs. Cell Abs. Cell Abs.
Abs: Aberrations
Positive Control: Cyclophosphamide 15          100 55 0.55 40 29 0.29 22 26 0.26 22 0 0 0
Vehicle Control: Dimethylsulfoxide 10          100 3 0.03 3 2 0.02 2 1 0.01 1 0 0 0
Test Chemical: Ethylenediamine tetraacetate, trisodium salt (EDTA)  25          100 1 0.01 1 1 0.01 1 0 0 0 0 0 0
50          100 4 0.04 4 2 0.02 2 1 0.01 1 1 0.01 1
75          100 4 0.04 4 2 0.02 2 2 0.02 2 0 0 0
100          100 3 0.03 3 1 0.01 1 2 0.02 2 0 0 0
Trend: 0.686 -0.156 1.164
Probability: 0.247 0.562 0.122
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
The substance Trisodium hydrogen ethylenediaminetetraacetate (CAS 150-38-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
other: Chromosomal aberration Assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix
Test concentrations with justification for top dose:
50; 75; 100 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Results of the Chromosome Aberrations Test for Na3EDTA.
Study Result: Negative
Activation Trial Trial Call
No Activation 1 Negative
Induced Rat Liver S9 2 Negative
Trial #:1   Activation: No Activation   Date: 10/17/1984   Harvest Time: 13.5 hrs   Trial Call: Negative  
Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
Abs. Per With Abs. Per With Abs. Per With Abs. Per With
Cell Abs. Cell Abs. Cell Abs. Cell Abs.
Abs: Aberrations
Vehicle Control: Dimethylsulfoxide 10          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
Positive Control: Mitomycin C 0.25       100 29 0.29 26 24 0.24 22 5 0.05 5 0 0 0
1          50 31 0.62 46 24 0.48 44 7 0.14 14 0 0 0
Test Chemical: Ethylenediamine tetraacetate, trisodium salt (EDTA)  25          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
50          100 2 0.02 2 1 0.01 1 1 0.01 1 0 0 0
75          100 5 0.05 5 3 0.03 3 2 0.02 2 0 0 0
100          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
Trend: 1.106 1.331 0.333
Probability: 0.134 0.092 0.37
Trial #:2   Activation: Induced Rat Liver S9   Date: 10/31/1984   Harvest Time: 14.0 hrs   Trial Call: Negative  
Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
Abs. Per With Abs. Per With Abs. Per With Abs. Per With
Cell Abs. Cell Abs. Cell Abs. Cell Abs.
Abs: Aberrations
Positive Control: Cyclophosphamide 15          100 55 0.55 40 29 0.29 22 26 0.26 22 0 0 0
Vehicle Control: Dimethylsulfoxide 10          100 3 0.03 3 2 0.02 2 1 0.01 1 0 0 0
Test Chemical: Ethylenediamine tetraacetate, trisodium salt (EDTA)  25          100 1 0.01 1 1 0.01 1 0 0 0 0 0 0
50          100 4 0.04 4 2 0.02 2 1 0.01 1 1 0.01 1
75          100 4 0.04 4 2 0.02 2 2 0.02 2 0 0 0
100          100 3 0.03 3 1 0.01 1 2 0.02 2 0 0 0
Trend: 0.686 -0.156 1.164
Probability: 0.247 0.562 0.122
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
other: Mouse lymphoma Assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats
Test concentrations with justification for top dose:
3000, 4000, 5000 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: methyl methanesulfonate; + S9 mix: methylcholanthrene
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Tables: Results of the mouse lymphoma test with Na3EDTA

