Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
There is no data available for the registered substance on genetic toxicity. However, there is data available for the source substances FeNaEDDHA and manganese salts.
Salmonella and E. coli tests in vitro (Ames Tests):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9), S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537, E. coli WP2 uvr A, OECD Guideline 471, GLP, +/-S9, negative
Manganese
MnCl2, S. typhimurium TA 98, TA 100, TA 1535, TA 1537,E. coli WP2 uvr A OECD Guideline 471, GLP, +/-S9, negative
In-vitro Mammalian Chromosome Aberation Test
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9), Chinese hamster ovary cells (CCL61), OECD Guideline 473, GLP, +/-S9, negative
Manganese
MnCl2, human peripheral lymphocytes, OECD Guideline 473, GLP, +/-S9, negative
In-vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9), L5178Y mouse lymphoma cells, OECD Guideline 476, GLP, +/-S9, negative
Manganese
MnCl2, L5178Y mouse lymphoma cells, OECD Guideline 476, GLP, +/-S9, negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS: precipitation observed, please refer to 'Any other information on results'
RANGE-FINDING/SCREENING STUDIES: The test substance was found to be non-toxic in the range-finding study.
COMPARISON WITH HISTORICAL CONTROL DATA: The historical control values were found to concur with the results from the study for both positive and vehicle controls.
ADDITIONAL INFORMATION ON CYTOTOXICITY: please refer to 'Any other information on results' - Conclusions:
- The test material has been found to be non-mutagenic under the test conditions reported. This provides evidence that no gene mutation can be associated with the manganese moiety of the target substance.
- Executive summary:
MnCl2was tested for gene mutation in a GLP compliant study according to OECD Guideline 471 in the strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A.The test material was found to cause no visible reduction in growth of the bacterial background lawn at any dose and was therefore tested up to the maximum dose level of 5000 µg/plate A particulate precipitate was at 1500 µg/plate and above. This was considered not to prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the test strains, with any dose of the test material with or without metabolic activation. All of the positive control substances induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains. This provides evidence that no gene mutation can be associated with the manganese moiety of the target substance.
- 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:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- lymphocytes: human, peripheral
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material was noted to precipitate at 315 µg/mL in the 4(20)-hour cultures without S9, and above 630 µg/mL in the 4(20)-hour cultures in the presence of S9. No precipitate was observed at the end of the exposure period in the 24-hour cultures.
RANGE-FINDING/SCREENING STUDIES: The preliminary toxicity test dose range was 4.92 to 1260 µg/mL. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test material was observed in the cultures at the end of the exposure, at and above 157.5 µg/mL in the 4(20)-hour exposure in the absence of S9 at and above 78.75 µg/mL in the 4(20)-hour exposure in the presence of S9 and at above 315 µg/mL in the 24 hour continuous exposure group. Haemolysis was observed at and above 315 µg/mL at harvesting in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1260 µg/mL in the 4(20)-hour exposure in the presence of metabolic activation and up to 157.5 µg/mL in the 4(20)-hour exposure in the absence of S9. The maximum dose with metaphases present in the 24-hour continuous exposure was 39.38 µg/mL. The test material induced clear evidence of toxicity in all of the exposure groups. - Conclusions:
- The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups in the absence or presence of a liver enzyme metabolising system or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro. This provides evidence that no cytogenicity can be associated with the manganese moiety of the target substance.
- Executive summary:
MnCl2 was tested for cytogenicity human peripheral lymphocytes in a GLP compliant study according to OECD Guideline 473. All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of test and of the activity of the metabolising system. The test material was found to be toxic to lymphocytes, and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included dose levels that induced approximately 50% mitotic inhibition. The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups in the absence or presence of a liver enzyme metabolising system or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro. This provides evidence that no cytogenicity can be associated with the manganese moiety of the target substance.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Test material-induced toxicity was noted at the highest dose level employed in the test.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of the test material was observed at and above 10 µg/mL in the 4-hour exposure groups in the absence of metabolic activation and at and above 20 µg/mL in the 4-hour exposure group in the presence of metabolic activation.
RANGE-FINDING/SCREENING STUDIES: All three exposure groups employed in the screening test exhibited a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. A precipitate of the test material was observed at and above 78.75 µg/mL in the 4-hour exposure group in the absence of metabolic activation, at and above 39.98 µg/mL in the 4-hour exposure group in the presence of metabolic activation, and at and above 19.69 µg/mL in the 24-hour exposure group in the absence of metabolic activation. In the mutagenicity experiments the maximum dose level was limited by test-material-induced toxicity. - Conclusions:
- The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test. This provides evidence that no gene mutation can be associated with the manganese moiety of the target substance.
- Executive summary:
MnCl2was tested for gene mutation in a GLP compliant study according to OECD Guideline 476. Doses ranging from 2.5 to 25 µg/mL were tested for 4 h in the presence and absence of metabolic activation. Doses from 0.31 to 15 µg/mL were tested for 24 h in the absence of metabolic activation. Test material-induced toxicity was noted at the highest dose levels employed in the test.The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test. This provides evidence that no gene mutation can be associated with the manganese moiety of the target substance.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
Six concentrations ranging from 20.6 - 5000 µg/plate were tested with strain Salmonella typhimurium TA100 and strain Escherichia coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. Although the test substance was applied as a fine homogeneous suspension, no precipitates were detectable on the plates. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and 5000 µg/plate without metabolic activation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria. - Conclusions:
- Based on the results of these experiment and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of S.thyphimurium and E.coli used.
