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Genetic toxicity in vitro

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Studies generally conducted to recognised training guidelines with GLP

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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
Study period:
2006-02-07, 2006-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 complete culture medium supplemented with 15 % horse serum (HS), 100 U/100 pg/mL Penicillin/Streptomycin, 220 pg/mL Sodium-Pyruvate, and 1.25 U/mL Amphotericin used as antifungal.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from Wistar rats (HanIbm) induced with phenobarbital and beta-naphthoflavone, mixed with a solution of co-factors.
Test concentrations with justification for top dose:
The cultures were evaluated at the following concentrations of the test item:
experiment I:
without S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL
with S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL

experiment II:
without S9 Mix: 1.3*; 2.5*; 5.0; 10.0; 20.0; 30.0; and 40.0 µg/mL
with S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL

experiment III:
without S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL

Following the expression phase of 72 hours the cultures marked with an asterisk were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: The test substance is insoluble in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation Migrated to IUCLID6: 19.5 µg/mL (4 h); 13.0 µg/mL (24 h)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation Migrated to IUCLID6: 4.5µg/mL
Details on test system and experimental conditions:
PRE-TEST:
A pre-test was performed in order to determine the concentration range of the main experiments.
1xE+7 cells were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed and finally resuspended in 30 mL complete culture medium for a 2-day growth period. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector (Mutation Research 31, 17-29, 1975).
Test item concentrations between 5.1 and 650 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Relevant toxic effects were observed at 325 µg/mL and above in the absence and at 81.3 µg/mL and above in the presence of metabolic activation (4 h treatment). Following the 24 h treatment without metabolic activation toxic effects occurred at 40.6 µg/mL and above.

MAIN TEST:
Experiment I
The treatment duration in the first experiment was 4 hours with and without metabolic activation.
Experiment II
The cells were treated with test item for 4 h with and for 24 h without metabolic activation. The experimental part without metabolic activation was terminated prior to the generation of any data on mutagenicity due to exceedingly severe toxic effects even at lower concentrations. Therefore, this experimental part was repeated as experiment IIA in a lower concentration range. The data of experiment IIA are reported as experiment II without metabolic activation.
Experiment III
This experiment was performed to verify a minor increase of the mutation frequency without metabolic activation in experiment I. Experiment III was performed in the absence of metabolic activation with a treatment time of 4 hours.

All experiments were performed in duplicate. Expression time of cells in growth medium was 48 - 72 h and selection time ranged from 10 to 15 days. The selection agent was Trifluorothymidine (TFT; 5 µg/mL). The number of replications was 2 and 2.0 cells/well in a 96-well microtiter plate were evaluated.

DETERMINATION OF CYTOTOXICITY:
Cytotoxicity was assessed using relative total growth and cloning efficiency 1= ln(number of empty wells on plates/192)/cells seeded per well.
Evaluation criteria:
EVALUATION OF RESULTS:
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
A positive response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth, and/or cloning efficiency 1 is less than 10 % of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies, clastogenic effects are indicated.

ACCEPTABILITY CRITERIA:
A mutation assay is considered acceptable if it meets the following criteria:
All plates, from either the cloning efficiency 2 (ln(mean number of empty wells per plate / 96) / cells seeded per well) or the TFT resistance-testing portion of the experiment are analysable.
- The absolute cloning efficiency 2 of the negative and/or solvent controls is > 0.5 (50%).
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range of our historical control data
- The positive controls induce significant (at least 2-fold) increases in the mutant frequencies. The cloning efficiencies and the relative total growth are greater than 10% of the concurrent vehicle control group.
Statistics:
A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance should be considered together.
The data generated at the second precipitating concentration (160 µg/mL) are not included in the statistical evaluation.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
A dose dependency of mutation frequency (Exp. I and II) was not considered biologically relevant. They either were not reproduced in the parallel culture (Exp. I) or absolute values of mutation frequency remained within the historical controls.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects were seen in experiment II at 30 and 40 µg/ml without and 160 µg/ml with S9-mix and in experiment III (without S9-mix) at 80 µg/ml and above.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
Precipitation was seen at 80 µg/mL and above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mouse lymphoma assay precipitation is problematic since the cells grow in suspension. At the end of the treatment, the cells are separated from the test item by centrifugation. A precipitate is likely to contaminate the cell-pellet at the centrifugation step. Thus, arbitrary amounts of test item are carried over to the following steps of the assay leading to non-defined exposure times of up to 48 hours to selection. Toxic or seemingly mutagenic artefacts are likely to occur under these conditions.

