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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assays (Ames test):


Evidence of mutagenic activity of JAU 6476 Chloromethylketone was seen. On Salmonella typhimurium TA 1535, TA 100, TA 98 and TA 102, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable.


 


Chromosomal abberation test mammalian cells:


In the absence and in the presence of S9 mix cultures treated with JAU 6476- Chloromethylketone showed biologically relevant and statistically significant increased numbers of aberrant metaphases.


 


In silico Ames Test:


Strong alert on mutagenicty when using state-of the-art prediction models (DEREK, Leadscope, VITIC) [Analysis April 07, 2021]

Link to relevant study records

Referenceopen allclose all

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:
Sept 2002 - Jan 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 473
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
purity 93.2%, Identity and stability tested analytically
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The karyotype of the V79 cells (modal number of chromosomes: 22) was confirmed on August 9, 2002.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 1 - 8 µg/ml
Concentration range in the main test (without metabolic activation): 0.5 - 4 µg/ml
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 4 hours

Expression time:
4 h

Selection time:
Experiment 1: 18 h (with and without metabolic activation)
Experiment 2: 30 h (with and without metabolic activation)

Fixation time:
2 h
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(>2 µg/ml)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 7 µg/ml)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations:
The substance showed clear cytotoxic effects. An indirect
cyctoxicity based mutagenic effect may be possible.

Mitotic index without S9 mix: In comparison to the solvent control, the mitotic indices in the treated cultures were relevantly reduced at 2 μg/ml and above. The cultures treated with mitomycin C showed no reduction in mitosis rate.


Mitotic index with S9 mix: In comparison to the solvent control, the treated cultures showed no relevant reduction of the mitosis rate. The positive control cyclophosphamide also reduced the mitosis rate.


Survival index without S9 mix: In comparison to the solvent control, the survival indices in the treated cultures were relevantly reduced at 2 μg/ml and above. The cultures treated with mitomycin C showed a reduction in survival rate.


Survival index with S9 mix: In comparison to the solvent control, the treated cultures showed a relevant reduction of the survival rate at 7 μg/ml and above. The positive control cyclophosphamide also reduced the survival rate.


Chromosome Aberrations


JAU 64 76-Chloromethylketone without S9 mix: Biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 18 or 30 hours culture time. The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase of metaphases with aberrations and demonstrated the sensitivity of the test system.


JAU 64 76-Chloromethylketone with S9 mix: Biologically relevant and statistically significant increases of metaphases with aberrations were detected after total culture times of 18 or 30 hours. The positive control cyclophosphamide induced statistically significant and biologically relevant increases of metaphases with aberrations and demonstrated the sensitivity of the test system and the activity of the used S9 mix.

Conclusions:
Based on the results of this test, JAU 64 76-Chloromethylketone is considered to be clastogenic for mammalian cells in vitro. Since nucleus disintegration was observed at 2 μg/ml (without S9 mix) and at 8 μg/ml (with S9 mix), an indirect cytotoxicity based effect may be possible.
Executive summary:

The clastogenic potential of JAU 64 76-Chloromethylketone was evaluated in a chromosome aberration test in vitro. lnitially Chinese hamster V79 cells were exposed in the absence of S9 mix for 4 hours to concentrations of 0.5, 1, 2, 3 and 4 μg/ml of JAU 6476-Chloromethylketone. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 2, 3 and 4 μg/ml were harvested 30 hours after the beginning of the treatment. In the presence of S9 mix cells were exposed for to concentrations of 1, 3, 6, 7 and 8 μg/ml of JAU 6476 Chloromethylketone. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 6, 7 and 8 μg/ml were harvested 30 hours after the beginning of the treatment. Based on their cytotoxicity concentrations were selected for reading of metaphases. Without S9 mix cytotoxic effects were observed at 2 μg/ml and above. With S9 mix cytotoxic effects were observed at 7 μg/ml and above. Precipitation of JAU 6476 -Chloromethylketone in the medium was not observed. Therefore, concentrations of 0.5, 1 and 2 μg/ml JAU 6476-Chloromethylketone were chosen for reading in the absence of S9 mix. In the presence of S9 mix 1, 6 and 8 μg/ml of JAU 6476-Chloromethylketone were employed. All of these cultures harvested 18 hours after the beginning of the treatment were included. In addition, cultures treated in the absence of S9 mix with 2 μg/ml and harvested 30 hours after the beginning of the treatment were used. The same was true for cultures treated in the presence of S9 mix with 8 μg/ml. In the absence and in the presence of S9 mix cultures treated with JAU 6476- Chloromethylketone showed biologically relevant and statistically significant increased numbers of aberrant metaphases. The positive controls mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity of the test system and the activity of the used S9 mix. Based on this test, JAU 64 76-Chloromethylketone is considered to be clastogenic for mammalian cells in vitro. Since nucleus disintegration was observed at 2 μg/ml (without S9 mix) and at 8 μg/ml (with S9 mix), an indirect cytotoxicity based effect may be possible.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct - Dec 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
92.1 % (analytical result dated August 30, 2000); stability in vehicle (DMSO) tested and confirmed
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix
Test concentrations with justification for top dose:
Concentration range (pre-test): 50-5000 µg/plate
Concentration range (main test): 0.75 - 96 µg/plate (due to the substance’s toxicity)
Vehicle / solvent:
Solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
Sufficient evidence available in the literature (e.g. Maron and Ames, 1983) and from own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: Nitrofurantoin (TA100); 4-nitro-1,2-phenylene diamine (4-NPDA) (TA1537, T98); 2-aminoanthracene (2-AA) (w S9 mix)
Rationale for test conditions:
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 ug or 5 ul per plate were used as the highest dose. At least four additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
In experiments without S9 mix buffer was used as replacement.
Evaluation criteria:
The toxicity of the substance was assessed in two ways. The first method was a
gross appraisal of background growth on the plates for mutant determination. If a
reduction in background growth was observed, it was indicated in the tables by the
letter “b” after the mutant count. Where only a single “b”, without any other values, is
noted for a concentration, this “b” represents three plates with reduced background
growth. (The same applies to the signs “c”, “v”, “p”, “n” or “%“, which may also be
used in the tables). Secondly, a toxic effect of the substance was assumed when
there was a marked and dose-dependent reduction in the mutant count per plate,
compared to the negative controls.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 6 µg/plate)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 6 µg/plate)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 6 µg/plate)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 6 µg/plate)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 6 µg/plate)
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 2: Results as mean values without S9 mix


































































































































































































