- Executive summary:
FeNaEDDHA was tested for mutagenic effects in vitro in histidine-required strains of Salmonella typhimurium and in a tryptophan-required strain of Escherichia coli. The following strains were used: S. thyphimurium TA 98, TA100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in bidistilled water and testen at 5 concentrations in the range of 312.5 - 5000 µg/plate in the presence and absence of metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 312.5 - 5000 µg/plate. The concentration of 5000 µg/plate represents the test limit dose. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as a positive control.
In both experiments, performed with and without metabolic activation, none of the tested concentrations of the test substance FeNaEDDHA led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.
- 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:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 ug/ml (without metabolic activation) or 125 ug/ml (with metabolic activation) could not be scored. - Conclusions:
- It was concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with FeNaEDDHA.
- Executive summary:
FeNaEDDHA was investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary cells in vitro in a GLP compliant study according to OECD Guideline 473 with and without extrinsic metabolic activation (S9). The test compound FeNaEDDHA was dissolved in DMSO and tested at each of the following conditions:
Experiments without metabolic activation:
- 18 hours treatment time:
original experiment: 7.81, 15.63 and 31.25 µg/mL
confirmatory experiment: 7.81, 15.63 and 31.25 µg/mL
- 42 hours treatment time: 7.81, 15.63 and 31.25 µg/mL
Final concentrations higher than 31.25 ug/mL of culture medium could not be scored due to solubility limitations. Mitomycin C (0.2 ug/mL) was used as a positive control in the 18 hours experiments.
Experiments with metabolic activation:
- 3 hours treatment followed by 15 hours recovery period:
original experiment: 31.25, 62.5 and 125 µg/mL
confirmatory experiment: 31.25, 62.5 and 125 µg/mL
- 3 hours treatment followed by 39 hours recovery, period: 31.25, 62.5 and 125 µg/mL
Final concentrations higher than 125 ug/mL of culture medium could not be scored due to solubility limitations. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/15 hours experiments.
In addition, DNA distribution of cultures treated under the above described conditions (18 hours only) was determined by flow cytometry. These measurements allow to analyse the influence of the test substance on the cell cycle of CHO cells. In both the experiments performed without and with metabolic activation no significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed. Flow cytometry experiments did not reveal any evidence for cell cycle disturbing activities of FeNaEDDHA either in the absence or in the presence of metabolic activation.
It was concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with FeNaEDDHA.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- please refer to field 'Any other information on results'
- Conclusions:
- Based on the result of two indipendently performed experiments and under the given experimental conditions, it is concluded that FeNaEDDHA and its metabolites did not show any mutagenic activity at the tk locus of mouse lymphoma L5178Y cells in the presence and absence of metabolic activation. The same result is expected for the target substance since it has the same constituents that act via the same mechanism as the source substance. No differences in the magnitude of the effects are expected.
- Executive summary:
FeNaEDDHA was tested for its ability to induce mutations at the tk locus (5-trifluoro-thymidine resistance) in L5178Y mouse lymphoma cells. The study consisted of a preliminary cytotoxicity range-finder and two independent experiments, each performed with and without metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial supernatant (S9 fraction).
In a first range-finder experiment the concentrations applied ranged from 0.49 to 1000.0 µg/mL separated by 2-fold intervals. Based on the results of the range finding study, 5 concentrations were chosen for the first mutagenicity experiment, separated by 4-fold intervals and ranging from 0.49 to 125.0 µg/mL in the part with and from 3.91 to 1000.0 µg/mL in the part without metabolic activation. At the highest concentrations the zero hour survival was 49.33% and 62.01% in the presence and absence of metabolic activation. In the confirmatory experiment, in the part with metabolic activation, the concentration range was increased ranging from 0.98 to 250.0 µg/mL. Without metabolic activation the same concentration range was used. With metabolic activation the relative survival at the highest concentration was 47.45%, without metabolic activation the value found was 81.90%. Negative (vehicle) and positive control treatments were included in each experiment with and without metabolic activation. The mutant frequencies of the negative controls were within normal ranges and the positive controls N-Nitrosodimethylamine (DMN, with metabolic activation) and Ethylmethansulfonate (EMS, without metabolic activation) produced statistically significant increases of mutant frequency. In the part performed with and without metabolic activation, no relevant increases in mutant frequency were observed after treatment with FeNaEDDHA.
