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Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

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:
dest. 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, TA 1537, TA 98 and 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
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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 restrictions
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
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:
other: -S 9: Mitomycin C; + S9: Cyclophosphamide
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
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 restrictions
Principles of method if other than guideline:
NTP-Standard Protocol
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 from the 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
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
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 without detailed documentation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
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: - S9: ethyl methane sulfonate; + S9: 3-methylcholanthrene
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
Positive controls validity:
valid
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
Positive controls validity:
valid

Table 1: Mutagenicity and Cytotoxicity of Na2EDTA

Concentration [µg/mL] S9 Relative growth Mutant frequency/ 106survivors
0 - 100 24
250 - 95.5 27
500 - 79.5 27
1000 - 87 20
1500 - 83.5 30
2000 - 65.5 29
0.25 µg/mL EMS - 42 693
0 + 100 48
250 + 78.5 55
500 + 95.5 56
1000 + 80.5 47
1500 + 107 40
2000 + 87.5 42
5 µg/mL MCA + 22 542
Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets generally accepted scientific principles
Principles of method if other than guideline:
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. Chemical-induced morphologically transformed cells are easily recognized and induced at relatively high frequencies in this assay.
GLP compliance:
no
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
other: BALB/c-3T3
Metabolic activation:
without
Test concentrations with justification for top dose:
First Experiment: 0.907; 1.36; 1.81; 2.72 mM
Second Experiment: 0.837; 1.67; 2.33; 2.79 mM
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
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
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
Positive controls validity:
valid

Na3EDTA was moderately cytotoxie 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 an limited activity transformation response in the first experiment, and an 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 bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to OECD 471 guideline study with acceptable restrictions (missing strain for crosslinking/oxidizing agents)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
missing strain for crosslinking/oxidizing agents
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA97, TA98, TA100, TA1535, and TA1537
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers S9 mix
Test concentrations with justification for top dose:
100; 333; 1000; 3333; 6666; 10,000 µg/plate
Vehicle / solvent:
Water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9 mix: sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine; +S9-mix: 2 aminoantracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
preincubation: 50 µL of the TS were mixed with 100 µL Salmonella culture and 500 µL S-9 mix of buffer

DURATION
- Preincubation period: 20 min
- Expression time (cells in growth medium): 2 days

Species / strain:
S. typhimurium, other: TA97, TA98, TA100, TA1535, and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Study, meets accepted scientific standard and is 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:
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 refered 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
Species / strain:
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:
With 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. With 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

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF AG, Experimental Toxicology and Ecology
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: 31-9412

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Purity test date: 31 October 1996
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle water over a period of 4 hours has been verified analytically
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Weight at study initiation: Mean 29 g
- Housing: Makrolon cages type Mill, in groups of 5
- Diet: Kliba, standardized pelleted feed, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water
Details on exposure:
- The low dose group was given 500 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 2.5 g/100 mL.
- The intermediate dose group was given 1000 mg test substance/kg body weight or 20 ml/kg body weight of a solution with a concentration of 5.0 g/100 mL.
- The top dose group was given 2000 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 10.0 g/100 mL.
Duration of treatment / exposure:
48 h (animals were treated twice at 24 h intervals)
Frequency of treatment:
twice
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
20 mg of cyclophosphamide (CPP)/kg body weight or 0 .15 mg of vincristine sulphate (VCR)/kg body weight, both, dissolved in purified water, were administered to male animals once, orally each in a volume of 10 mL/kg body weight.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:


METHOD OF ANALYSIS:
2,000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters have been evaluated:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter )
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed .
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range .

A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
- The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
As clinical signs only piloerrection were observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- After two administrations of the highest dose of 2000 mg/kg body weight, 0.4 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours.
- In the two lower dose groups, rates of micronuclei of about 0.6 ‰ (1000 mg/kg group) and 0.7 ‰ (500 mg/kg group) were detected.
- With 12.7 %o the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg body weight.
- With 81 .7%o the positive control vincristine for induction of spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i .e. 10.5 ‰.
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups

Thus, the test substance Trilon BD did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control value and was within the historical control range. Nor were large micronuclei (d > D/4) observed either in the negative control group or in the three dose groups of Trilon BD.

