Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assey test according to OECD guideline 471 using Salmonella typhimurium and Escherichia coli, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used with and without metabolic activation.


 


Results of a gene mutation assay in mammalian cells according to OECD 476 did also not indicate mutation potential of the test substance.


 


The test item tested in a chromosome aberration assay in mammalian cells according to OECD guideline 473 up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells. Thus, the test item is considered clastogenic in this system.


Conclusion: The test item is considered not mutagenic but clastogenic.

Link to relevant study records

Referenceopen allclose all

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:
2014-03-18 to 2014-04-03
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:
adopted 21st July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. Main DNA target GC.
Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver induced by Phenobarbital (PB) and β-naphthoflavone (BNF)
Test concentrations with justification for top dose:
5000; 1581, 500, 158, 50 and 15.8 μg/plate
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Test item is good soluble in DMSO
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
E.coli and all of Salmonella strains with S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537 without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535 without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
Salmonella TA98 without S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) and pre-incubation
The Experiment I (Initial Mutation Test) of the main study the plate incorporation method was used. In the Experiment II (Confirmatory Mutation Test) the pre-incubation method was applied and the concentrations examined were the same as investigated in the Experiment I.

DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth; Toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment.
Evaluation criteria:
Evaluation of Experimental Data

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least


Conditions for the Validity of the Test

The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.
Statistics:
None
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the Confirmatory Mutation Test, the absence of revertant growth and reduced background lawn development was obtained in S. typhimurium TA1537 at 5000 μg/plate (-S9 Mix).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the Confirmatory Mutation Test, the absence of revertant growth and reduced background lawn development was obtained in S. typhimurium TA1535 at 5000 μg/plate (-S9 Mix) and in S. typhimurium TA1535 at 1581 μg/plate (-S9 Mix).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment for Toxicity

The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the toxicity test the concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
The obtained revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA100, in the whole examined concentration range of 5000-5 μg/plate, with addition of metabolic activation (+S9 Mix).
Slightly higher revertant colony counts were obtained in S. typhimurium TA98 at the concentrations of 50 μg/plate, with addition of metabolic activation (+S9 Mix) and in TA100 in the concentration range of 5000-5 μg/plate, without metabolic activation (-S9 Mix).

COMPARISON WITH HISTORICAL CONTROL DATA: The obtained changes, slightly lower or higher revertant counts remained in the corresponding historical control data and biological variability range of the applied test system.

Table 1: Summary of results of initial mutation test


 

















































































































































































































































































































































































































Initial Mutation Test (Plate Incorporation Test)



Concentrations (mg/plate)



Salmonella typhimuriumtester strains



Escherichia coli



TA 98



TA 100



TA 1535



TA 1537



WP2uvrA



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean values of revertants per plate Mutation rate (MR)



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Untreated Control



13.7



0.84



23.7



1.09



87.3



1.10



135.3



1.24



5.7



0.74



10.7



1.03



8.7



1.00



7.0



0.91



25.3



1.01



25.7



0.94



DMSO Control



16.3



1.00



21.7



1.00



79.7



1.00



109.0



1.00



7.7



1.00



10.3



1.00



8.7



1.00



7.7



1.00



25.0



1.00



27.3



1.00



Ultrapure Water Control











90.3



1.00







7.0



1.00















30.0



1.00







5000



11.3



0.69



21.0



0.97



114.0



1.43



132.7



1.22



10.0



1.30



9.3



0.90



12.3



1.42



10.3



1.35



20.7



0.83



26.3



0.96



1581



13.7



0.84



19.3



0.89



102.7



1.29



124.0



1.14



7.3



0.96



12.3



1.19



7.3



0.85



9.7



1.26



25.3



1.01



28.0



1.02



500



15.3



0.94



20.0



0.92



88.0



1.10



122.0



1.12



8.7



1.13



11.3



1.10



8.0



0.92



9.0



1.17



21.7



0.87



29.0



1.06



158



13.0



0.80



22.3



1.03



85.0



1.07



117.0



1.07



6.0



0.78



9.0



0.87



7.0



0.81



7.0



0.91



25.7



1.03



33.0



1.21



50



11.3



0.69



21.0



0.97



90.0



1.13



120.7



1.11



7.3



0.96



9.0



0.87



9.0



1.04



6.3



0.83



23.0



0.92



29.7



1.09



15.8



13.3



0.82



22.0



1.02



87.7



1.10



114.0



1.05



6.3



0.83



10.3



1.00



8.7



1.00



8.7



1.13



27.7



1.11



26.0



0.95



NPD (4mg)



173.3



10.61







































SAZ (2mg)











948.0



10.49







729.3



104.19























9AA (50mg)



























452.7



52.23















MMS (2mL)



































826.0



27.53







2AA (2mg)







1725.3



79.63







1856.0



17.03







209.3



20.26







175.0



22.83











2AA (50mg)







































284.3



10.40



 


Table 2: Summary of results of confirmatory mutation test


 

















































































































































































































































































































































































































Confirmatory Mutation Test (Pre-Incubation Test)



Concentrations (mg/plate)



Salmonella typhimuriumtester strains



Escherichia coli



TA 98



TA 100



TA 1535



TA 1537



WP2uvrA



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean values of revertants per plate Mutation rate (MR)