Referenceopen allclose all
Table 1: Range finding study – toxicity assay
With (+) or Without (-) S9-mix |
Strain |
Dose (µg/plate) |
||||||||||
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
||
- |
TA100 |
75 |
81 |
78 |
96 |
89 |
77 |
85 |
74 |
70 |
86P |
84P |
+ |
TA100 |
77 |
96 |
84 |
85 |
74 |
75 |
91 |
88 |
74 |
70P |
73P |
- |
WP2uvrA- |
25 |
23 |
25 |
23 |
27 |
30 |
23 |
35 |
31 |
22P |
27P |
+ |
WP2uvrA- |
37 |
24 |
27 |
34 |
29 |
25 |
36 |
32 |
38 |
30P |
24P |
P - Precipitate |
||||||||||||
Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls), Experiment 1
Number of Revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
93 |
15 |
22 |
14 |
10 |
96 (95) |
19 (18) |
20 (22) |
16 (17) |
13 (12) |
97 |
21 |
24 |
20 |
13 |
Table 3: Spontaneous Mutation Rates (Concurrent Negative Controls), Experiment 2
Number of Revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
123 |
22 |
33 |
22 |
12 |
123 (118) |
25 (23) |
25 (29) |
25 (24) |
12 (12) |
109 |
21 |
29 |
24 |
13 |
Table 4: Test Results, Experiment 1 – Without Metabolic Activation
Test Period |
From 19 July 2009 |
To 22 July 2009 |
|||
Test Substance (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|
0 |
95 111 (102) 99 8.3# |
16 20 (18) 19 2.1 |
23 25 (23) 20 2.5 |
26 21 (23) 23 2.5 |
15 13 (14) 13 1.2 |
50 |
112 96 (104) 104 8.0 |
16 20 (19) 20 2.3 |
18 25 (22) 23 3.6 |
21 22 (22) 24 1.5 |
14 13 (12) 10 2.1 |
150 |
115 110 (109) 102 6.6 |
21 13 (18) 21 4.6 |
26 15 (20) 19 5.6 |
16 20 (21) 27 5.6 |
12 11 (12) 13 1.0 |
500 |
103 92 (95) 90 7.0 |
18 19 (17) 14 2.6 |
21 23 (25) 30 4.7 |
21 21 (21) 20 0.6 |
15 15 (12) 15 0.0 |
1500 |
110 P 95 P (103) 104P 7.5 |
19P 18P (18) 16P 1.5 |
22 24P (23) 22P 1.2 |
22P 23P (25) 26P 3.2 |
13P 15P (12) 9P 3.1 |
5000 |
88P 117P (105) 111P 15.3 |
18P 21P (19) 18P 1.7 |
26P 21P (24) 24P 2.5 |
25P 23P (25) 26P 1.5 |
9P 12P (12) 16P 3.5 |
Name Concentration No. colonies per plate |
ENNG |
ENG |
ENNG |
4NQO |
9AA |
3 |
5 |
2 |
0.2 |
80 |
|
475 526 (492) 476 29.2 |
99 150 (115) 95 30.7 |
145 145 (145) 145 0.0 |
123 118 (119) 115 4.0 |
345 462 (400) 394 58.8 |
|
ENNG – N-ethyl-N’-nitro-N-nitrosoguanidine 4NQO – 4-Nitroquinoline-1-oxide 9AA – 9-Aminoacridine P – Precipitate # - Standard deviation |
|||||
Table 5: Test Results, Experiment 1 – With Metabolic Activation
Test Period |
From 19 July 2009 |
To 22 July 2009 |
|||
Test Substance (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|
0 |
97 96 (98) 100 8.3# |
12 12 (11) 9 1.7 |
23 29 (28) 32 4.6 |
25 24 (23) 20 2.6 |
14 9 (12) 14 2.9 |
50 |
117 101 (106) 100 9.5 |
11 9 (10) 10 1.0 |
31 25 (25) 19 6.0 |
20 21 (20) 18 1.5 |
12 12 (12) 12 0.0 |
150 |
97 102 (101) 103 3.2 |
10 9 (9) 9 0.6 |
25 23 (25) 27 2.0 |
26 22 (24) 24 2.0 |
15 10 (12) 11 12.6 |
500 |
115 74 (94) 92 20.6 |
13 8 (10) 9 2.6 |
29 19 (23) 21 5.3 |
31 20 (24) 20 6.4 |
16 15 (15) 13 1.5 |
1500 |
90 P 82 P (88) 91P 4.9 |
12P 9P (11) 13P 2.1 |
26P 24P (23) 20P 3.1 |
19P 18P (24) 20P 6.4 |
11P 15P (12) 9P 3.1 |
5000 |
92P 104P (95) 90P 7.6 |
12P 9P (11) 11P 1.5 |
24P 24P (23) 20P 2.3 |
20P 24P (22) 22P 2.0 |
12P 9P (11) 13P 2.1 |
Name Concentration No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
1 |
2 |
10 |
5 |
2 |
|
2248 2526 (2506) 2743 248.1 |
209 151 (179) 177 29.1 |
172 225 (184) 154 36.9 |
206 184 (197) 202 11.7 |
278 217 (247) 247 30.5 |
|
2AA – 2-Aminoanthracene BP – Benzo(a)pyrene P – Precipitate # - Standard deviation |
|||||
Table 6: Test Results, Experiment 2 – Without Metabolic Activation
Test Period |
From 19 July 2009 |
To 22 July 2009 |
|||
Test Substance (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|
0 |
101 95 (104) 115 10.3# |
20 16 (19) 20 2.3 |
21 24 (23) 25 2.1 |
26 26 (25) 23 1.7 |
11 16 (14) 15 2.6 |
50 |
106 97 (104) 106 5.2 |
21 21 (21) 21 0.0 |
18 26 (24) 29 5.7 |
20 26 (25) 2 9 4.6 |
8 15 (12) 13 3.6 |
150 |
113 107 (111) 114 3.8 |
24 20 (21) 20 2.3 |
22 29 (25) 23 3.8 |
20 19 (22) 27 4.4 |
10 8 (9) 10 1.2 |
500 |
96 104 (102) 106 5.3 |
16 22 (17) 14 4.2 |
23 24 (24) 25 1.0 |
23 25 (26) 30 3.6 |
15 8 (9) 10 1.2 |
1500 |
118P (118) 119P 1.0 117 * |
24P 20P (23) 26P 3.1 |
21P 24P (22) 22P 1.5 |
29P 21P (25) 26P 4.0 |
10P 11P (10) 9P 1.0 |
5000 |
101P 104P (105) 110P 4.6 |
22P 23P (22) 21P 1.0 |
27P 24P (24) 21P 3.0 |
26P 25P (25) 24P 1.0 |
12P 13P (11) 9P 2.1 |
Name Concentration No. colonies per plate |
ENNG |
ENG |
ENNG |
4NQO |
9AA |
3 |
5 |
2 |
0.2 |
80 |
|
295 312 (309) 320 12.8 |
209 222 (212) 206 8.5 |
416 492 (462) 477 40.3 |
246 217 (228) 220 15.9 |
1078 2042 (1426) 1157 535.2 |
|
ENNG – N-ethyl-N’-nitro-N-nitrosoguanidine 4NQO – 4-Nitroquinoline-1-oxide 9AA – 9-Aminoacridine P – Precipitate # - Standard deviation * p≤0.05 |