No inhibition of erythropoiesis, induced by the treatment of mice with Trilon BD was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Micronuclei induction in germ cells of the mouse was evaluated after after i.p. application. Due to the very alkaline character of the test and its application very close to the gonads the route of application chosen in this test is not suitable and results obtained are considered to be equivocal.
Principles of method if other than guideline:
Micronuclei induction in germ cells of the mouse were evaluated after after i.p. application. In a second set of experiments chromosomal aberrations in mouse spermatogonia were determined.
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy
- Age at study initiation: 8 -12 weeks
Route of administration:
intraperitoneal
Details on exposure:
- Application volume: 10 ml/kg bw

Duration of treatment / exposure:
24 or 48 h (2 treatment sacrifice intervals)

Frequency of treatment:
single treatment
Dose / conc.:
186 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 control animals
3 for the 24 h interval
at least 4 for the 48 h interval

Control animals:
yes
Positive control(s):
5.5 mg/kg bw Adriamycin
Tissues and cell types examined:
spermatids
Evaluation criteria:
The presence of MN was assessed on both Golgi and Cap phases of spermatid development, by scoring a minimum of 1000 Golgi phase spermatids per animal, and the Cap phase cells observed at the same time.
Sex:
male
Genotoxicity:
positive
Remarks:
see table 1
Toxicity:
not specified
Negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Frequency of MN in early spermatids

Golgi phase

Cap phase

Total

+MN (‰ ± SE)

Total

+MN (‰ ± SE)

Controls

6,201

5 (0.8 ± 0.4)

5,836

3 (0.5 ± 0.3 )

ADM 24 hr

3,327

0

4,493

3 (0 .7 ± 0 .4)

ADM 48 hr

4,023

18 (4 .5 ± 1. 1)***

4,070

17 (4.2 ± 1.0)** *

EDTA 24 hr

2,993

9 (3.0 ± 1.0)*

3,436

2 (0 .6 ± 0 .4)

EDTA 48 hr

4,010

15 (3 .8 ± 1 .0)***

3,541

2 (0 .9 ± 0 .4 )

It was speculated by the authors whether EDTA induced MN either because of an S-independent clastogenic action of the compound under study, or for chromosome lagging.

CHROMOSOMAL ABERRATIONS

- No induction of chromosomal aberration by EDTA

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
In vivo mouse micronucleus assay
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy
- Age at study initiation: 8 -12 weeks
Route of administration:
intraperitoneal
Details on exposure:
- Application volume: 10 mL/kg bw
Duration of treatment / exposure:
24 or 48 h (2 treatment sacrifice intervals)
Frequency of treatment:
single treatment
Dose / conc.:
186 mg/kg bw/day
No. of animals per sex per dose:
6 control animals
3 for the 24 h interval
at least 4 for the 48 h interval
Control animals:
yes
Positive control(s):
1 mg/kg bw Mitomycin C
Tissues and cell types examined:
bone marrow; polychromatic erythrocytes
Details of tissue and slide preparation:
Bone marrow smears were prepared following a standard protocol (MacGregor et al., 1987).
Evaluation criteria:
The frequency of MN induced in bone marrow was estimated by scoring 2000 polychromatic erythrocytes (PCE) per animal at both time intervals. The frequency of normochromatic erythrocytes (NCE) was also assessed, to verify the possible cytotoxicity of each tested dose through the PCE/NCE ratio.
Statistics:
The frequencies of MN observed in each experimental group were pooled and the significance of differences between treated and control groups were evaluated by using the G test. The mean frequency of aberrations per cell and the PCE/NCE ratio were compared by the Student t-test after square root transformation of values.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid

Table 1: Frequency of MN in Bone Marrow PCEs After Treatment With EDTA at Two Time Intervals