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Untreated Control



16.7



1.09



23.3



1.06



108.7



1.32



139.3



1.23



9.3



0.93



17.7



1.20



6.3



0.79



7.3



0.79



31.3



1.12



42.0



1.09



DMSO Control



15.3



1.00



22.0



1.00



82.3



1.00



113.7



1.00



10.0



1.00



14.7



1.00



8.0



1.00



9.3



1.00



28.0



1.00



38.7



1.00



Ultrapure Water Control











78.7



1.00







9.3



1.00















40.0



1.00







5000



5.3



0.35



14.3



0.65



44.7



0.54



100.0



0.88



0.0



0.00



12.7



0.86



0.0



0.00



11.3



1.21



23.3



0.83



29.7



0.77



1581



13.0



0.85



20.7



0.94



82.7



1.00



112.7



0.99



5.7



0.57



14.3



0.98



7.3



0.92



9.0



0.96



32.7



1.17



39.7



1.03



500



13.7



0.89



20.0



0.91



95.3



1.16



114.3



1.01



10.3



1.03



13.0



0.89



6.3



0.79



7.0



0.75



35.0



1.25



43.7



1.13



158



14.3



0.93



23.0



1.05



89.7



1.09



115.7



1.02



10.0



1.00



11.0



0.75



6.7



0.83



8.3



0.89



31.3



1.12



41.0



1.06



50



13.7



0.89



20.3



0.92



84.7



1.03



127.3



1.12



10.3



1.03



13.3



0.91



7.7



0.96



7.0



0.75



28.0



1.00



43.3



1.12



15.8



14.3



0.93



24.3



1.11



87.3



1.06



122.3



1.08



11.7



1.17



14.0



0.95



8.7



1.08



8.0



0.86



24.0



0.86



40.0



1.03



NPD (4mg)



178.0



11.61







































SAZ (2mg)











1230.0



15.64







497.3



53.29























9AA (50mg)



























365.0



45.63















MMS (2mL)



































1096.0



27.40







2AA (2mg)







709.3



32.24







1310.7



11.53







120.3



8.20







111.7



11.96











2AA (50mg)







































261.7



6.77



 


Table 3: Historical control data for Revertants/Plate (for the period of 2008-2013)


































































































































































































































































































































































 



Bacterial strains



Historical control data of untreated control



‑S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



22.6



110.1



10.1



7.5



24.7



SD



3.3



31.0



1.4



2.7



4.2



Minimum



11



66



2



2



11



Maximum



40



162



23



19



41



+S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



29.6



117.7



12.0



8.7



34.2



SD



3.6



22.9



1.6



2.3



3.5



Minimum



13



76



4



2



20



Maximum



48



170



24



20



54



 



Bacterial strains



Historical control data of DMSO


control



‑S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



22.1



106.0



9.9



7.4



23.8



SD



3.1



29.3



1.2



2.8



3.5



Minimum



11



66



3



2



10



Maximum



40



156



23



18



42



+S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



28.6



117.4



12.1



8.4



34.2



SD



3.4



22.1



1.6



2.2



3.8



Minimum



17



73



3



2



17



Maximum



49



168



25



18



55



 



Bacterial strains



Historical control data of Water


control



‑S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



23.8



110.3



9.9



6.5



25.2



SD



6.8



30.9



1.4



3.4



3.8



Minimum



9



70



2



1



12



Maximum



43



159



24



16



45



+S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



29.8



118.8



11.8



7.8



35.1



SD



7.4



23.0



1.5



3.4



4.9



Minimum



10



81



3



2



19



Maximum



48



174



24



20



55



 



Bacterial strains



Historical control data of positive controls



‑S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



199.6



904.8



792.5



457.5



700.3



SD



35.1



107.9



106.0



111.8



58.9



Minimum



165



477



332



110



341



Maximum



248



1953



1278



1439



1236



+S9



 



TA98



TA100



TA1535



TA1537



E. coli



Average



199.6



1340.5



173.3



152.1



280.8



SD



35.1



347.7



37.6



23.3



80.0



Minimum



248



491



87



68



144



Maximum



165



2869



603



465



520


Conclusions:
The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with the test item was conducted according to the OECD guideline 471 and GLP. The test item was suspended respectively dissolved in dimethyl sulfoxide (DMSO). Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential. in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate. The tested test item concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.


 


In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study were fulfilled.


 


No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. Slight, unequivocal inhibitory effect of the test item was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in the examined Salmonella typhimurium strains at the concentration of 5000 μg/plate in absence of exogenous metabolic activation (-S9 Mix).