General Remarks

Toxicity

Relevant toxic effects, indicated by relative cloning efficiency 1 or a relative total growth of less than 50 % in both parallel cultures, were observed in the second experiment at 30 and 40 μg/mL without metabolic activation and at 160 μg/mL with metabolic activation. In the third experiment relevant toxic effects were noted in the precipitating range at 80 μg/mL and above.

In the experimental parts using 4 hour treatment, the dose range was limited by the solubility of the test item. Two precipitating concentrations were analyased. In the experimental part using continuous treatment for 24 hours without metabolic activation (experiment II), cytotoxic effects were dose limiting.

 

Mutagenicity

The data with metabolic activation do not indicate a possible mutagenic potential of the test item. Generally elevated but not dose dependent levels of the mutation frequency observed in the first culture of the first experiment with metabolic activation were based on the relatively low solvent control. Compared to the corresponding negative control no relevant increase occurred.

The data generated in the absence of metabolic activation with a treatment period of 4 hours showed an isolated increase of the mutation frequency at the maximum concentration of 160 μg/mL in the presence of precipitation.

Following 24 hours of treatment, no increase at all was noted even at severely cytotoxic concentrations in the soluble range. The increase at precipitating concentrations occurred in the second culture of the first experiment without metabolic activation and in the first culture of the third experiment. The threshold of twice the colony count of the corresponding solvent control was exceeded. No comparable increase occurred in the parallel cultures under identical conditions. Both of the increased levels of the mutation frequency are judged as biologically irrelevant precipitation artifacts.

 

Controls

In this study the range of the negative and solvent controls was from 34 up to 209 mutant colonies per 106cells; the range of the groups treated with the test item was from 32 up to 538 mutant colonies per 106 cells. Both solvent controls did not quite reach the historical range of solvent controls in the second experiment without metabolic activation. However, this effect was judged as biologically irrelevant fluctuation since both corresponding negative controls remained within the range of historical negative controls.

 

Statistics

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The data generated at the second precipitating concentration of 160 μg/mL are not included in the statistical evaluation. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in experiment I culture I with and without metabolic activation. In experiment II such a trend was observed in both cultures without metabolic activation. However, the significant trends mentioned above were not considered biologically relevant since they either were not reproduced in the parallel culture (experiment I) or the absolute values of the mutation frequency remained within the historical range of negative and solvent controls (experiment II).

Table 2. Summary of results.


conc. μg/mL S9 mix relative cloning efficiency 1 relative total growth mutant colonies/ 106 cells induction factor relative cloning efficiency 1 relative total growth mutant colonies/ 106 cells induction factor
Column 1 2 3 4 5 6 7 8 9 10
Experiment 1/ 4 h treatment culture I culture II
Neg. control wtth medium
- 100.0 100.0 148
100.0 100.0 103
Solv. control with DMSO
- 100.0 100.0 165 1.0 100.0 100.0 181 1.0
Pos. Control with MMS 19.5 - 67.7 25.9 703 4.8 49.2 25.6 449 4.3
Test item 5.0 - 112.8 culture was not continued* 106.6 culture was not continued*
Test item 10.0 - 108.2 151.2 90 0.5 101.6 61.3 387 2.1
Test item 20.0 - 106.0 112.5 117 0.7 79.8 55.3 261 1.4
Test item 40.0 - 94.6 107.5 169 1.0 104.9 70.6 262 1.4
Test item 80.0 (p) - 71.7 85.8 217 1.3 122.4 60.1 292 1.6
Test Item 160.0 (p) - 56.8 72.5 280 1.7 81.0 44.6 538 3.0
Neg. control with medium
+ 100.0 100.0 209
100.0 100.0 120
Solv. control with DMSO
+ 100.0 100.0 106 1.0 100.0 100.0 66 1.0
Pos. control with CPA 4.5 + 49.9 67.8 703 3.4 57.0 69.8 336 2.8
Test item 5.0 + 100.0 culture was not continued* 73.6 culture was not continued*
Test item 10.0 + 83.9 102.1 193 1.8 63.0 156.5 43 0.7
Test item 20.0 + 87.0 84.6 230 2.2 79.0 102.7 73 1.1
Test item 40.0 + 107.0 95.3 207 2.0 84.9 146.5 37 0.6
Test item 80.0 (p) + 82.4 67.6 251 2.4 67.6 134.9 45 0.7
Test item 160.0 (p) + 88.6 51.2 237 2.2 75.7 52.6 80 1.2
Experiment II / 24 h treatment culture I culture II
Neg. control with medium
- 100.0 100.0 52
100.0 100.0 46
Solv. control with DMSO
- 100.0 100.0 34 1.0 100.0 100.0 37 1.0
POS. Control with MMS 13.0 - 35.7 29.2 756 14.6 42.8 19.1 392 8.5
Test item 1.3 - 116.4 culture was not continued* 114.4 culture was not continued*
Test item 2.5 - 93.7 culture was not continued* 102.5 culture was not continued*
Test item 5.0 - 100.0 59.0 48 1.4 87.5 67.6 51 1.4
Test item 10.0 - 95.7 48.7 69 2.0 91.3 72.8 38 1.1
Test item 20.0 - 86.4 30.3 62 1.8 79.4 59.4 35 1.0
Test item 30.0 - 40.6 27.7 65 1.9 43.4 24.2 65 1.8
Test item 40.0 - 5.8 9.0 45 1.3 9.9 9.4 38 1.0
Experiment II / 4 h treatment