|||||
Table 7: Test Results, Experiment 2 – With Metabolic Activation
Test Period |
From 19 July 2009 |
To 22 July 2009 |
|||
Test Substance (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|
0 |
108 106 (108) 109 1.5# |
10 9 (11) 13 2.1 |
25 27 (26) 26 1.0 |
22 21 (22) 22 0.6 |
16 16 (15) 12 2.3 |
50 |
93 91 (106) 107 8.7 |
15 9 (12) 13 3.1 |
22 22 (25) 31 5.2 |
21 26 (22) 22 0.6 |
13 15 (15) 16 1.5 |
150 |
98 89 (99) 110 10.5 |
13 8 (10) 13 3.1 |
22 23 (22) 31 0.6 |
19 24 (22) 22 2.5 |
14 14 (12) 7 4.0 |
500 |
91 106 (102) 110 10.0 |
12 14 (12) 9 2.5 |
30 29 (30) 30 0.6 |
26 21 (23) 22 2.6 |
16 14 (14) 12 2.0 |
1500 |
81P 100P (96) 110P 10.0 |
9P 13P (10) 9P 2.3 |
25P 31P (27) 25P 3.5 |
25P 25P (24) 22P 1.7 |
9P 16P (14) 16P 4.0 |
5000 |
90P 84P (92) 102P 9.2 |
11P 10P (10) 8P 1.5 |
26P 23P (25) 27P 2.1 |
24P 21P (22) 22P 1.5 |
12P 15P (12) 10P 2.5 |
Name Concentration No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
1 |
2 |
10 |
5 |
2 |
|
2474 2427 (2510) 2629 105.7 |
374 332 (332) 291 41.5 |
342 264 (313) 333 42.7 |
261 620 (457) 490 181.8 |
497 494 (498) 504 5.1 |
|
2AA – 2-Aminoanthracene BP – Benzo(a)pyrene P – Precipitate # - Standard deviation |
|||||
The test material was found to cause no visible reduction in growth of the bacterial background lawn at any dose and was therefore tested up to the maximum dose level of 5000 µg/plate A particulate precipitate was at 1500 µg/plate and above. This was considered not to prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for and of the bacterial strains, with any dose of the test material, with or without metabolic activation. In the TA100 revertant colony, a small but statistically significant increase was observed on the 1500 µg/plate in Experiment 2 (increase of less than 1.5 times). However these were within the range specified by the Standard Test Method, this increase proved non-reproducible over two separate experiments. This was concluded to have no biological or toxicological relevance. All of the positive control substances induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of test and of the activity of the metabolising system. The test material was found to be toxic to lymphocytes, and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included dose levels that induced approximately 50% mitotic inhibition.
Please refer to attached document, Appendix 1 for full tabulated results
Table 2: Results from the preliminary toxicity test
Dose (µg/mL) |
%RSG (-S9) 4-Hour Exposure |
%RSG (+S9) 4-Hour Exposure |
%RSG (-S9) 24-Hour Exposure |
0 |
100 |
100 |
100 |
4.92 |
102 |
89 |
33 |
9.84 |
96 |
100 |
11 |
19.69 |
89 |
89 |
1 |
39.38 |
72 |
75 |
0 |
78.75 |
2 |
57 |
0 |
157.5 |
9 |
1 |
0 |
315 |
1 |
0 |
0 |
630 |
0 |
0 |
0 |
1260 |
0 |
0 |
0 |
Table 3: Summary of results for main experiment, 4 hour exposure
Treatment (µg/mL) |
4-Hours –S9 |
Treatment (µg/mL) |
4-Hours +S9 |
||||
%RSG |
RTG |
MF§ |
%RSG |
RTG |
MF§ |
||
0 |
100 |
1.00 |
81.37 |
0 |
100 |
1.00 |
74.20 |
2.5† |
97 |
|
|
20 |
91 |
1.04 |
64.08 |
5 |
95 |
1.06 |
73.26 |
40 |
68 |
0.86 |
78.58 |
10 |
91 |
0.94 |
96.72 |
60 |
43 |
0.41 |
116.75 |
20 |
101 |
1.24 |
90.28 |
80 |
37 |
0.32 |
96.12 |
40 |
44 |
0.46 |
104.53 |
100 |
32 |
0.22 |
104.65 |
60 |
19 |
0.08 |
128.81 |
120 |
26 |
0.15 |
104.12 |
80 |
17 |
0.13 |
91.23 |
140 |
25 |
0.18 |
100.39 |
120† |
14 |
|
|
160‡ |
13 |
0.04 |
42.87 |
Linear trend |
NS |
Linear trend |
|||||
EMS |
|
|
|
CP |
|
|
|
400 |
71 |
0.62 |
656.51 |
2 |
55 |
0.22 |
1662.80 |
† Not plated for viability or 4-TFT resistance MF§ 5-TFT resistant mutants/106viable cells 2 days after treatment NS Not significant ‡ Treatment excluded from test statistics due to toxicity |
|||||||
Table 4: Summary of results for main experiment, 24 hour exposure
Treatment (µg/mL) |
4-Hours –S9 |
||
%RSG |
RTG |
MF§ |
|
0 |
100 |
1.00 |
103.16 |
0.31 |
97 |
0.99 |
91.33 |
0.63 |
105 |
0.97 |
79.60 |
1.25 |
95 |
1.07 |
50.22 |
2.5 |
76 |
0.81 |
100.92 |
5 |
41 |
0.38 |
173.75* |
7.5 |
14 |
0.11 |
372.63* |
10† |
7 |
|
|
15† |
4 |
|
|
Linear trend |
*** |
||
EMS |
|
|
|
150 |
54 |
0.32 |
1211.25 |
† Not plated for viability or 4-TFT resistance * p<0.05 *** p<0.001 |
|||
Analytical control:
To confirm that the cells were actually exposed to the intened test concentrations and to confirm the stability of the test substance in the vehicle used, determination of the concentration of the test substance in solution was performed by UV/VIS-spectroscopy. The values found by analysis of the different samples were in agreement with the intended concentrations (between 90.2 and 96.1 %), thus demonstrating a sufficient stability of the test substance in the vehicle.