Nonactivation Trial: 1 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: H2O 0          64 87 113 59 69
63 112 132 70
65 108 133 68
64# 92 148.5 78
Test Chemical: 60          65 107 177 91 83
74 109 165 74
70          58 110 97 56 68
67 113 162 81
80          67 100 131 65 59
63# 116 99 52
90          58 86 119 69 72
63 93 140 75
100          71 111 142 67 73
66 101 159 80
Positive Control: MMS 15          49 42 293 198 181*
53 45 261 164
Trial Notes:
Nonactivation Trial: 2 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          78 103 24 10 18
93 91 50 18
103 104 57 18
84 102 63 25
Test Chemical: 1000          80 42 61 25 23
76 51 45 20
2000          87 53 52 20 21
88 52 61 23
3000          79 38 47 20 21
77 50 50 22
4000          93 32 80 29 27
65 30 49 25
5000          79 23 49 21 22
76 29 54 24
Positive Control: MMS 15          52 25 146 93 93*
38 18 107 93
Trial Notes:
Nonactivation Trial: 3 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          69 94 88 43 38
62 109 48 26
58 87 79 46
76 110 84 37
Test Chemical: 1000          61 59 95 52 57
55 60 101 61
2000          68 73 111 55 49
61 61 77 42
3000          62 64 110 59 60
52 50 95 61
4000          61 45 74 40 42
61 51 81 45
5000          50 34 68 45 46
55 37 77 47
Positive Control: MMS 15          23 24 163 235 217*
26 23 156 200
EMS 250          41 61 427 347 325*
52 59 474 302
Trial Notes:
Induced S9 Trial: 1 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          108 93 101 31 36
102 109 98 32
109 101 119 36
97 97 130 45
Test Chemical: 1000          105 74 137 43 47
100 78 152 51
2000          82 58 101 41 42
97 67 126 43
3000          99 56 159 54 44
109 62 113 35
4000          99 55 106 36 38
85 45 101 40
5000          77 36 124 54 55
85 36 145 57
Positive Control: MCA 2.5        72 44 615 286 305*
70 40 680 323
Trial Notes:
Induced S9 Trial: 2 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          60 96 36 20 20
64 110 35 18
56 92 48 29
66 103 25 13
Test Chemical: 1000          52 61 41 26 21
63 75 31 16
2000          67 63 56 28 27
62 61 48 26
3000          61 40 41 22 31
71 48 85 40
4000          66 41 56 28 29
57 35 51 30
5000          70 34 65 31 28
59 36 45 25
Positive Control: MCA 2.5        34 19 231 229 232*
34 17 237 236
Trial Notes:
Footnotes:
Asterisks(*) indicate significant responses.
r = rejected value due to quality control criteria
# = reduced sample size because of the loss of one culture dish due to contamination
MMS = methyl methanesulfonate
MCA = methylcholanthrene
DMSO = dimethylsulfoxide (solvent)
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
The substance Trisodium hydrogen ethylenediaminetetraacetate (CAS 150-38-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
other: Mouse lymphoma Assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats
Test concentrations with justification for top dose:
3000, 4000, 5000 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: methyl methanesulfonate; + S9 mix: methylcholanthrene
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Tables: Results of the mouse lymphoma test with Na3EDTA

Nonactivation Trial: 1 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: H2O 0          64 87 113 59 69
63 112 132 70
65 108 133 68
64# 92 148.5 78
Test Chemical: 60          65 107 177 91 83
74 109 165 74
70          58 110 97 56 68
67 113 162 81
80          67 100 131 65 59
63# 116 99 52
90          58 86 119 69 72
63 93 140 75
100          71 111 142 67 73
66 101 159 80
Positive Control: MMS 15          49 42 293 198 181*
53 45 261 164
Trial Notes:
Nonactivation Trial: 2 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          78 103 24 10 18
93 91 50 18
103 104 57 18
84 102 63 25
Test Chemical: 1000          80 42 61 25 23
76 51 45 20
2000          87 53 52 20 21
88 52 61 23
3000          79 38 47 20 21
77 50 50 22
4000          93 32 80 29 27
65 30 49 25
5000          79 23 49 21 22
76 29 54 24
Positive Control: MMS 15          52 25 146 93 93*
38 18 107 93
Trial Notes:
Nonactivation Trial: 3 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          69 94 88 43 38
62 109 48 26
58 87 79 46
76 110 84 37
Test Chemical: 1000          61 59 95 52 57
55 60 101 61
2000          68 73 111 55 49
61 61 77 42
3000          62 64 110 59 60
52 50 95 61
4000          61 45 74 40 42
61 51 81 45
5000          50 34 68 45 46
55 37 77 47
Positive Control: MMS 15          23 24 163 235 217*
26 23 156 200
EMS 250          41 61 427 347 325*
52 59 474 302
Trial Notes:
Induced S9 Trial: 1 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          108 93 101 31 36
102 109 98 32
109 101 119 36
97 97 130 45
Test Chemical: 1000          105 74 137 43 47
100 78 152 51
2000          82 58 101 41 42
97 67 126 43
3000          99 56 159 54 44
109 62 113 35
4000          99 55 106 36 38
85 45 101 40
5000          77 36 124 54 55
85 36 145 57
Positive Control: MCA 2.5        72 44 615 286 305*
70 40 680 323
Trial Notes:
Induced S9 Trial: 2 Experiment Call: Negative and Non-Toxic
Conc. Cloning Relative Total Mutant Colonies Mutant Frequency AVG Mutant Frequency
µg/mL Efficiency Growth
Vehicle Control: FOP 0          60 96 36 20 20
64 110 35 18
56 92 48 29
66 103 25 13
Test Chemical: 1000          52 61 41 26 21
63 75 31 16
2000          67 63 56 28 27
62 61 48 26
3000          61 40 41 22 31
71 48 85 40
4000          66 41 56 28 29
57 35 51 30
5000          70 34 65 31 28
59 36 45 25
Positive Control: MCA 2.5        34 19 231 229 232*
34 17 237 236
Trial Notes:
Footnotes:
Asterisks(*) indicate significant responses.
r = rejected value due to quality control criteria
# = reduced sample size because of the loss of one culture dish due to contamination
MMS = methyl methanesulfonate
MCA = methylcholanthrene
DMSO = dimethylsulfoxide (solvent)
Endpoint:
in vitro transformation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
A cell transformation assay was performed at pH 6.7 using Syrian hamster embryo cells.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
mammalian cell line, other: Syrian hamster embryo (SHE)
Metabolic activation:
without
Test concentrations with justification for top dose:
24 h exposure: 25; 50; 75; 100 µg/mL
7-day exposure: 25; 50; 75; 100; 125; 150 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
DURATION
Upon treatment of the cultures with test chemical, they were either refed with control medium after a 24-h exposure and then left undisturbed for the 7-day clonal expression period or exposed to chemical for the entire 7-day clonal expression period.