 


The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-14 to 2017-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline"
Principles of method if other than guideline:
There are deviations from the guidelines regarding the confirmation of negative results and the maximum recommended concentration.
Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).
The maximum recommended concentration was 2000 μg/mL, according to current Guideline (OECD 476, 29 July 2016) instead of 5000 μg/mL.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)

MEDIA USED
- Type and identity of media:
Ham's F12 medium (F12-10)
supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).
During the 5 hour treatment with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL of 6-thioguanine (6-TG)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
without S9: 125, 250, 500, 1000, 2000, µg/mL (Solvent (DMSO) 10 µL/mL; positive control 1.0 µL/mL)
with S9: 125, 250, 500, 1000, 2000, µg/mL (Solvent (DMSO) 10 µL/mL; positive control 20.0 µL/mL)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5x10^6 cells per dish

DURATION
- Preincubation period: 24 h
- Exposure duration: 5 h
- Expression time: 19 h
- Selection time: 8 d

SELECTION AGENT: hypoxanthine Ham's (F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency; relative total growth
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (based 95% control limit).
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one- way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 (1000 and 2000 µg/mL); with S9 (250, 500, 1000 and 2000 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: Bone
- Evaporation from medium: No
- Precipitation: Not observed

RANGE-FINDING/SCREENING STUDIES: Yes

HISTORICAL CONTROL DATA
Please refer to "any other information including tables"

Table 1: Main mutation assay summary of results with S9












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control a



202.0



±



2.0



100



100



0



2



2



2



0



6



100



6.00



Pos. control
(EMS 1.0 µL/mL) a



55.3



±



3.21



27



63



202



198



188



211



202



1001



63



1588.89**



TEST ITEM



 



125g/mL a



198.7



±



1.53



98



99



1



1



2



0



0



4



99



4.04



250g/mL a



199.3



±



1.53



99



99



1



0



1



2



1



5



99



5.05



500g/mL a



199.3



±



3.51



99



98



2



1



0



1



1



5



98



5.10



1000g/mL a



181.0



±



2.00



90



99



1



1



1



2



1



6



99



6.06



2000g/mL a



149.3



±



2.08



74



98



2



2



1



1



0



6



98



6.12



 


 












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control b



201.0



±



2.0



100



100



4



0



0



1



1



6



100



6.00



Pos. control
(EMS 1.0 µL/mL) b



55.7



±



1.15



28



63



194



199



196



189



209



987



63



1566.67**



TEST ITEM



 



125g/mL b



199.3



±



1.15



99



99



2



1



1



1



0



5



99



5.05



250g/mL b



199.7



±



2.08



99



99



1



0



4



2



0



7



99



7.07



500g/mL b



199.0



±



2.00



99



99



2



2



2



0



0



6



99



6.06



1000g/mL b



182.3



±



2.08



91



98



0



1



0



1



2



4



99



4.04



2000g/mL b



149.7



±



1.53



74



98



1



1



1



2



0



5



99



5.05



 












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control c



201.0



±



2.00



100



100



0



1



2



2



2



7



100



7.00



Pos. control
(EMS 1.0 µL/mL) c



50.3



±



1.15



25



65



189



193



197



201



189



969



65



1490.77**



TEST ITEM



 



125g/mL c



197.3



±



2.31



98



100



1



1



3



0



0



5



100



5.00



250g/mL c



200.3



±



1.53



100



98



1



1



2



1



0



5



98



5.10



500g/mL c



196.0



±



1.00



98



99



3



2



0



1



0



6



99



6.06



1000g/mL c



186.3



±



2.08



93



98



1



1



1



2



2



7



98



7.14



2000g/mL c



151.3



±



4.04



75



98



1



0



0



3



2



6



98



6.12



 


 












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control d



200.7



±



1.15



100



100



1



3



0



1



1



6



100



6.00



Pos. control
(EMS 1.0 µL/mL) d



53.3



±



1.53



27



64



196



192



194



187



184



953



64



1489.06**



TEST ITEM



 



125g/mL d



198.0



±



1.73



99



100



3



0



2



0



1



6



100



6.00



250g/mL d



199.7



±



1.53



100



99



0



2



1



1



0



4



98



4.08



500g/mL d



198.0



±



1.00



99



99



0



1



4



0



1



6



99



6.06



1000g/mL d



186.7



±



2.31



93



99



1



2



0



1



1



5



98



5.10



2000g/mL d



151.7



±



2.89



76



99



1



0



1



2



2



6



98



6.12



 


a = parallel of first culture 


b = parallel of first culture.


c = parallel of second culture.


d = parallel of second culture.


 


abs.C.E. = Absolute Cloning Efficiency


EMS= Ethyl methanesulfonate


** = p < 0.01 to the concurrent negative control and to the historical control


 


 


 


Table 2: Main mutation assay summary of results without S9


 












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control a



199.7



±



0.58



100



100



1



1



2



1



1



6



100



6.00



Pos. control
(DMBA 20 µg/mL) a



123.3



±



2.08



62



69



104



109



114



112



105



544



69



788.41**



TEST ITEM



 



125g/mL a



191.3



±



1.53



96



98



1



1



2



2



1



7



97



7.22



250g/mL a



178.7



±



1.53



89



99



1



1



2



1



0



5



99



5.05



500g/mL a



169.0



±



1.73



85



98



1



2



3



0



2



8



97



8.25



1000g/mL a



140.7



±



1.15



70



98



0



1



2



1



2



6



98



6.12



2000g/mL a



119.0



±



1.00



60



97



1



1



3



1



2



8



97



8.25



 


 












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control b



200.0



±



1.73



100



100



2



0



2



1



2



7



100



7.00



Pos. control
(DMBA 20 µg/mL) b



123.7



±



1.53



62



70



122



102



110



100



107



541



70



772.86**



TEST ITEM



 



