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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CHIMEXANE CL was found not to be mutagenic in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, either with or without S9 mix.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 December 2007 to 30 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Name: Chimexane CL - Source and lot/batch No.of test material: CHIMEX / 0132889- Physical state: Yellowish limpid liquid- Date of receipt: 03 December 2007- Expiration date of the lot/batch: February 2008- Purity test date: 25 October 2007STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Stored at room temperature
Target gene:
His+ for S. typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) S9 fraction; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254 (500 mg/kg bw) by the intraperitoneal route.
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST (Direct plate incorporation method): 10, 100, 500, 1000, 2500 and 5000 μg/plate for the TA 98, TA 100 and TA 102 strains, with and without S9 mixMUTAGENICITY EXPERIMENTSFirst experiment (Direct plate incorporation method): 1.95, 3.91, 7.81, 15.6, 31.3 and 62.5 μg/plate without S9 in all strains 7.81, 15.6, 31.3, 62.5, 125 and 250 μg/plate with S9 for the TA 98 and TA 1537 strains 3.91, 7.81, 15.6, 31.3, 62.5 and 125 μg/plate with S9 for the TA 100, TA 1535 and TA 102 strainsSecond experiment (Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)):0.98, 1.95, 3.91, 7.81, 15.6 and 31.3 μg/plate without S9 for the TA 102 strain 0.49, 0.98, 1.95, 3.91, 7.81 and 15.6 μg/plate without S9 for TA 98, TA 100, TA 1535 and TA 1537 7.81, 15.6, 31.3, 62.5, 125 and 200 μg/plate with S9 for the TA 98 and TA 1537 strains 3.91, 7.81, 15.6, 31.3, 62.5 and 125 μg/plate with S9 for the TA 100, TA 1535 and TA 102 strainsThird experiment (Direct plate incorporation method):7.81, 15.6, 31.3, 62.5, 125 and 200 μg/plate with S9 for the TA 1537 strain
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water for injections- Formulation procedureAll dose-levels were expressed as active item taking into account of the dry extract of 30.8%.The test item was dissolved in the vehicle at concentrations of:50 mg/mL for the preliminary toxicity test,2.5 mg/mL for the first experiment,2 mg/mL for the second experiment,20 mg/mL for the third experiment.The preparations were made immediately before use.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Water for injections
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Water for injections
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Anthramine
Remarks:
With S9 mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Strains of S. typhimurium were obtained from B.N. Ames' Laboratory (University of California, Berkeley or Oakland Research Institute, USA).METHOD OF APPLICATION: in agar (plate incorporation); preincubationDURATION- Preincubation period: 60 minutes at 37 °C- Incubation period: 48-72 h at 37 °C for both direct plate incorporation and preincubation methodsNUMBER OF REPLICATIONS: - Preliminary toxicity test: One plate/dose for vehicle and test item- Mutagenicity experiments: 3 plates/dose for test item, vehicle and positive controlsDETERMINATION OF CYTOTOXICITY- Method: Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.OTHER: - After 48-72 h of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk, UK). Manual counting was used as needed. - The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
None
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Water solubility: The test item was freely soluble in the vehicle (water for injections) at 50 mg/mL.- Precipitation: YesPRELIMINARY TOXICITY TEST- A moderate to important precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 100 μg/plate, with S9 mix and at 5000 μg/plate without S9 mix. Without S9 mix, a strong toxicity was induced at dose-levels ≥ 100 μg/plate, in the three strains tested. With S9 mix, a moderate to strong toxicity was induced at dose-levels ≥ 100 μg/plate, in the TA 100 strain and ≥ 500 μg/plate in the TA 98 and TA 102 strains.MUTAGENICITY EXPERIMENTSExperiments without S9 mix:- No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. - A moderate to strong toxicity was induced in all tester strains, generally at dose-levels ≥ 7.81 μg/plate. - The test item did not induce any noteworthy increase in the number of revertants, in either experiment, in any of the five strains.Experiments with S9 mix:- No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.- A moderate to strong toxicity was induced in all tester strains, generally at dose-levels ≥ 125 μg/plate in the direct plate incorporation method and ≥ 62.5 μg/plate in the preincubation method.- In the first experiment (direct plate incorporation method), a slight increase, without any clear evidence of a dose-relationship, of the number of revertants (up to 4.4-fold the vehicle control mean value) was noted in the TA 1537 strain. No noteworthy increase in the number of revertants was noted in this strain using the preincubation method. In order to check the reliability of the slight increase observed in the first experiment, a confirmatory experiment was undertaken under the same experimental conditions. No noteworthy increase in revertants was induced in this confirmatory experiment and therefore the previous observation was considered of no biological relevance.- In the remaining tester strains, the test item did not induce any noteworthy increase in the number of revertants, in either experiment.HISTORICAL CONTROL DATA (means and standard deviation)- Positive historical control data: 594 ± 67, 966 ± 362.4, 191 ± 38.6, 675 ± 122.2 and 1971 ± 333.8 for TA 1535 (NaN3), TA 1537 (9AA), TA 98 (2 NF), TA 100 (NaN3) and TA 102 (MMC) strains, respectively (without S9); 244 ± 86.3, 126 ± 33.2, 1451 ± 344.9, 971 ± 316.3, 414 ± 153.7 and 2467 ± 807 for TA 1535 (2 AM), TA 1537 (2 AM), TA 98 (2 AM), TA 100 (2 AM), TA 100 (BaP) and TA 102 (2 AM) strains, respectively (with S9)- Negative (solvent/vehicle) historical control data: 18 ± 5.9, 7 ± 2.6, 23 ± 7.4, 116 ± 23.8 and 360 ± 52.2 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (without S9); 16 ± 4.4, 10 ± 3.8, 29 ± 6.3, 110 ± 21 and 486 ± 67.9 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (with S9)Note:sodium azide (NAN3); 9-Aminoacridine (9 AA); 2-Nitrofluorene (2 NF); Mitomycin C (MMC); 2-Anthramine (2 AM); Benzo(a)pyrene (BaP)