There were no known or occurrences in this study that were considered to have affected the quiality or integrity of the test data.
Table 1: Original experiment
With(+) or without(-) S9 Mix |
Test substance concentration (µg/plate) |
Number of revertants (number of colonies/plate) |
|||||
Base-pair substitution type |
Frameshift type |
||||||
TA 100 |
TA 1535 |
UP2 uvrA |
TA 102 |
TA 98 |
TA 1537 |
||
S9 Mix (+) |
Solvent control |
173 183 185 (180) |
33 16 21 (23) |
36 33 36 (35) |
357 360 282 (333) |
53 40 51 (48) |
8 16 7 (10) |
312.50 |
185 197 187 (190) |
19 18 25 (21) |
40 41 43 (41) |
330 331 321 (327) |
48 41 52 (47) |
7 8 8 (8) |
|
625.00 |
181 195 157 (178) |
21 20 19 (20) |
36 40 49 (42) |
344 357 337 (346) |
45 68 60 (58) |
8 16 13 (12) |
|
1250.00 |
192 196 180 (189) |
28 25 15 (23) |
25 28 30 (28) |
342 328 331 (334) |
51 52 50 (51) |
13 7 12 (11) |
|
2500.00 |
171 182 189 (181) |
25 19 18 (21) |
44 43 32 (40) |
379 365 360 (368) |
57 48 51 (52) |
9 14 6 (10) |
|
5000.00 |
177 180 173 (177) |
27 13 17 (19) |
39 48 40 (42) |
336 376 354 (355) |
52 44 52 (49) |
16 7 6 (10) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
S9 Mix (-) |
Solvent control |
161 127 168 (152) |
24 17 24 (22) |
41 41 24 (35) |
222 255 206 (228) |
44 27 36 (36) |
18 13 8 (13) |
312.50 |
134 149 157 (147) |
25 19 27 (24) |
27 32 41 (33) |
128 194 218 (180) |
29 26 27 (27) |
8 19 16 (14) |
|
625.00 |
151 161 174 (162) |
27 25 24 (25) |
29 31 49 (36) |
293 351 314 (319) |
36 44 41 (40) |
12 15 17 (15) |
|
1250.00 |
137 140 162 (146) |
19 24 18 (20) |
26 25 37 (29) |
276 308 368 (317) |
44 33 38 (38) |
14 12 16 (14) |
|
2500.00 |
157 151 168 (159) |
17 19 20 (19) |
33 40 28 (34) |
234 256 236 (242) |
30 41 30 (34) |
17 15 13 (15) |
|
5000.00 |
149 159 164 (157) |
21 24 15 (20) |
30 30 36 (32) |
72 141 240 (151) |
32 21 38 (30) |
16 16 14 (15) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Positive control requiring S9 Mix |
Name |
2-Amino-anthracene |
Cyclophosphamide |
2-Amino-anthracene |
2-Amino-anthracene |
2-Amino-anthracene |
2-Amino-anthracene |
Concentration (µg/plate) |
2.50 |
400.00 |
50.00 |
20.00 |
2.50 |
2.50 |
|
Number of colonies/plate |
1284 916 1466 (1222) |
243 344 410 (332) |
737 847 894 (826) |
789 1028 1500 (1106) |
595 925 740 (753) |
139 167 147 (151) |
|
Positive control not requiring S9 Mix |
Name |
Sodium azide |
Sodium azide |
4-N00 |
Hitomycin-C |
2-Nitro-fluorene |
9-Amino-acridine |
Concentration (µg/plate) |
5.00 |
5.00 |
2.00 |
2.00 |
20.00 |
150.00 |
|
Number of colonies/plate |
834 831 676 (780) |
953 928 900 (927) |
552 620 488 (553) |
1497 1364 571 (1144) |
1805 1808 1686 (1766) |
1704 1357 1707 (1589) |
|
Notes: 1. When inhibition is found against growth of the bacteria, mark the applicable value with an asterix 2. Fill the average number of colonies in each concentration in the () 3. "Number of revertants"-Fi11 in the observed value and average value in order beginning with low concentration of the test substance |
|||||||
Toxicity test / Selection of concentrations
The selection of the highest scorable concentrations could not primarily be based on cytotoxicity data. Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 µg/mL (without metabolic activation) or 125 µg/mL (with metabolic activation) could not be scored. An inhibition in mitotic activity by 62.3% could be observed in the original experiment performed with metabolic activation (3h/15h) at the highest scorable concentration of 125 µg/mL. In the respective confirmatory experiment cytotoxicity was noted at the non-scorable concentration of 250 µg/mL only. In the absence of metabolic activation toxicity appeared only after 42 hours treatment at the highest concentration of 1000 µg/mL (non-scorable).