Neg. control with medium
+ 100.0 100.0 57
100.0 100.0 56
Solv. control with DMSO
+ 100.0 100.0 64 1.0 100.0 100.0 52 1.0
Pos. control with CPA 4.5 + 19.1 33.5 385 6.7 20.3 26.7 284 5.5
Test item 5.0 + 82.4 culture was not continued* 124.8 culture was not continued*
Test item 10.0 + 100.0 141.7 32 0.5 77.0 106.6 60 1.2
Test item 20.0 + 72.0 111.2 52 0.8 74.4 98.2 54 1.0
Test item 40.0 + 73.2 89.4 51 0.8 87.8 62.2 79 1.5
Test item 80.0 (p) + 73.2 72.6 42 0.7 97.7 93.3 48 0.9
Test item 160.0 (p) + 36.3 37.4 46 0.7 87.8 42.8 49 0.9
Experiment III / 4 h treatment culture I culture II
Neg. control with medium
- 100.0 100.0 196
100.0 100.0 144
Solv. control with DMSO
- 100.0 100.0 144 1.0 100.0 100.0 154 1.0
Pos. Control with MMS 19.5 - 77.5 24.4 429 2.2 92.6 11.7 381 2.6
Test item 5.0 - 78.8 culture was not continued* 103.5 culture was not continued*
Test item 10.0 - 87.9 85.2 127 0.9 83.8 81.2 182 1.2
Test item 20.0 - 60.2 98.6 163 1.1 100.0 72.3 173 1.1
Test item 40.0 - 62.2 89.7 147 1.0 98.3 87.7 153 1.0
Test item 80.0 (p) - 53.1 17.6 160 1.1 70.2 38.0 156 1.0
Test item 160.0 (p) - 39.1 20.6 347 2.4 96.7 33.2 195 1.3

* culture was not continued since a minimum of four concentrations is required by the guidelines

(p) precipitation visible to the naked eye

Table 3. HISTORICAL DATA

Number of mutant colonies per 106 cells
4 h treatment

Negative control Positive control Solvent control

without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix

MMS DMNA CPA
range: 41 - 208 41 -197 212 – 3321 203 - 847 209 – 1269 41 - 204 40 - 205
Mean value: 107 110 479 389 390 106 110
Standard deviation 37 38 281 203 170 39 35

Table 3. HISTORICAL DATA (cont.)

Number of mutant colonies per 106 cells
24 h treatment

Negative control Positive control Solvent control

without S9 mix without S9 mix (MMS) without S9 mix
range: 40-212 243 - 2431 40 - 202
Mean value: 109 863 107
Standard deviation 40 461 39
Conclusions:
The test material did not invoke a mutagenic response under the conditions of the test.
Executive summary:

In this OECD 476 test with GLP certification, mouse lymphoma cells L5178Y were treated with test article at doses up to 160 µg/mL using DMSO as a vehicle. The test design consisted of two main experiments using two parallel cultures.The cells were treated with the test item for 4 h with and without metabolic activation in the first experiment. In the second experiment the duration of treatment was 4 h with and 24 h without metabolic activation.A confirmatory experiment was added to verify mutagenicity data generated in the first experiment without metabolic activation. Experiment III was solely performed in the absence of metabolic activation under experimental conditions identical to the first experiment without metabolic activation.

