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Genetic toxicity: in vitro

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007
Reference Type:
other company data
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-benzyl-2-dimethylamino-4'-morpholinobutyrophenone
EC Number:
404-360-3
EC Name:
2-benzyl-2-dimethylamino-4'-morpholinobutyrophenone
Cas Number:
119313-12-1
Molecular formula:
C23 H30 N2 O2
IUPAC Name:
2-benzyl-2-dimethylamino-4'-morpholinobutyrophenone
Details on test material:
- Physical state: Solid (light yellow)
- Molecular weight: 366.51 g/mol
- Analytical purity: 99.9 mol %
- Lot/batch No.: 37973FC4AA
- Expiration date of the lot/batch: 2010-12-01
- Stability under test conditions: Several hours at room temperature, in the refrigerator and in the freezer
- Storage condition of test material: At room temperature, light protected
- Solubility in water: 5.9 mg/l

Method

Target gene:
Thymidine Kinase Locus
Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.