Original mutagenicity study
In the experiment performed without metabolic activation (experiment 1; 18 hours treatment), 2.0% of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentrations of 7.81 µg/mL, 15.63 µg/mL and 31.25 µg/mL 1.0%, 2.0% and 1.5% of cells with specific chromosomal aberrations were found.
In the experiment performed with metabolic activation (experiment 2; 3 hours treatment/15 hours recovery), 1.0% of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 31.25 µg/mL, 62.5 µg/mL and 125 µg/mL the respective values were 2.5%, 2.0% and 5.0%. The value obtained with the highest concentration of the second experiment showed a statistically significant difference when compared with the respective negative control. The value however is below the critical limit required for a positive response and it is within the historical range for negative controls. Furthermore no increase at all was observed in the respective confirmatory experiment. The slight increase in the frequency of aberrant metaphases is therefore considered to be spontaneous in origin.
Flow cytometry
The influence of the test substance on the cell cycle of CHO cells was tested at the concentrations selected for chromosome analysis. The DNA distribution was determined by flow cytometry and compared with the profile of the respective control culture. In the absence and in the presence of metabolic activation a shift in the DNA distribution profile could not be detected. Therefore, no evidence of a cell cycle disturbance by the test substance was obtained in the CHO cells.
Analytical results
The test material in suspension was analysed by UV/VIS-spectroscopy to confirm the intended concentrations to be used in the mutagenicity tests and the stability of the test substance in the vehicle used. The concentration values found were 120.6 and 124.1% of the calculated concentrations, thus indicating a sufficient stability of the test substance in the vehicle.
TABLE 1: MITOTIC INDEX VALUES / CYTOTOXICITY |
|||||
| Original study, experiment 1 18h treatment without metabolic activation | |||||
Cells scored |
Mitosis | M.I. % | Frequency % of control | ||
| Solvent control | 2000 | 263 | 13.15 | 100 |
|
| CGA65047SG100 (A-5787A) | |||||
| 1000 µg/mL | 2000 | 250 | 12.50 | 95.06 | |
| 500 µg/mL | 2000 | 234 | 11.70 | 88.97 | |
| 250 µg/mL | 2000 | 268 | 13.40 | 101.90 | |
| 125 µg/mL | 2000 | 273 | 13.65 | 103.80 | |
| 62.5 µg/mL | 2000 | 282 | 14.10 | 107.22 | |
| 31.25 µg/mL | 2000 | 290 | 14.50 | 110.27 | |
| 15.63 µg/mL | 2000 | 300 | 15.00 | 114.07 | |
| 7.81 µg/mL | 2000 | 310 | 15.50 | 117.87 | |
| Original study, experiment 2 3h treatment with metabolic activation/ 15h recovery | |||||
Cells scored |
Mitosis | M.I. % | Frequency % of control | ||
| Solvent control | 2000 | 220 | 11.00 | 100.00 | |
| CGA65047SG100 (A-5787A) | |||||
| 1000 µg/mL | 2000 | 114 | 5.70 | 51.82 | |
| 500 µg/mL | 2000 | 70 | 3.50 | 31.82 | |
| 250 µg/mL | 2000 | 62 | 3.10 | 28.18 | |
| 125 µg/mL | 2000 | 83 | 4.15 | 37.73 | |
| 62.5 µg/mL | 2000 | 218 | 10.90 | 99.09 | |
| 31.25 µg/mL | 2000 | 224 | 11.20 | 101.82 | |
| 15.63 µg/mL | 2000 | 238 | 11.90 | 108.18 | |
| 7.81 µg/mL | a) | ||||
a) When three subsequent concentrations with a frequency of 70% mitosis or more in relation to the solvent control are found, the evaluation of the lower concentrations is omitted.