Precipitation of the test item and decrease in cloning efficiency was observed at doses of 80 and 160 µg/mL. A biologically irrelevant increase in mutation frequency was observed in some dishes of the first experiment. They were not reproduced in the parallel culture and considered as artifacts caused by precipitates. An increase in mutation frequency was also observed in the second experiment (without metabolic activation, 24 h). Since absolute values of the mutation frequency remained within the historical range of negative and solvent controls, this effect was considered to be biologically irrelevant. Therefore, the test article is considered to be non-mutagenic to mammalian cells in vitro.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-04-07, 1987-05-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1981
Deviations:
yes
Remarks:
Only four S. Typhimurium strains instead of five were used.
Principles of method if other than guideline:
The experiments were performed in a room with red light illumination, in order to avoid any influence of UV-light on the test material.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (All strains are histidine-auxotrophic mutants)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
0.3 mL S9 fraction of liver from rats (Tif: RAIf(SPF)) induced with Aroclor 1254 and 0.7 mL of a solution of cofactors.
Test concentrations with justification for top dose:
20, 78, 313, 1250, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: test article is insoluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without activation
Positive control substance:
other: Daunorubicin-HCl
Remarks:
strain TA 98: 5 and 10 µg/plate
Positive controls:
yes
Remarks:
without activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
strain TA 100: 0.125 and 0.25 µg/plate
Positive controls:
yes
Remarks:
without activation
Positive control substance:
sodium azide
Remarks:
strain TA 1535: 2.5 and 5.0 µg/plate
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without activation
Positive control substance:
9-aminoacridine
Remarks:
strain TA 1537: 50 and 100 µg/plate
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
strains TA 98, TA 100, TA 1537: 5 µg/plate
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with activation
Positive control substance:
cyclophosphamide
Remarks:
strain TA 1535: 250 µg/plate
Details on test system and experimental conditions:
DETERMINATION OF CYTOTOXICITY
In a preliminary toxicity test with strain TA 100 without metabolic activation (plate incorporation), nine concentrations of test article ranging from 0.08 to 5000 µg/plate were tested to determine the highest concentration to be used in the mutagenicity assay.

MUTAGENICITY TEST
All experiments were performed in triplicate. The test was performed as agar plate incorporation test for 48 h in the dark at 37 +/- 1.5 °C. The selection based on growth in absence of histidine.
Evaluation criteria:
The test substance is considered to be positive when:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537;
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable.

A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
Statistics:
Arithmetic mean and standard deviation
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 313 µg/plate and above the test article precipitated in soft agar.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Table 1.

Table1. Toxicity test on strain TA 100 without microsomal activation. Colonies per plate and arithmetic mean (m).

Concentration μg/plate Experiment
1 2 mean
Control 113 113 113
0.08 104 109 107
0.31 115 138 127
1.2 114 120 117
4.9 108 121 115
19.5 111 124 118
78.1 104 115 110
312.5 114 124 125
1250.0 123 127 125
5000.0 113 116 115
Positive controls (4-nitroquinoline-N-oxide) (μg/mL)
Control 105 111 108
0.125 883 930 907
0.250 1387 1483 1435

Table 2. Mutagenicity test* without microsomal activation: number of back mutant colonies per plate, arithmentic mean (m) and standard deviation (SD)

Concentration μg/plate STRAIN
TA 98
TA 100  TA 1535
TA 1537 
Experiment
1 2 3 mean (SD) 1 2 3 mean (SD) 1 2 3 mean (SD) 1 2 3 mean (SD)
Control 19 8 18 15 (6) 125 138 161 141 (18) 13 12 14 13 (1) 5 13 6 8 (4)
20 26 18 17 20 (5) 140 164 165 156 (14) 20 14 19 18 (3) 4 2 5 4 (2)
78 19 19 16 18 (2) 139 173 170 161 (19) 7 18 16 14 (6) 12 8 5 8 (4)
313 21 17 13 17 (4) 132 149 132 138 (10) 9 18 13 13 (5) 5 6 5 5 (1)
1250 11 15 14 13 (2) 152 124 153 143 (16) 14 9 15 13 (3) 3 4 4 4 (1)
5000 12 16 13 14 (2) 149 140 132 140 (9) 8 11 13 11 (3) 6 4 5 5 (1)
Positive controls (μg/plate)

daunorubicin-HCl











Control 15 18 12 15 (3)











5 914 794 788 832 (71)











10 481 530 400 470 (66)
















4-nitroquinoline-N-oxide







Control



151 153 134 146 (10)







0.125



560 567 606 578 (25)







0.25



1144 892 1065 1034 (129)
















sodium azide



Control







15 14 5 11 (6)



2.5







484 448 524 485 (38)



5







836 776 750 787 (44)
















9 aminoacridine
Control











12 5 9 9 (4)
50











14 17 26 19 (6)
100











206 194 261 220 (36)

* representative study

Table 3. Mutagenicity test* with microsomal activation: number of back mutant colonies per plate, arithmentic mean (m) and standard deviation (SD)