125g/mL b



193.0



±



1.00



97



98



1



0



3



0



2



6



98



6.12



250g/mL b



180.3



±



1.53



90



98



1



1



2



1



1



6



98



6.12



500g/mL b



170.3



±



2.08



85



98



2



1



1



1



1



6



98



6.12



1000g/mL b



142.3



±



0.58



71



99



1



0



2



3



2



8



99



8.08



2000g/mL b



121.0



±



1.00



61



98



1



2



2



1



1



7



98



7.14



 


 












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control c



201.7



±



1.53



100



100



3



0



1



1



2



7



100



7.00



Pos. control
(DMBA 20 µg/mL) c



122.7



±



0.58



61



71



100



105



102



117



109



533



70



761.43**



TEST ITEM



 



125g/mL c



194.0



±



3.61



96



98



1



2



1



1



0



5



98



5.10



250g/mL c



178.7



±



1.53



89



99



1



2



1



1



1



6



99



6.06



500g/mL c



169.7



±



0.58



84



98



1



2



2



1



2



8



98



8.16



1000g/mL c



142.7



±



1.15



71



98



1



1



2



2



0



6



98



6.12



2000g/mL c



123.0



±



1.00



61



98



3



1



1



2



1



8



97



8.25



 


 












































































































































NON
ACTIVATION
TEST
CONDITION



SURVIVAL TO TREATMENT



REL. POPU-
LATION
GROWTH (%)
OF CONTROL



MUTANT COLONIES
DISH NUMBER



TOTAL
MUTANT
COLONIES



ABSOLUTE
C.E.
%



MUTANT
FREQ.
X 10-6



MEAN COLONY
NUMBERS.D.



PERCENT
VEH. CONTROL



1



2



3



4



5



Solvent control d



201.3



±



2.08



100



100



2



0



1



4



0



7



100



7.00



Pos. control
(DMBA 20 µg/mL) d



121.7



±



1.53



60



71



104



116



112



108



112



552



70



788.57**



TEST ITEM



 



125g/mL d



194.7



±



4.16



97



99



1



1



1



0



3



6



98



6.12



250g/mL d



180.3



±



1.53



90



98



1



1



1



1



2



6



98



6.12



500g/mL d



171.0



±



1.73



85



98



1



3



1



0



0



5



98



5.10



1000g/mL d



144.0



±



1.00



72



99



2



1



3



0



1



7



98



7.14



2000g/mL d



124.0



±



1.00



62



98



2



0



0



2



1



5



98



5.10



 


 


Table 3: historical control data of solvent control (2015 – 2016)


 













































 



Without S9 mix



With S9 mix



5-hour treatment



5-hour treatment



Mean



6.23



6.55



SD



0.57



0.89



Range



4.90 - 8.82



4.12 – 11.76



Lower 95% confidence interval



4.94



4.53



Upper 95% confidence interval



7.53



8.57



n



10



10



 


 


 


Table 4: historical control data of positive control













































 



Without S9 mix


EMS



With S9 mix


DMBA



5-hour treatment



5-hour treatment



Mean



1526.96



755.62



SD



27.21



15.16



Range



1357.81 – 1636.92



690.00 - 810.29



Lower 95% confidence interval



1465.41



721.32



Upper 95% confidence interval



1588.50



789.92



n



10



10



EMS    =         Ethyl methanesulphonate


DMBA =         7,12-Dimethylbenzanthracene


SD      =         Standard deviation


n         =         number of experiments


Range: minimum value-maximum value


 

Conclusions:
The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells. Thus, the test item was not mutagenic under the conditions of this study.
Executive summary:

The test item, dissolved in Dimethyl sulfoxide (DMSO), was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476 and GLP. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.


5-hour treatment period without S9-mix:


125, 250, 500, 1000 and 2000 μg/mL


5-hour treatment period with S9-mix:


125, 250, 500, 1000 and 2000 μg/mL


 


In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)).


Phenotypic expression was evaluated up to 8 days following exposure.


In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.


There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.


The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data.


The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to maximum recommended concentration.


Thus, the test item was not mutagenic under the conditions of this study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-09 to 2017-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Lot. No.: 10H016
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations.
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Modal number of chromosomes: 2n = 22

MEDIA USED
- Type and identity of media: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated fetal bovine serum (final concentration 10 %).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Experiment A
with S9: 125, 250, 1000 µg/mL (solvent control 10 µL/mL; positive control 5 µg/mL)
without S9: 125, 250, 500 µg/mL (solvent control 5 µL/mL; positive control 1 µg/mL)

Experiment B
with S9: 125, 250, 500, 1000 µg/mL (solvent control 10 µL/mL; positive control 5 µg/mL)
without S9: 31.3, 62.5, 125.0 µg/mL (solvent control 1.25 µL/mL; positive control 0.4 µg/mL)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10^5 cells per replicate

DURATION
- Exposure duration: Experiment A: 3h; experiment B: 3h (with S9); 20 h without S9
- Expression time (cells in growth medium): 20h

SPINDLE INHIBITOR: colchicine (0.2 μg/mL)

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300

DETERMINATION OF CYTOTOXICITY
- Method: Relative increase in cell count (RICC)

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Statistics:
For statistical analysis CHI^2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RICC < 50 at 500 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
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:
RICC < 50 at 1000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RICC < 50 at 125 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: No
- Precipitation: Not observed

RANGE-FINDING/SCREENING STUDIES: Yes

HISTORICAL CONTROL DATA: Please refer to "Any other information on results incl. tables"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC, when cytokinesis block is not used

Table 1: Mean percentage of cells with structural chromosome aberrations experiment A















































































































Concentration
(µg/mL)



S9 mix



Treatment
time



Harvesting time



Mean aberrant cells/150 cells



incl. gaps



excl. gaps



Solvent control
(DMSO)



-



3 h



20 h



6



3



Test item



125 µg/mL



-



3 h



20 h



7



3



250 µg/mL



-



3 h



20 h



8



5



500 µg/mL



-



3 h



20 h



9



5



Pos. Control (EMS)



-



3 h



20 h



35**



26**



Solvent control
(DMSO)



+



3 h



20 h



6



3



Test item



125 µg/mL



+



3 h



20 h



9



3



250 µg/mL



+



3 h



20 h



8



3



500 µg/mL



+



3 h



20 h



11



7



1000 µg/mL



+



3 h



20 h



18*



12*



Pos. Control (Cycl.)



+



3 h



20 h



49**



40**



 


 


Table 2: mean percentage of cells with structural chromosome aberrations experiment B







































































































Concentration
(µg/mL)



S9 mix



Treatment
time



Harvesting time



Mean aberrant cells/150cells



incl. gaps



excl. gaps



Solvent control
(DMSO)



-



20 h



20 h



6



3



Test item



31.3 µg/mL



-



20 h



20 h



8



4



62.5 µg/mL



-



20 h



20 h



8



3



125 µg/mL



-



20 h



20 h



8



5



Pos. Control (EMS)



-



20 h



20 h



47**



38**



Solvent control
(DMSO)



-



20 h



28 h



7



3



Test item



31.3 µg/mL



-



20 h



28 h



8



4



62.5 µg/mL



-



20 h



28 h



6



3



125 µg/mL



-



20 h



28 h



12



8



Pos. Control (EMS)



-



20 h



28 h



42**



35**



Positive control (-S9): Ethyl methanesulfonate (1.0L/mL)


Positive control (+S9): Cyclophosphamide (5.0g/mL)


*: p<0.05


**: p<0.01


 


 


Table 3: mean percentage of cells with structural chromosome aberrations experiment B




































































Concentration
(µg/mL)



S9 mix



Treatment
time



Harvesting time



Mean aberrant cells/150 cells



incl. gaps



excl. gaps



Solvent control
(DMSO)



+



3 h



28 h



6



3



Test item



125 µg/mL



+



3 h



28 h



10



5



250 µg/mL



+



3 h



28 h



9



4



500 µg/mL



+



3 h



28 h



16*



7



1000 µg/mL



+



3 h



28 h



16*



12*



Pos. Control (Cycl.)



+



3 h



28 h



47**



40**



 


 


 


Historical control data


 


Table 4: 3h/20h treatment/sampling time without S9-mix


 























































 



number of aberrant cells/ 150 cells



negative control



positive control
(Ethyl methanesulfonate)



incl. Gaps



excl. Gaps



incl. Gaps



excl. Gaps



Mean



5.70



2.56



40.20



30.85



SD



0.63



0.59



3.83



3.41



Lower confidence interval



4.27



1.31



31.53



23.13



Upper confidence interval



7.13



3.99



48.87



38.57



n



10



10



10



10



 


Table 5: 3h/20h treatment/sampling time with S9-mix























































 



number of aberrant cells/ 150 cells



negative control



positive control
(Cyclophosphamide)



incl. Gaps



excl. Gaps



incl. Gaps



excl. Gaps



Mean



5.57



2.80



46.40



39.65



SD



0.74



0.55



2.10



1.83



Lower confidence interval



4.07



1.56



41.66



35.51



Upper confidence interval



7.43



4.04



51.14



43.79



n



10



10



10



10



 


 


Table 6: 20h/20h treatment/sampling time without S9-mix


 























































 



number of aberrant cells/150 cells



negative control



positive control
(Ethyl methanesulfonate)



incl. Gaps



excl. Gaps



incl. Gaps



excl. Gaps



Mean



5.70



2.85



45.20



37.85



SD



1.00



0.59



2.32



2.13



Lower confidence interval



3.44



1.51



39.94



33.02



Upper confidence interval



7.96



4.19



50.46



42.68



n



10



10



10



10



 


 


Table 7: 20h/28h treatment/sampling time without S9-mix























































 



number of aberrant cells/ 150cells



negative control



positive control
(Ethyl methanesulfonate)



incl. Gaps



excl. Gaps



incl. Gaps



excl. Gaps



Mean



5.41



2.65



45.95



37.59



SD



0.65



0.60



1.70



1.92



Lower confidence interval



3.93



1.30



42.11



33.23



Upper confidence interval



6.89



4.00



49.79



41.94



n



10



10



10



10



 


Table 8: 3h/28h treatment/sampling time with S9-mix























































 



number of aberrant cells/ 150 cells



negative control



positive control
(Cyclophosphamide)



incl. Gaps



excl. Gaps



incl. Gaps



excl. Gaps



Mean



5.45



2.75



45.90



38.90



SD



0.67



0.59



2.07



3.29



Lower confidence interval



3.93



1.41



41.21



31.47



Upper confidence interval



6.97



4.09



50.59



46.33



n



10



10



10



10



 


 

Conclusions:
The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.
Thus, the test item is considered clastogenic in this system.
Executive summary:

The test item was tested in a Chromosome Aberration Assay in V79 cells according to OECD guideline 473 and GLP. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:


 


Experiment A with 3/20 h treatment/sampling time


without S9 mix: 125, 250 and 500 μg/mL


with S9 mix: 125, 250, 500 and 1000 μg/mL


Experiment B with 20/20 h treatment/sampling time


without S9 mix: 31.3, 62.5 and 125 μg/mL


Experiment B with 20/28 h treatment/sampling time


without S9 mix: 31.3, 62.5 and 125 μg/mL


Experiment B with 3/28 h treatment/sampling time


with S9 mix: 125, 250, 500 and 1000 μg/mL


 


In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, in the absence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.


In Experiment A, biologically significant increase in the number of cells showing structural chromosome aberrations at the cytotoxic concentration of 1000 μg/mL, in the presence of metabolic activation. There were statistical differences between treatment and concurrent solvent and historical control groups, too. At concentrations of 500 and 1000 μg/mL dose-response relationships were noted in the number of cells showing structural chromosome aberrations and endoreduplication.


 


In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. A 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused biologically and statistically significant increase in the number of cells showing structural chromosome aberrations and endoreduplication at concentrations of 1000 μg/mL. In the number of cells showing structural chromosome aberrations and endoreduplication a dose-response relationships were observed at concentrations of 500 and 1000 μg/mL.


 


There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.


The validity of the test was shown as the concurrent positive controls Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations and were compatible with the historical control range.


 


The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.


The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.


Thus, the test item is considered clastogenic in this system. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In an in vivo erythrocyte micronucleus assay according to OECD guideline 474, the test item did not induce micronuclei in bone marrow cells of the mouse. Therefore, the test item is considered to be non-clastogenic under the experimental conditions reported.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-11-24 to 2021-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The NMRI mouse is a recommended test system for the Mammalian Erythrocyte Micronucleus Test
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc.
- Age at study initiation: 6 – 10 weeks
- Weight at study initiation: 30.4 - 36.7 g
- Assigned to test groups randomly: Yes
- Housing: Singly in Makrolon Type II / III cages with wire mesh top and granulated soft wood bedding
- Diet: Ad libitum (certified 2018C Teklad Global 18% protein rodent diet)
- Water: Ad libitum (tap water)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 45-65
- Air changes (per hr): At least 8
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 2 (positive control animals) or from day 1 to day 3 (negative control and treated animals)
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals and its ability to formulate a suitable dosing preparation since the test item is not stable in water.
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The preparations were made freshly before each dosing occasion.
Duration of treatment / exposure:
Animals used for micronuclei evaluation: 48 hours (2 applications at an interval of 24 hours + 24 hours post treatment period)

Animals used for plasma sampling: 25 hours and 28 hours (2 applications at an interval of 24 hours + 1 hour or 4 hours post treatment period)
Frequency of treatment:
Animals were treated twice (test item treatment + negative control) at an interval of 24 hours or once (positive control)
Post exposure period:
24 hours
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle
No. of animals per sex per dose:
6 animals per sex per dose for micronucleus evaluation and 6 high dose animals (3 animals 1 hour after last application and 3 animals 4 hours after last substance application) and 3 vehicle animals for plasma sampling
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Oral (gavage)
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose was selected on the basis of a pre-experiment with two animals per sex at a dose level of 2000 mg/kg bw under identical conditions as in the mutagenicity study concerning animal strain, vehicle, route, frequency, and volume of administration. As no deaths or severe suffering occured, 2000 mg/kg bw was selected as the highest concentration recommended in the guideline.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei. To describe a cytotoxic effect, the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides. All animals per test group were evaluated as described.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test substance is classified as positive in the assay if
a) at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control;
b) this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test and
c) any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95 % control limits).

Providing that all acceptability criteria are fulfilled, a test substance is considered clearly negative in the assay if:
a) none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control;
b) there is no dose-related increase at any sampling time when evaluated by an appropriate trend test;
c) all results are inside the distribution of the historical negative control data (e.g., Poisson-based 95 % control limits), and
d) bone marrow exposure to the test substance(s) occurred.