None

Conclusions:
Under the test conditions, test substance is not mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to test substance at the following concentrations:

PRELIMINARY TOXICITY TEST (Direct plate incorporation method): 10, 100, 500, 1000, 2500 and 5000 μg/plate for the TA 98, TA 100 and TA 102 strains, with and without S9 mix

MUTAGENICITY EXPERIMENTS

First experiment (Direct plate incorporation method):

1.95, 3.91, 7.81, 15.6, 31.3 and 62.5 μg/plate without S9 in all strains

7.81, 15.6, 31.3, 62.5, 125 and 250 μg/plate with S9 for the TA 98 and TA 1537 strains

3.91, 7.81, 15.6, 31.3, 62.5 and 125 μg/plate with S9 for the TA 100, TA 1535 and TA 102 strains

Second experiment (Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)):

0.98, 1.95, 3.91, 7.81, 15.6 and 31.3 μg/plate without S9 for the TA 102 strain

0.49, 0.98, 1.95, 3.91, 7.81 and 15.6 μg/plate without S9 for TA 98, TA 100, TA 1535 and TA 1537 strains

7.81, 15.6, 31.3, 62.5, 125 and 200 μg/plate with S9 for the TA 98 and TA 1537 strains

3.91, 7.81, 15.6, 31.3, 62.5 and 125 μg/plate with S9 for the TA 100, TA 1535 and TA 102 strains

Third experiment (Direct plate incorporation method):

7.81, 15.6, 31.3, 62.5, 125 and 200 μg/plate with S9 for the TA 1537 strain

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254 (500 mg/kg bw) by the intraperitoneal route. Vehicle and positive control groups were also included in mutagenicity tests.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines. The selected dose-levels ranged from 0.49 to 62.5 μg/plate and from 3.91 to 250 μg/plate, without and with S9 mix, respectively. No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. Without S9 mix, a moderate to strong toxicity was induced in all tester strains, generally at dose-levels ≥ 7.81 μg/plate. With S9 mix, a moderate to strong toxicity was induced in all tester strains, generally at dose-levels ≥ 125 μg/plate in the direct plate incorporation method and ≥ 62.5 μg/plate in the preincubation method.

The test item did not induce any noteworthy increase in the number of revertants which could be considered as biologically relevant, in any of the five strains, either with or without S9 mix.

 

Under the test conditions, test substance CHIMEXANE CL did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhymurium.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to the OECD guideline 471, on compliance with GLP, CHIMEXANE CL was found not to be mutagenic in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without metabolic activation.

Justification for selection of genetic toxicity endpoint.

Only one study available for this endpoint.

Justification for classification or non-classification

As Chimexane CL was negative in an in vitro reverse gene mutation assay in bacteria, it is not classified as mutagenic according to CLP Regulation (EC) N° (1272-2008).