Concentration μg/plate STRAIN
TA 98 TA 100 TA 1535 TA 1537
Experiment 1 2 3 mean (SD) 1 2 3 mean (SD) 1 2 3 mean (SD) 1 2 3 mean (SD)
Control 30 37 38 35 (4) 180 157 164 167 (12) 14 21 14 16 (4) 8 6 13 9 (4)
20 26 26 20 24 (3) 185 172 148 168 (19) 17 19 17 18 (1) 9 8 14 10 (3)
78 30 29 20 26 (6) 177 187 165 176 (11) 13 18 19 17 (3) 14 3 13 10 (6)
313 24 41 29 31 (9) 172 164 173 170 (5) 16 14 13 14 (2) 16 12 12 13 (2)
1250 28 24 25 26 (2) 197 192 171 187 (14) 9 11 12 11 (2) 6 9 8 8 (2)
5000 13 20 22 18 (5) 157 158 146 151 (7) 7 8 17 11 (6) 6 11 7 8 (3)
Positive controls (μg/plate)

2-aminoanthracene 2-aminoanthracene



2-aminoanthracene
Control 36 17 25 26 (10) 161 163 148 157 (8)



8 12 8 9 (2)
5 1409 1287 762 1153 (344) 1779 1514 1416 1570 (188)



240 224 177 214 (33)









cyclophosphamide



Control







6 17 12 12 (6)



250







409 325 414 383 (50)



* representative study

Conclusions:
The test material did not invoke a mutagenic response under the conditions of the test.
Executive summary:

In this study (OECD 471) conducted to generally accepted scientific standards with GLP, first a preliminary toxicity test with strain TA 100 (without metabolic activation) of the test material (EC 404-360-3) was conducted. Nine concentrations from 0.08 to 5000 µg/plate were tested without metabolic activation in order to determine the concentrations to be used for the main test.

For the main test, five concentrations ranging from 20 -5000 µg/plate (with a 0 µg/plate for control) were tested on different strains of Salmonella typhimurium with and without metabolic activation provided by rats' livers using acetone as a vehicle. Stastically, no mutagenic activity was observed over the duration of the test compared to the controls, thus according to the CLP regulation 1272/2008, this test material is not genotoxic.

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
Study period:
1989-11-07, 1990-02-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient Mixture F-12 supplemented with 10% fetal calf serum + Penicillin/Streptomycin 100 units / mL / 100 µg / mL (Gibco AG, Basle, Switzerland).
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
0.15 mL S9 fraction of liver from rats (Tif: RAIf(SPF)) induced with Aroclor 1254 and 0.2 mL of a solution of cofactors in 0.65 mL medium.
Test concentrations with justification for top dose:
ORIGINAL STUDY
Experiment 1, 2, 3, 4, 5: 1.72, 3.44, 6.88, 13.75, 27.5, 55.0, 110.0, 220.0 µg/ml

CONFIRMATORY STUDY
Experiment 1, 2: 13.75, 27.5, 55.0, 110.0, 220.0 µg/ml
Experiment 3: 6.88, 13.75, 27.5, 55.0, 110.0 µg/ml
Experiment 4: 0.86, 1.72, 3.44, 6.88, 13.75 µg/ml

For further details see table 1.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The test substance is insoluble in water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: 0.1 and 0.04 µg/mL
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
0.15 mL S9 fraction of liver from RAI rats induced with Aroclor 1254 and 0.2 mL of a solution of cofactors and 0.65 ml medium. Migrated to IUCLID6: 40.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Preincubation period of cells: 24 - 57 h

Exposure scenario in the original study:
Experiment 1: with activation, Duration of treatment: 3 hours, Recovery: 14 hours
Experiment 2: with activation, Duration of treatment: 3 hours, Recovery: 21 hours
Experiment 3: without activation, Duration of treatment: 14 hours, Recovery: 0 hours
Experiment 4: without activation, Duration of treatment: 24 hours, Recovery: 0 hours
Experiment 5: without activation. Duration of treatment: 48 hours, Recovery: 0 hours

Exposure scenario in the confirmatory study:
Experiment 1: with activation, Duration of treatment: 3 hours, Recovery: 14 hours
Experiment 2: with activation, Duration of treatment: 3 hours, Recovery: 21 hours
Experiment 3: without activation, Duration of treatment: 14 hours, Recovery: 0 hours
Experiment 4: without activation, Duration of treatment: 24 hours, Recovery: 0 hours

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid 0.4 µg/mL

NUMBER OF METAPHASE EVALUATED:
200 in treatment groups, 50 in positive controls

NUMBER OF CELLS EVALUATED:
2000

DETERMINATION OF CYTOTOXICITY (performed as integral part of the mutagenicity test by mitotic index)
For the determination of the mitotic index the preparations from the various cultures were examined first, uncoded. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells per concentration.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Evaluation criteria:
200 metaphase figures with 19 to 21 chromosomes from cultures of two falcon flasks in the vehicle control and in the treated groups, and at least 50 metaphase cells in the positive controls were examined for the following aberrations:
a) specific aberrations: breaks, exchanges, deletions, fragments
b) unspecific aberrations: gaps, premature chromosome condensation, and chromosome decay
c) numerical aberrations (metaphases with >21 chromosomes) will be registered, yet reported only in case of deviations.
Statistics:
A test substance is considered to be active in this test system if in comparison to the negative control a marked increase in the number of specific chromosomal aberrations appears or if an increased number of exchange figures appear together with a high number of other specific chromosomal aberrations such as breaks and fragments.
A concentration-related response in the number of aberrations should be demonstrable.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Chromosome aberration and polyploidy
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
mitotic index decrease (table 2).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
REMARK
In the original study, in all experiments performed, the number of cells with specific chromosomal aberrations in the treatment groups showed no marked increase in comparison with the negative control. The incidence of changes observed is within the range of spontaneous aberrations inherent in this particular cell line used as compared to the historical data. The confirmatory study confirmed the results of the original study.

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: TK 11319 is insoluble
- Precipitation: Addition of the stock solutions to the cell cultures produced turbidity in the media at the final concentrations of 220.0 and 110.0 µg/mL without and with activation.
Remarks on result:
other: strain/cell type: CHO-K1 CCL61
Remarks:
Migrated from field 'Test system'.

Table 2. Mitotic Index values (MI) Original study

Experiment 1. 3 hours with metabolic activation (recovery 14 h)

Cells scored Mitosis Frequency % of control MI (%)
Solvent control 2000 155 100 7.75
Test compound (μg/mL) 220.0 2000 54 34.8 2.7
110.0 2000 65 41.9 3.25
55.0 2000 78 50.3 3.9
27.5 2000 117 75.5 5.85
13.8 2000 145 93.5 7.25
6.9 2000 150 96.8 7.5
3.4 *


1.7 *


Experiment 2. 3 hours with metabolic activation (recovery 21 h)

Cells scored Mitosis Frequency % of control M.I. (%)
Solvent control 2000 174 100 8.7
Test compound (μg/mL) 220.0 2000 71 40.8 3.55
110.0 2000 99 56.9 4.95
55.0 2000 116 66.7 5.8
27.5 2000 145 83.3 7.25
13.8 2000 135 77.6 6.75
6.9 2000 152 87.4 7.6
3.4 *


1.7 *


Experiment 3. 14 hours without metabolic activation

Cells scored Mitosis Frequency % of control M.I. (%)
Solvent control 2000 135 100 6.75
Test compound (μg/mL) 220.0 2000 20 14.8 1.0
110.0 2000 35 25.9 1.75
55.0 2000 35 25.9 1.75
27.5 2000 42 31.1 2.1
13.8 2000 58 43 2.9
6.9 2000 87 64.4 4.35
3.4 2000 120 88.9 6.0
1.7 2000 136 100.7 6.8
Experiment 4. 24 hours without metabolic activation

Cells scored Mitosis Frequency % of control M.I. (%)
Solvent control 2000 91 100 4.55
Test compound (μg/mL) 220.0 2000 1 1.1 0.05
110.0 2000 18 19.8 0.9
55.0 2000 18 19.8 0.9
27.5 2000 16 17.6 0.8
13.8 2000 66 72.5 3.3
6.9 2000 73 80.2 3.65
3.4 2000 75 82.4 3.75
1.7 *


Experiment 5. 48 hours without metabolic activation

Cells scored Mitosis Frequency % of control M.I. (%)
Solvent control 2000 79 100 3.95
Test compound (μg/mL) 220.0 2000 4 5.1 0.2
110.0 2000 10 12.7 0.5
55.0 2000 7 8.9 0.35
27.5 2000 10 12.7 0.5
13.8 2000 19 24.1 0.95
6.9 2000 64 81 3.2
3.4 2000 82 103.8 4.1
1.7 2000 93 117.7 4.65

* When 3 subsequent concentrations with a frequency of 70 % mitosis or more in relation to the negative control are found, the evaluation of the lower concentrations is omitted.

Conclusions:
The test material did not invoke a mutagenic response under the conditions of the test.
Executive summary:

In this OECD 473 test with GLP compliance, chinese hamster ovary cells CCL61 were treated with test article at doses up to 220 µg/ml using DMSO as a vehicle. The original test design consisted of five main experiments using two parallel cultures. The cells were treated with the test item for 3 h with metabolic activation and a recovery time of 14 hours and 21 hours (experiments 1 and 2 respectively). For experiments 3 -5, the treatment was with no metabolic activation or recovery time, and the durations of the treatments were 14 hours, 24 hours and 48 hours respectively.