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations.
Statistics:
Statistical methods (as appropriate, Mann-Whitney Test, linear regression analysis) were used as an aid in evaluating the results.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 2000 mg/kg bw per application
- Solubility: Soluble in corn oil at all tested concentrations
- Clinical signs of toxicity in test animals: One animal showed partially closed eyes 2-4 hours after the first treatment. Two animals showed piloerection 2-4 hours after the first treatment. The animals did not express any clinical symptoms after the second treatment.
- Evidence of cytotoxicity in tissue analysed: The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item did not have any cytotoxic properties in the bone marrow.
- Rationale for exposure: The maximum recommended dose outlined in OECD guideline 474 was used in the pre-experiment as no severe toxicity was expected based on information from the acute oral toxicity and the repeated dose toxicity studies.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: See "Any other information on results incl. tables"
- Ratio of PCE/NCE: See "Any other information on results incl. tables"
- Appropriateness of dose levels and route: The dose levels were chosen according to the maximum recommended dose outlined in OECD guideline 474. This dose induced no severe suffering or deaths in the pre-experiment and was thus considered well tolerated in the main experiment. The substance was considered to have the highest bioavailability via the oral route as the substance rapidly hydrolyses in contact with aqueous solutions (e.g. stomach fluid) and its hydrolysis products are thus considered to become bioavailable via the oral route.
- Statistical evaluation: See "Any other information on results incl. tables"

Table 1: Summary of micronucleus test results











































































Test
Group



Dose
mg/kg
b.w.



Sampling
time
(after 2nd application)



Mean MN/4000 PCE



SD  MN/4000 PCE



Range



Ratio
 PCE /total Ery



% ratio
Vehicle



min



max



Vehicle



0



24



7.0



3.1



2



11



0.732



100.00



Dose 1



500



24



4.5



2.3



1



8



0.750



102.46



Dose 2



1000



24



5.7



1.8



3



8



0.714



97.54



Dose 3



2000



24



5.7



2.4



3



10



0.716



97.81



Positive



40



24



115.7



32.9



61



144



0.729



99.59



 


 Table 2: Biometry. Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw. The Holm-Bonferroni Adjustment method was used to correct for the Familiywise error rate of multiple comparisons.




































Negative control versus test group



Significance



p



p adjusted



500 mg test item/kg b.w.; 24 h



-



0.139



0.417



1000 mg test item/kg b.w.; 24 h



-



0.325



0.650



2000 mg test item/kg b.w.; 24 h



-



0.418



0.650



Positive Control - 40 mg CPA/kg b.w.; 24 h



+



0.005



0.020



-      =     not significant
+     =     significant


Furthermore, a linear regression analysis was performed (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) in order to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group.


A trend was judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.7372 was obtained, demonstrating that there was no dose dependent increase of mean micronuclei values.


 


Table 3: Content of the test item in dose formulation samples (corn oil)































































Sample ID


 



Nominal test concentration


[mg/mL]



Timing



Test item


analysed
[mg/mL]



% of nominal test concentration


[%]



1 a



Control



After the
first application



n.d.



n.a.



2 a



50



45.2



90



3 a



100



94.8



95



4 a



200



185



93



5 a



Control



After the second application



n.d.



n.a.



6 a



50



45.8



92



7 a



100



94.2



94



8 a



200



188



94



n.d.: not detectable (< 7.5 mg/mL); n.a.: not applicable


 


Table 4: Contents of the hydrolysis products 2,2-dimethyl-3-oxopropyl acetate and 3-aminomethyl-3,5,5-trimethylcyclohexylamine in blood plasma































































Sample ID *


 



Timing



Treatment

[mg/kg]



2,2-dimethyl-3-oxopropyl acetate analysed
[mg/L]



3-aminomethyl-3,5,5-trimethylcyclohexylamine analysed
[mg/L]



P-1 a/b



1 h after second treatment



Control (Vehicle)



n.d.



n.d.



P-2 a/b



n.d.



n.d.



P-3 a/b



n.d.



n.d.



P-4 a/b



2000 mg/kg



n.d.



< LOQ



P-5 a/b



n.d.



< LOQ



P-6 a/b



n.d.



< LOQ



P-7 a/b



4 h after second treatment



2000 mg/kg



n.d.



14.5



P-8 a/b



n.d.



9.02



P-9 a/b



n.d.



< LOQ



* two aliquots of each sample were provided by the test facility: aliquot a was used for analysis of 2,2-dimethyl-3-oxopropyl acetate analysed, aliquot b was used for analysis of 3-aminomethyl-3,5,5-trimethylcyclohexylamine; n.d.: not detectable (< 2.4 mg/L); < LOQ: below limit of quantification (i.e. < 8 mg/L)

Conclusions:
In conclusion, in can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-genotoxic in this in vivo micronucleus assay.
Executive summary:

A study according to OECD guideline 474 and GLP was peformed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The dose volume administered orally (twice) was 10 mL/kg b.w. The administered volume of the positive control was 10 mL/kg. 24 h after the second administration of the test item, the bone marrow cells were collected for micronuclei analysis. Six males per test group were evaluated for the occurrence of micronuclei. Per animal, 4000 polychromatic erythrocytes were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item, the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated: 500, 1000, and 2000 mg/kg bw.