PCE

NCE

PCE/NCE ratio

Total

+MN ( ‰ ± SE)

Total

+MN ( ‰ ± SE)

PCE/NCE

Controls

11,810

21 (1 .8 ± 0.4)

12,014

14 (1.2 ± 0.3)

1.09 ± 0.09

MMC 24 hr

6,149

93 (15.1 ± 1.6)

6,085

22 (3.6 ± 0.8)

1.01 ± 0.05

MMC 48 hr

8,302

89 (10.7 ± 1.1)

9,881

70 ( .1 ± 0.8)

0.87 ± 0.08

EDTA 24 hr

12,153

18 (1 .5 ± 0.3)

13,820

15 (1 .1 ± 0 .3)

0 .94 ± 0.1 1

EDTA 48 hr

8,019

20 (2 .5 ± 0.6)

7,514

15 (2 .0 ± 0.5)

1 .05 ± 0 .09

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Documentation insufficient for assessment
GLP compliance:
no
Type of assay:
mammalian germ cell cytogenetic assay
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy
Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
10 mL/kg bw

Duration of treatment / exposure:
6 h and 5 days
Frequency of treatment:
single treatment
Dose / conc.:
93 mg/kg bw/day (actual dose received)
Dose / conc.:
186 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (within 2 independent experiments)
Control animals:
yes, concurrent no treatment
Tissues and cell types examined:
secondary spermatocytes
Sex:
male
Genotoxicity:
negative
Remarks:
Clinical observations are not reported
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable

EDTA did not induce significant increases in aneuploidy in secondary spermatocytes at both exposure levels and meiotic stages tested.

Additional information

IN VITRO

Na3EDTA was negative in a reverse gene mutation assay using bacteria Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 as well as E. Coli WP2uvrA without and with uninduced and arochlor induced liver S9 from male Fischer 344 rats, B6C3F1 mice or Syrian hamsters. The substance was tested up to concentrations of 10,000 µg/plate (Dunkel, 1985).

Similar results were obtained by Zeiger (1988), who tested up tp 10,000 µg/plate Na3EDTA on Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 with and without Arochlor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers S9 mix.

Several mammalian cell line gene mutations assays are available. In a mouse lymphoma assay with Na3EDTA, the mutant frequency was not increased at concentrations of 3000, 4000, 5000 µg/mL and a treatment time of 4 h. The assay was conducted with and without metabolic activation and no cytotoxicity was detected (NTP, 1984).

Another mouse lymphoma assay with Na2EDTA was also negative at concentrations of 250, 500, 1000, 1500 and 2000 µg/mL with and without metabolic activation. However, 2000 µg/mL Na2EDTA reduced the relative growth to 65.5 % compared to the control in cells without metabolic activation. This was not the case if cells were treated with S9 mix (Whittaker, 2001).

Cell transformation assays with Na2 and Na3 salts of EDTA were negative.

One test was performed on SHE cells with exposure of up to 100 µg/mL for 24 h or 150 µg/ml Na2EDTA for up to 7 days without metabolic activation (LeBoef, 1996).

Additionally a cell transformation assay using BALB/c-3T3 cells was performed without metabolic activation. Cells were exposed to up to up to 770 µg/mL Na3EDTA for 48 h without metabolic activation (Matthews, 1993).

IN VIVO

Several in vivo tests for genotoxicity on somatic cells have been performed. The key study which was performed according to OECD 474 guideline and GLP, concluded that no micronuclei were induced in polychromatic erythrocytes of NMRI mice after repeated oral administration (twice with a 24-hour interval between administrations) of 500, 1000 and 2000 mg/kg bw Na2EDTA. As clinical sign only piloerection was observed after the second administration of 2000 mg/kg. No lethal effects or cytotoxicity (PCE/NCE ratio) were induced. Only males (5 per group) were used because no distinct symptomatic differences between males and females were noticed in a pre-test (BASF SE, 2000).