A confirmatory experiment was added to verify data generated in the first set of experiments at doses up to 220 µg/ml

. These additional experiments were performed with metabolic activation with a treatment duration of three hours (experiments 1 and 2) and a recovery period of 14 and 21 hours (experiments 1 and 2 respectively). Experiments were also performed without metabolic activation with a treatment duration of 14 and 24 hours (experiments 3 and 4 respectively) without a recovery period.

In the original study, in all experiments performed, the number of cells with specific chromosomal aberrations in the treatment groups showed no marked increase in comparison with the negative control. The incidence of changes observed is within the range of spontaneous aberrations inherent in this particular cell line used as compared to the historical data. The confirmatory study confirmed the results of the original study. Thus according to this study, EC 404 -360 -3 is not genotoxic.

Genetic toxicity in vivo

Description of key information

Study conducted to recognised training guidelines with GLP

Link to relevant study records
Reference
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
Study period:
1987-04-06, 1987-09-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln, Switzerland
- Age at study initiation: Females 6 - 10 weeks; males 4 - 9 weeks
- Weight at study initiation: Females 23-32 g; males 26-35 g
- Assigned to test groups randomly: yes
- Diet: Ad libitum, standard diet: NAFAG No.924
- Water: Ad libitum
- Acclimation period: 4 - 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 51 - 74
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Carboxymethylcellulose (CMC) 0.5 % in water
- Justification for choice of solvent/vehicle: TK 11319 is insoluble in water
- Concentration of test material in vehicle: 5000 mg/kg bw in 20 mL/kg bw
Duration of treatment / exposure:
Single dose exposure
Frequency of treatment:
Single dose
Post exposure period:
16, 24, and 48 hours
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
Remarks:
In a preliminary toxicity test the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay.
No. of animals per sex per dose:
8 per sex per group
Control animals:
yes, concurrent no treatment
Positive control(s):
A dose of 64 mg/kg bw of cyclophosphamide was used as positive control. The route of administration was oral per gavage.
Tissues and cell types examined:
Bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a polished cover glass. The preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water (1/1) for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6 %) for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in xylene and mounted. 1000 polychromatic erythrocytes (PCE) per animal were used to determine the incidence of micronucleated PCE. A total of 1000 erythrocytes were counted for determination of the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE -ratio).

Statistics:
The significance of difference is assessed by χ2-test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No significant differences in the PCE/NCE-ratio between treated groups and controls.
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1. Effect of test article on bone marrow cells of chinese hamster: arithmetic mean per sex and group.


Sacrifice (h) 

16 24 48

Control TK 11319 Control TK 11319 Control TK 11319
sex of the animals
polychromatic erythrocytes (P) 626 520 550 551 489 471 482 486 555 539 459 485
normochromatic erythrocytes (N) 374 480 450 449 511 529 518 514 445 461 541 515
ratio P/N 1.7 1.1 1.2 1.2 1.0 0.9 0.9 0.9 1.2 1.2 0.8 0.9
P with micronuclei 0.2 0.2 0.4 0.2 0.6 1.0 1.0 0.6 0.8 0.8 0.6 0.6
percent of P with micronuclei 0.02 0.02 0.04 0.02 0.06 0.10 0.10 0.06 0.08 0.08 0.06 0.06

Table 1 (cont.). Effect of test article on bone marrow cells of chinese hamster: arithmetic mean per sex and group (positive control).


Sacrifice (h)

24

Control Cyclophosphamide (64 mg/kg)
sex of the animals
polychromatic erythrocytes (P) 489 471 452 450
normochromatic erythrocytes (N) 511 529 548 550
ratio P/N 1.0 0.9 0.8 0.8
P with micronuclei 0.6 1.0 27.0 32.2
percent of P with micronuclei 0.06 0.10 2.70 3.22
Conclusions:
The substance tested in the in vivo Micronucleus Test with Chinese Hamster (method similar to OECD 474) up to 5000 mg/kg bw was not genotoxic.
Executive summary:

In this guideline (OECD 474) study conducted to GLP standards, the test material (EC 404-360-3) was tested in vivo on chinese hamsters (male and female) aged 4 -10 weeks. The test substance was administered via oral gavage in a vehicle of 0.5% carboxymethylcellulose (concentration of test material in vehicle was 5000 mg/kg bw in 20 ml/kg bw). 8 animals of each sex per dose were tested, with various sacrifice times (16, 24 and 48 hours). After sacrifice, bone marrow was harvested from the shafts of both femurs with fetal calf serum for analysis. As well as a control, there was a positive control using 64 mg/kg bw of cyclophosphamide (also administered via oral gavage). The bone marrow tissue was then examined for incidence of polychromatic erythrocytes (PCE) - an increase in frequency being an indicator of induced chromosome damage. No PCE was observed for the test material, therefore EC 404 -360 -3 is not genotoxic.