Compound



Group



concentration



TA 1535



TA 100



TA 1537



TA 98



TA102



Test substance



6-10



0



9



93



6



27



170



0.75



10



259



4



29



272



1.5



11



388



6



26



268



3



20



588



5



29



298



6



25



894



6



39



381



12



25



1082



4



45



373



 



24



8



813



1



23



9



 



48



0



-           



0



0



-



Sodium azide



 



573



-



-



-



-



NF



 



-



241



-



-



-



4-NPDA



 



-



-



78



146



-



Cumene



 



 



-



-



-



-



367



Test substance



11-15



0



16



92



8



34



237



0.75



15



236



7



40



345



1.5



18



324



7



36



409



3



31



576



6



44



455



6



35



915



6



54



484



12



39



1095



4



51



522



 



24



30



406



2



44



253



 



48



-



0



-



-



0



Sodium azide



 



793



-



-



-



-



NF



 



-



248



 -



-



-



4-NPDA



 



-



-



83



141



-



Cumene



 



 



-



-



-



-



417



Table 3: Results as mean values with S9 mix










































































































































































Compound



Group



concentration



TA 1535



TA 100



TA 1537



TA 98



TA102



Test substance



6-10



0



9



113



9



37



242



1.5



11



231



8



43



316



3



14



312



8



54



334



6



16



486



6



47



399



12



18



875



9



75



516



24



42



1323



8



77



597



 



48



23



1240



5



87



613



 



96



-



344



-



17



379



2-AA



 



188



1481



330



1559



480



Test substance



11-15



0



13



118



9



39



298



1.5



13



209



9



36



386



3



16



266



6



44



439



6



23



488



6



62



473



12



37



901



12



72



551



24



53



1226



12



101



629



 



48



53



944



8



87



592



 



96



-



-



2



23



502



2-AA



 



239



1545



396



1529



602



 

Conclusions:
positive with metabolic activation and
positive without metabolic activation
Executive summary:

JAU 6476 Chloromethylketone was investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 ug per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. Doses up to and including 6 ug per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 96 ug per plate for assessment purposes. Evidence of mutagenic activity of JAU 6476 Chloromethylketone was seen. On Salmonella typhimurium TA 1535, TA 100, TA 98 and TA 102, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable. The lowest reproducible effective dose was 0.75 ug per plate for Salmonella typhimurium TA 100, 3 ug per plate for TA 1535 and TA 102, and 12 ug per plate for TA 98. The Salmonella/microsome test thus showed JAU 6476 Chloromethylketone to have a mutagenic effect. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In addition to the experimental Ames test an in silico prediction of genotoxicity was performed for assessing the possible DNA reactivity of the substance.


With regard to the results of the prediction models (DEREK, Leadscope, VITIC) the substance raised a concern with respect to possible mutagenicity [April 07, 2021].


















Derek Version: Derek Nexus: 6.1.0, Nexus: 2.3.0, Derek KB 2020 1.0 
Leadscope Version: 3.0.0-30,  ICH-M7: Bacterial Mutation v2 
VITIC Version: 2.6.1  

Justification for classification or non-classification

Based on the positive in vitro study results for genetic toxicity (gene mutations in bacteria and chromosomal abberations in mammalian cells) combined with a strong in silico prediction alert allocation to category 2 (H341) according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, is warranted.