In another well-conducted in vivo micronucleus assay Na2EDTA was negative in bone marrow cells of mice (strain: BALB/c). Mice were treated with a single intraperitoneal dose of 186 mg/kg bodyweight; the sampling times were 24 hours and 48 hours after treatment. The tested dose was near to the LD50 value. No cytotoxic effects (PCE/NCE) were induced; information about clinical signs or lethal effects were not given. Only males (3 per 24-hour group; 4 per 48-hour group) were used (Russo & Levis, 1992).

Additionally, in vivo mutagenicity tests have been performed on rodent germ cells. Na2EDTA induced micronuclei in germ cells at the late stages of spermacytogenesis of mice (strain: BALB/c) after intraperitoneal administration of a very high dose of 186 mg/kg bw in the range of the i.p. LD50 value.

The frequency of micronuclei was analysed in Golgi phase and Cap phase, representing the two earliest phases of spermatids development. The sampling times were 24 hours and 48 hours after administration. Na2EDTA induced micronuclei in Golgi phase spermatids (0.30 % and 0.38 % micronucleated spermatids at 24 hours and 48 hours sampling as compared to 0.08 % in controls); in Cap phase spermatids negative results were obtained. Toxicity data were not given. Aneuploidy is discussed as most probable origin of micronuclei produced by NA2EDTA in secondary spermatocytes because the substance generally induced micronuclei of larger size in comparison with other substances.

Micronuclei induction in germ cells was evaluated after after i.p. application in this study. Due to the very alkaline character of the test item and its application very close to the gonads the route of application chosen in this test is not suitable and results obtained are considered to be equivocal.

In addition, Na2EDTA does not induce chromosomal aberrations in the spermatogonial phase, the most suitable germ cell population to detect chromosomal aberrations. An in vivo chromosomal aberration assay with mouse spermatogonia (strain: BALB/c) led to a negative result after a single i.p. administration of 186 mg/kg bw Na2EDTA. The sampling time was 24 hours sampling time after administration (Russo & Levis, 1992).

Zordan et al. (1990) investigated aneugenic properties of Na2EDTA in primary and secondary spermatocytes of mouse (strain: BALB/c). After single i .p. administration of 93 and 186 mg/kg bw no increases in aneuploid spermatocytes were observed. The sampling was 6 hours and 5 days after administration; higher doses resulted in lethality.


Short description of key information:
Several in vitro and in vivo tests using Na2EDTA are available. Additional information of EDTA free acid and other Na salts of EDTA have been used for the overall risk assessment. Na salts of EDTA were tested negative in several ames tests. Na salts of EDTA were tested negative in several mouse lymphoma assays. Only one mouse lymphoma assay using edetic acid was positive. However, it was not clear whether this effect was due to the test substance or the pH change. Several other in vitro tests have been performed, and in general EDTA was not genotoxic in vitro.
In vivo, somatic cells in mice (bone marrow cells) showed negative results with respect to the endpoints micronuclei, aneuploidy and sister chromatid exchanges. In germ line cells negative results were obtained for induction of structural chromosomal aberrations in spermatogonia, for induction of aneuploidy in primary and secondary spermatocytes, and also for induction of dominant lethals. A positive result was obtained in a micronucleus test with spermatids after ip application, indicating that aneugenic effects may be induced in specific phases of spermatogenesis (late spermacytogenesis). The effect was linked to the use of an extremely high dose in the LD50 range. Since the induction of aneuploidy is based on a threshold mode of action, the potential for induction of aneuploidy will not be expressed at low doses. Furthermore, the effects may be indirect, resulting from the lower intracellular bioavailability of essential elements. On balance, EDTA and its sodium salts may show a low aneuploidogenic potential at extremely high doses. On the basis of the various negative findings and the assumption of a threshold mode of action for aneugens, it can be concluded that EDTA and its sodium salts are not mutagenic for humans.
This result was also confirmed by the independent evaluation of the MAK Commission for the Investigation of Health Hazards of Chemical Compounds in the work area (MAK, 46. Lieferung, 2009).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.