Additional information

Two Ames tests are available. Concentrations of 20, 78, 313, 1250, and 5000 µg/plate were tested in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, with and without metabolic activation (Ciba-Geigy 1987) according to OECD 471 (1981). The same concentrations were tested in a previous screening study in Salmonella strains TA 98, TA 100 and TA 1537, with and without metabolic activation (Ciba-Geigy 1986); the test conduct was similar to OECD 471.

In the first study no increased mutation frequency was observed both in the absence and presence of S9-mix. Up to the highest concentration no cytotoxicity was seen. In the second study also no increased mutation frequency was observed in the absence of S9-mix. In the presence of S9-mix, a slight (not doubled) and not significant increase of mutant colonies was observed at concentrations of 313 µg/plate and above for strain TA 100 only. Since the mutation frequency was not doubled in comparison to control and no dose-relationship was seen the effect is considered of no biological relevance. Up to the highest concentration no cytotoxicity was seen.

Thus, the test article was not genotoxic when tested in the Ames test under the chosen conditions.

In a guideline study conducted according to OECD 473 (1983) doses of 1.72, 3.44, 6.88, 13.75, 27.5, 55.0, 110.0, 220.0 µg/ml were tested for the induction of chromosome aberrations in Chinese hamster ovary cells in vitro with and without S9-mix (Ciba-Geigy 1990). The number of cells with chromosomal aberrations in the treatment groups showed no marked increase in comparison with the negative control. Thus, under the given experimental conditions, no evidence of mutagenic effects was obtained in Chinese hamster ovary cells in vitro after treatment with the test article.

Mouse lymphoma cells L5178Y were treated with test article according to OECD 476 (RCC 2006). The test design consisted of two main experiments using two parallel cultures. The cells were treated with the test item for 4 h with and without metabolic activation in the first experiment. In the second experiment the duration of treatment was 4 h with and 24 h without metabolic activation. A confirmatory experiment was added to verify mutagenicity data generated in the first experiment without metabolic activation. The cultures were evaluated at concentrations ranging from 10 to 160 µg/ml with and without S9-mix in the first experiment. In the second experiment the concentrations were 5 to 40 µg/ml without and 10 to 160 µg/ml with S9-mix. The confirmatory experiment without S9-mix was conducted with 10 to 160 µg/ml.

Precipitation of the test item and decrease in cloning efficiency was observed at doses of 80 and 160 µg/ml in all experiments. A biologically irrelevant increase in mutation frequency was observed in some dishes of the first experiment with S9-mix, which could not be reproduced; the effect was therefore considered as artifact. An increase in mutation frequency was also observed in the second experiment (without S9-mix). Since absolute values of the mutation frequency remained within the historical range of negative and solvent controls, this effect was also considered to be biologically irrelevant. Therefore, the test article was not mutagenic in the mouse lymphoma test.

An in vivo micronucleus test with Chinese Hamster was conducted similar to OECD 474, at a dose of 5000 mg/kg bw (Ciba-Geigy 1987).The number of polychromatic erythrocytes with micronuclei in the treatment group showed no increase in comparison to the negative control. Thus, under the given experimental conditions, no evidence of mutagenic effects was obtained in Chinese hamster in vivo.

Under in vitro and in vivo conditions, the substance is not mutagenic to bacteria and to mammalian cells in culture with and without metabolic activation and is not clastogenic in mammalian cells. Under in vivo conditions the substance is not clastogenic in Chinese hamster.

 


Short description of key information:
Valid studies on the in vitro and in vivo genetic toxicity are available. Two Ames tests were performed, the more recent according to OECD 471 (Ciba-Geigy 1987) and the older as a screening study with three strains (Ciba-Geigy 1986). A chromosome aberration assay with Chinese Hamster Ovary Cells was conducted according to OECD 473 (Ciba-Geigy 1990) and a forward mutation assay in mouse lymphoma L5178Y cells according to OECD 476 (RCC, 2006) is also available. For in vivo mutagenicity testing, a micronucleus test with Chinese Hamster similar to OECD 474 was done (Ciba-Geigy1987). None of the assays revealed positive results.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive (EC 944/2013).