| TABLE 2 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 1 | ||||||||||
| 18 h treatment without metabolic activation | ||||||||||
| Treatment | total no of cells examined | % cells with specific aberrations # | total number of cells with aberrations | |||||||
| gaps | ct del | ct exc | cs del | cs exc | mab | pol | end | |||
| Solvent control | 200 | 2.0 | 1 | 2 | 1 | 1 | 7 | |||
| CGA 65047 SG 100 (CA-5787 A) | ||||||||||
| 7.81 µg/mL | 200 | 1.0 | 3 | 1 | 1 | 3 | ||||
| 15.63 µg/mL | 200 | 2.0 | 2 | 1 | 2 | 1 | 3 | |||
| 31.25 µg/mL | 200 | 1.5 | 3 | 2 | 1 | 6 | ||||
| positive control (Mito-C, 0.2 µg/mL | 50 ) | 24.0*** | 5 | 6 | 6 | 2 | ||||
| TABLE 3 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 2 | ||||||||||
| 3 h treatment with metabolic activation / 15 h recovery | ||||||||||
| Treatment | total no of cells examined | % cells with specific aberrations # | total number of cells with aberrations | |||||||
| gaps | ct del | ct exc | cs del | cs exc | mab | pol | end | |||
| Solvent control | 200 | 1.0 | 6 | 2 | 8 | |||||
| CGA 65047 SG 100 (CA-5787 A} | ||||||||||
| 31.25 µg/mL | 200 | 2.5 | 6 | 2 | 2 | 1 | 3 | 1 | ||
| 62.5 µg/mL | 200 | 2.0 | 13 | 1 | 3 | 9 | ||||
| 125 µg/mL | 200 | 5.0* | 10 | 6 | 3 | 1 | 5 | 1 | ||
| positive control (CPA, 20 µg/mL) | 100 | 17.0*** | 2 | 6 | 9 | 3 | 3 | |||
Legend to Tables2 -3 |
|
| ctdel | Chromatid deletions (including deletions, breaks, fragments) |
| ct exc | Chromatid exchanges (including triradials, quadriradials, endfusions and acentric rings) |
| csdel | Chromosome deletions (including deletions, breaks, fragments) |
| cs exc | Chromosome exchanges (including dicentrics, polycentrics, centric and acentric rings) |
| mab | Multiple aberrations: metaphases containing more than10 aberrations of different types or more than 5 aberrations of one particular type (excluding gaps) |
| gaps | Chromatid and chromosome type gaps |
| pol | Polyploid metaphases (>30 centromers) |
| end | Endoreduplications |
| CPA | Cyclophosphamide |
| Mito-C | Mitomycin-C |
| *) | Statistical significance:0.05 =P> 0.01 |
| Statistical significance:0.01 =P> 0.001 | |
| Statistical significance: P< 0.001 | |
| #) | %cells with aberrations excluding gaps and numerical alterations(pol,end) |
Toxicity
In the cytoxicity range-finder experiment, 12 concentrations of the test substance were tested. In the part with metabolic activation, the concentrations ranged from 0.49 to 1000.0 µg/mL separated by 2 fold intervals. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. In the part with metabolic activation, down to the concentration of 31.25 µg/mL, a growth inhibiting effect greater than 50.0% in comparison to the negative control could be seen. No relevant cytotoxicity could be detected after treatment with the test chemical in the part without metabolic activation.
Accordingly, five concentrations were selected for the original mutagenicity experiment. In the part with metabolic activation the following concentrations were selected: 0.49, 1.95, 7.81, 31.25 and 125.0 µg/mL. At the top concentration of 125.0 µg/mL the mean zero hour survival value was 49.33%. The relative total growth at the end ofthe expression period revealed a mean value of 31.40%. In the part without metabolic activation the concentrations appplied were: 3.91, 15.63, 62.50, 250.0 and 1000.0 µg/mL. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. The highest concentration showed a mean relative survival of 62.01%. The mean relative total growth value was 80.31%. All concentrations were selected to determine viability and TFT-resistance two days after treatment.
In the confirmatory mutagenicity experiment, in the part with metabolic activation, the concentration range was increased. The concentrations applied were: 0.98, 3.91, 15.63, 62.50 and 250.0 µg/mL. In the part without metabolic activation the same concentration range as in the original experiment was selected. The mean relative survival value obtained in the part with metabolic activation was 47.45% at the highest concentration. The mean relative total growth value obtained after the two days expression period was 42.05%. The zero hour survival value in the part without metabolic activation was 81.90%. The relative total growth determined after the expression revealed a value of 75.74%. All concentrations were selected to determine viability and TFT-resistance 2 days after treatment.
Mutagenicity
In the presence and absence of metabolic activation, in the original and confirmatory experiments, reproducible, statistically significant and dose-related relevant increases in mutant frequencies were not observed. In the original experiment, in the part with metabolic activation, the highest concentration of 125.0 µg/mL was excluded from statistical analysis due excessive heterogeneity in the duplicate survival plates. In the part without metabolic activation the concentration of 15.36 µg/mL was excluded from statistical anlysis due to heterogeneity in the survival plates. Nevertheless, the mutant frequency values found at these concentrations are lying within the normal range and do not influence the validity of the study in any respect.
Large and small colonies
For the negative and positive controls the number of wells containing small colonies and the number of wells containing large colonies were scored. With metabolic activation the mean proportion of small colonies in the negative controls ranged from 40.0 to 44.4%, whereas with the positive controls (DMN) an increased proportion of 55.4 to 59.5% was observed. Without metabolic activation the proportion of small colonies in the negative controls ranged from 67.3 to78.0%, whereas with the positive controls (EMS) a proportion of 42.0 to 53.3% was observed.
Small and large colony numbers are not reported for CGA 65 047 SG 100, (A-5787 A) as the data did not fulfil the criteria for a positive response.