NUMBER OF REPLICATIONS:2


Statistics:
one-tailed Fisher's Exact
Key result
Species / strain:
mammalian cell line, other: Syrian hamster embryo (SHE)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
After a 24-h exposure, EDTA (in culture medium) was tested at concentrations up to 100 µg/mL (49% RPE) and failed to induce a statistically significant
increase in MTF compared with controls. After a 7-day exposure, EDTA was tested at concentrations up to 150 µg/mL (14% RPE) and failed to induce a
statistically significant increase in transformation frequency compared with controls (see table 1)

Table 1: Results of the cell transformation assay:

RPE (%) Number of MT/ Total Colonies MTF (%)
7-day Control 100 3/1269 0.24
50 µg/mL 95 2/1187 0.17
75 µg/mL 88 0/736 0.00
100 µg/mL 75 0/932 0.00
125 µg/mL 51 1/424 0.24
150 µg/mL 14 0/180 0.00
24 h Control 100 7/1293 0.54
25 µg/mL 102 7/1317 0.53
50 µg/mL 94 13/1182 1.10
75 µg/mL 77 4/995 0.40
100 µg/mL 49 10/1225 0.82

RPE = relative plating efficiency; MT = morphologically transformed colonies; MTF = morphological transformation frequency

Endpoint:
in vitro transformation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The substance Disodium dihydrogen ethylenediaminetetraacetate (CAS 139-33-3) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
A cell transformation assay was performed at pH 6.7 using Syrian hamster embryo cells.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
mammalian cell line, other: Syrian hamster embryo (SHE)
Metabolic activation:
without
Test concentrations with justification for top dose:
24 h exposure: 25; 50; 75; 100 µg/mL
7-day exposure: 25; 50; 75; 100; 125; 150 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
DURATION
Upon treatment of the cultures with test chemical, they were either refed with control medium after a 24-h exposure and then left undisturbed for the 7-day clonal expression period or exposed to chemical for the entire 7-day clonal expression period.

NUMBER OF REPLICATIONS:2


Statistics:
one-tailed Fisher's Exact
Key result
Species / strain:
mammalian cell line, other: Syrian hamster embryo (SHE)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
After a 24-h exposure, EDTA (in culture medium) was tested at concentrations up to 100 µg/mL (49% RPE) and failed to induce a statistically significant
increase in MTF compared with controls. After a 7-day exposure, EDTA was tested at concentrations up to 150 µg/mL (14% RPE) and failed to induce a
statistically significant increase in transformation frequency compared with controls (see table 1)

Table 1: Results of the cell transformation assay:

RPE (%) Number of MT/ Total Colonies MTF (%)
7-day Control 100 3/1269 0.24
50 µg/mL 95 2/1187 0.17
75 µg/mL 88 0/736 0.00
100 µg/mL 75 0/932 0.00
125 µg/mL 51 1/424 0.24
150 µg/mL 14 0/180 0.00
24 h Control 100 7/1293 0.54
25 µg/mL 102 7/1317 0.53
50 µg/mL 94 13/1182 1.10
75 µg/mL 77 4/995 0.40
100 µg/mL 49 10/1225 0.82

RPE = relative plating efficiency; MT = morphologically transformed colonies; MTF = morphological transformation frequency

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification