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Toxicological information

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
No indepedent repeat experiment for incubation with S9 mix. Dose with S9 mix should have been slightly higher. It resulted only in 24% viability instead of the required 10 - 20% viability.
GLP compliance:
yes (incl. certificate)
Remarks:
The report includes GLP certificates from the German, Dutch and Indian Authorities. All certificates are valid for the period of the study.
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
tk - thymidinkinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Arochlor 1254 induced rat live
Test concentrations with justification for top dose:
17, 34, 68 and 136 µg/ml (3h, with S9)
55, 110, 220, 440 µg/ml (3h and 24h, without S9)
Vehicle / solvent:
DMSO

The results of the solubility test of Study No. 04733 for the same compound indicated that the test item forms a workable suspension with DMSO at the required concentration of 250000 /lg/mL. Also, DMSO is one of the organic solvents compatible with the test system. Hence, DMSO was selected as the vehicle to prepare the stock and dilutions ofthe test item as weil as the positive controls.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h and 24h
- Expression time (cells in growth medium): 48h

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT) at 4 mug/mL

NUMBER OF REPLICATIONS: tests were done in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: cell growth
Evaluation criteria:
There are several criteria for determining a positive result, such as a concentration related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. A test item, for which the results do not meet the above criteria is considered non mutagenic in this system.
Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells
used. A positive concentration response that is reproducible is most meaningful. Negative results indicate that, under the test conditions the test
substance does not induce gene mutations in the cultured mammalian cells used.
Statistics:
The number of negative contraI values are assumed to follow Poisson Distribution. Test of significance of difference between negative control values over different dose groups and the positive control was carried out using a method of analysis suggested by M.R.Thomas (Cole and Arlett, 1984), based on chi2. All analysis and significance tests were evaluated at 5% level of signiftcance (P<0.05).

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG was 24%
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Moderatetyl cytotoxic: RTG was 29 (24h) and 39 (3h). The next highest concentration was found to be non scorable based on precipitates upon centrifugation in pretests.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the presence of metabolie activation at the end of exposure to treatment, the pH of the test medium ranged between 7.05 to 7.22 with 7.10 in the DMS0 control. Similarly, in the absence of metabolic activation, the pH of the treatment medium ranged between 7.05 and 7.20 Wilh 7.21 in the DMSO control at the end of exposure to treatment.
- Precipitation: from 800 µg/ml onward, in presence and absence of metabolic activation

RANGE-FINDING/SCREENING STUDIES:
Preliminary test results of Study No. 04733 for the same test item indicated heavy precipitation of the test item at and above 2400 µg/ml. This precipitation interfered with the observation of the cells. Hence, the test item was checked fo it precipitation in the treatment medium (TM) up to the maximum concentration of 1600 µg/ml of the medium. At and abave 800 µg/mL, the test item precipitated In the form of a pellet in the centrifuge tubes both in the presence and absence of metabolic activation in the 3-hour as weil as in the 24-hour exposure regime. Hence cell counts could not be determined. Based on these observations, it was decided to test up to 136 µg/mL in the presence of metabolie aetivation for the 3-hour exposure and up to 440 µg/mL for the 3-hour as well as the 24-hour exposure in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, solvent and positive control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity test, the test item was toxic to the cells at and above 200 µg/mL when exposed for 3 hours in the presence of metabolic activation resulting in dead and disfigured cells. There was evidence of growth inhibition as relative total growth (RTG) at 100 µg/mL. However, this growth inhibition was not significant. In the 3-hour as well as the 24-hour exposure in the absence of metablic activation, there was evidence of growth inhibition as relativ total growth (RTG) at 400 µg/mL. However, this growth inhibition was not significant.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Presence of Metabolic Activation (Experiment 1)

Group Test item concn. (µg/mL) Non-selective medium     Selective medium    
P(0) individual plates
M.F x 10-6 M.F/survivor x 10-6 Induced M.F x 10-6 RTG
P(0) C.E. RCE R1 R2 Total
G1 DMSO (200 µI) 16/96 90 100 78/96 83/96 161/192 88 0.98 - 100
G2 17 18/96 84 93 79/96 84/96 163/192 82 0.98 - 69
G3 34 16/96 90 100 86/96 81/96 167/192 70 0.78 - 60
G4 68 20/96 78 88 89/96 80/96 169/192 64 0.81 - 43
G5 136 22/96 74 82 79/96 87/96 166/192 73 0.99 - 24
G6 CPA 12 23/96 71 80 23/96 36/96 59/192 590+ 8.26 7.28 43

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 2)

Group Test item concn. (µg/mL) Non-selective medium     Selective medium    
P(0) individual plates
M.F x 10-6 M.F/survivor x 10-6 Induced M.F x 10-6 RTG
P(0) C.E. RCE R1 R2 Total
G1 DMSO (200 µI) 14/96 96 100 78/96 84/96 162/192 85 0.88 - 100
G2 55 16/96 90 93 81/96 85/96 166/192 73 0.81 - 80
G3 110 16/96 90 93 83/96 80/96 163/192 82 0.91 0.03 62
G4 220 18/96 84 87 78/96 79/96 157/192 101 1.20 0.32 51
G5 440 18/96 84 87 76/96 78/96 154/192 110 1.32 0.44 39
G6 MMS 20 21/96 76 79 42/96 33/96 75/192 470+ 6.18 5.30 50

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 3)

Group Test item concn. (µg/mL) Non-selective medium     Selective medium    
P(0) individual plates
M.F x 10-6 M.F/survivor x 10-6 Induced M.F x 10-6 RTG
P(0) C.E. RCE R1 R2 Total
G1 DMSO (200 µJ) 15/96 93 100 80/96 83/96 163/192 82 0.88 - 100
G2 55 15/96 93 100 79/96 81/96 160/192 91 0.98 0.10 82
G3 110 18/96 84 90 86/96 76/96 162/192 85 1.01 0.13 74
G4 220 19/96 81 87 77/96 80/96 157/192 101 1.24 0.36 44
G5 440 20/96 78 85 81/96 84/96 165/192 76 0.97 0.08 27
G6 MMS20 20/96 78 85 36/96 32/96 68/192 519+ 6.62 5.74 29

M.F = Mutant Frequency

C.E = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

+ = Significantly higher than control (p ≤ 0.05)

Applicant's summary and conclusion

Conclusions:
The study indicated that the test item does not have the potential to cause gene mutation at the tk locus at the concentrations tested and under the conditions of testing.