Table 1: Summary of the mutagenicity test (Experiment with metabolic activation)
| Treatment | Zero hour survival % | Relative total growth (RTG) % |
| Negative control DMN 2 µL/mL | 100.00 78.35 |
100.00 48.88 |
| CGA 65 047 SG 100 (A-5787 A): | ||
| 125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL |
49.33 103.76 93.79 85.79 75.22 |
31.40 86.83 64.80 63.13 60.61 |
| Treatment | Mutant frequency X 10E-6 | Significance (P) |
| Negative control DMN 2 µL/mL | 24.58 387.77 |
# |
| CGA 65 047 SG 100. (A-5787 A): | ||
| 125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL |
54.08 27.73 25.17 36.01 27.83 |
§ NS NS NS NS |
| Test for linear trend: NS | ||
Table 2: Summary of the mutagenicity test (Experiment without metabolic activation)
| Treatment | Zero hour survival % | Relative total growth (RTG) % |
| Negative control EMS 1 µL/mL | 100.00 1.82 |
100.00 2.54 |
| CGA 65 047 SG 100 (A-5787 A): | ||
| 1000.00 µg/mL 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL 3.91 µg/mL |
62.01 74.51 100.13 134.44 144.04 |
80.31 109.86 96.75 92.87 115.83 |
| Treatment | Mutant frequency x 10E-6 | Significance (P) |
| Negative control EMS 1 µL/mL | 33.76 841.91 |
# |
| CGA 65 047 SG 100 (A-5787 A): | ||
| 1000.00 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL 3.91 µg/mL |
38.61 22.40 35.37 40.38 39.89 |
NS NS NS § NS |
| Test for linear trend | NS |
§ excluded from experiment;
NS not significant.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Salmonella and E. coli tests in vitro (Ames Tests):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to induce mutagenic effect in histidine-requiring strains of Salmonella typhimurium (TA 98, TA 100, TA 102, TA 1535, TA 1537) and in a tryptophan-requiring strain of Escherichia coli (WP2 uvr A) (OECD Guideline 471) (CIBA-GEIGY, 1994b; Report No. 931146a). The test compound was dissolved in bidistilled water and tested at five concentrations ranging from 312.5 to 5000 µg/plate with and without metabolic activation. Suitable positive controls were used for each strain. All experiments were repeated in order to confirm the results. The results revealed no increased incidence of mutants by the test item with and without metabolic activation. Therefore, it was concluded that the test compound did not show mutagenic activity in S. typhimurium and E. coli. The positive controls induced mutagenic activity. In conclusion, Fe(Na)EDDHA provoked no mutagenic activity in this test system.
Manganese
MnCl2 was tested for gene mutation in a GLP compliant study according to OECD Guideline 471 in the strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A (Thompson & Bowles 2009). The test material was found to cause no visible reduction in growth of the bacterial background lawn at any dose and was therefore tested up to the maximum dose level of 5000 µg/plate A particulate precipitate was at 1500 µg/plate and above. This was considered not to prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the test strains, with any dose of the test material with or without metabolic activation. All of the positive control substances induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
In-vitro Mammalian Chromosome Aberation Test
EDDHA
The test compound Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke clastogenic effects in Chinese hamster ovary cells (CCL61) in vitro (OECD TG 473) (CIBA-GEIGY, 1994c; Report No. 931147). The compound was dissolved in DMSO and tested without metabolic activation at concentrations of 0, 7.81,15.63 and 31.25 µg/mL for 18 and 42 hours. With metabolic activation (liver S9 fraction from Aroclor 1254 induced rat liver) concentrations of 0, 31.25, 62.5 and 125 µg/mL were applied for 3 hours followed by 15 hours recovery or 3 hours followed by 39 hours recovery. Higher concentrations could not be reached due to solubility limitations. Three independent experiments of each with and without metabolic activation were performed. Two replicate culture per concentration and 200 cells per concentration were evaluated.
The results showed in both experiments with and without metabolic activation no increased number of metaphases with chromosomal aberrations. In contrast, the positive controls (Mitomycin 0.2 µg/mL and Cyclophosphamide 20 µg/mL) induced clastogenic effects. In conclusion, the test substance provoked no clastogenic activity in this test in vitro.
Manganese
MnCl2 was tested for cytogenicity in a GLP compliant study according to OECD Guideline 473 (Morris & Durward 2009). All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of test and of the activity of the metabolising system. The test material was found to be toxic to lymphocytes, and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included dose levels that induced approximately 50% mitotic inhibition. The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups in the absence or presence of a liver enzyme metabolising system or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
In-vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke mutations at the tk locus in L5178Y mouse lymphoma cells in vitro (OECD Guideline 476) (CIBA-GEIGY, 1994a; Report No. 931146b). The test compound was dissolved in DMSO. The range finding experiments showed that 1000 µg/mL was the highest concentration which could be used. Higher concentrations (greater than 100 mg/mL) produced precipitates in the vehicle. In the presence of metabolic activation (liver S9 -fraction from Aroclor 1254 treated rats) the two highest concentrations revealed cytotoxicity. In absence of metabolic activation no toxicity was noted.
For the mutagenicity experiment, concentrations ranging from 0 to 125 µg/mL with metabolic activation and from 0 to 1000 µg/mL without metabolic activation were used. In the confirmatory experiment with metabolic activation concentrations ranging from 0 to 250 µg/mL were applied. The same concentrations (0 to 1000 µg/mL) were used in the confirmatory experiment without metabolic activation. Corresponding positive controls (N-Nitrosodimethylamine, with metabolic activation and Ethylmethansulfonate without metabolic activation) were included. The mouse lymphoma cells were treated for 4 hours. After two days expression time, mutations at the tk locus were selected by resistance to 5-trifluorothymidine. Two types of colonies were selected, large colonies (base-pair substitutions and deletions) and small colonies (chromosome aberrations). The results showed no increased incidence of mutations at the tk locus of mouse lymphoma L5178Y cells in presence or absence of metabolic activation. Positive controls showed mutagenic activity. In conclusion, Fe(Na)EDDHA was not mutagenic in this test system in vitro.
Manganese
MnCl2 was tested for gene mutation in a GLP compliant study according to OECD Guideline 476 (Flanders 2009). Doses ranging from 2.5 to 25 µg/mL were tested for 4 h in the presence and absence of metabolic activation. Doses from 0.31 to 15 µg/mL were tested for 24 h in the absence of metabolic activation. Test material-induced toxicity was noted at the highest dose levels employed in the test.The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Justification for classification or non-classification
Based on the available data the registered substance is not subject to classification and labelling according to the Regulation (EC) No 1272/2008.
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