The highest dose (maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. The animals treated with the test item and the vehicle control as well as with the test item did not exhibit any clinical symptoms. Accuracy of dose formulations was confirmed within phase number 20I13137-01-RATX. Recoveries were between 90 and 95% of nominal concentrations. Furthermore, within the same phase, bioavailability was confirmed by analytical detection of the test item in plasma (namely, the analytes 2,2-dimethyl-3-oxopropyl acetate (CAS 16184-79-5) and 3-aminomethyl-3,5,5-trimethylcyclohexylamine (CAS 2855-13-2)). 2,2-dimethyl-3-oxopropyl acetate could not be detected in plasma at any time point, whereas 3-aminomethyl-3,5,5-trimethylcyclohexylamine was present in plasma in test item treated animals and could be quantified in two of three animals at the 4 h after the second application time point (9.02 and 14.5 mg/L, respectively). In the third animal with blood withdrawal 4 h after the second application, 3-aminomethyl-3,5,5-trimethylcyclohexylamine could also be detected, but at a level which was below the Limit of Quantification (LOQ, <8 mg/L) but above the Limit of Detection (LOD, >2.4 mg/L). In conclusion, exposure of the blood and consequently also the bone marrow could be demonstrated. At 1 h after the second application, 3-aminomethyl-3,5,5-trimethylcyclohexylamine concentration in plasma was below LOQ in the three animals sacrificed at that time point.


After treatment with the test item, the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item not exert any cytotoxic effects in the bone marrow (see table below).


Table 1: Summary of micronucleus test results











































































Test
Group



Dose
mg/kg
b.w.



Sampling
time
(after 2nd application)



Mean MN/4000 PCE



SD  MN/4000 PCE



Range



Ratio
 PCE /total Ery



% ratio
Vehicle



min



max



Vehicle



0



24



7.0



3.1



2



11



0.732



100.00



Dose 1



500



24



4.5



2.3



1



8



0.750



102.46



Dose 2



1000



24



5.7



1.8



3



8



0.714



97.54



Dose 3



2000



24



5.7



2.4



3



10



0.716



97.81



Positive



40



24



115.7



32.9



61



144



0.729



99.59



 


In comparison to the corresponding vehicle controls, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei after administration of the test item with any dose level used. The concurrent vehicle control range (2-11 MN per 4000 PCE) was well within the range of the historical control data (0-18 MN per 4000 PCE). 40 mg/kg b.w. orally administered cyclosphosphamide was used as positive control which induced a substantial and statistically significant increase in cells with micronuclei (range: 61-144 MN per 4000 PCE), which was well within the range of the historical control data (range: 36-249 MN per 4000 PCE).


In conclusion, in can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-clastogenic in this in vivo micronucleus assay.

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

Additional information

Genetic toxicity in vitro


 


Bacterial Reverse Mutation Assay


The Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with the test item was conducted according to the OECD guideline 471and GLP. The test item was suspended respectively dissolved in dimethyl sulfoxide (DMSO). Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential. in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate. The tested test item concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.


In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. Slight, unequivocal inhibitory effect of the test item was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in the examined Salmonella typhimurium strains at the concentration of 5000 μg/plate in absence of exogenous metabolic activation (-S9 Mix). The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.


 


Gene mutation in mammalian cells


The test item, dissolved in Dimethyl sulfoxide (DMSO), was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476 and GLP. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.


5-hour treatment period without S9-mix:


125, 250, 500, 1000 and 2000 μg/mL


5-hour treatment period with S9-mix:


125, 250, 500, 1000 and 2000 μg/mL


In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)).


Phenotypic expression was evaluated up to 8 days following exposure.


In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.


There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.


The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data.


The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to maximum recommended concentration.


Thus, the test item was not mutagenic under the conditions of this study.


 


In vitro mammalian chromosome aberration test


The test item was tested in a Chromosome Aberration Assay in V79 cells according to OECD guideline 473 and GLP. The test item was dissolved in DMSO and the following concentration were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:


 


Experiment A with 3/20 h treatment/sampling time


without S9 mix: 125, 250 and 500 μg/mL


with S9 mix: 125, 250, 500 and 1000 μg/mL


Experiment B with 20/20 h treatment/sampling time


without S9 mix: 31.3, 62.5 and 125 μg/mL


Experiment B with 20/28 h treatment/sampling time


without S9 mix: 31.3, 62.5 and 125 μg/mL


Experiment B with 3/28 h treatment/sampling time


with S9 mix: 125, 250, 500 and 1000 μg/mL


 


In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, in the absence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.


In Experiment A, biologically significant increase in the number of cells showing structural chromosome aberrations at the cytotoxic concentratio of 1000 μg/mL, in the presence of metabolic activation. There were statistical differences between treatment and concurrent solvent and historical control groups, too. At concentrations of 500 and 1000 μg/mL dose-response relationships were noted in the number of cells showing structural chromosome aberrations and endoreduplication.


 


In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. A 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused biologically and statistically significant increase in the number of cells showing structural chromosome aberrations and endoreduplication at concentrations of 1000 μg/mL. In the number of cells showing structural chromosome aberrations and endoreduplication a dose-response relationships were observed at concentrations of 500 and 1000 μg/mL.


 


There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.


The validity of the test was shown as the concurrent positive controls Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations and were compatible with the historical control range.


 


The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells. Thus, the test item is considered clastogenic in this system. 


 


Genetic toxicity in vivo


In vivo Mammalian Erythrocyte Micronucleus Test


A study according to OECD guideline 474 and GLP was peformed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The dose volume administered orally (twice) was 10 mL/kg b.w. The administered volume of the positive control was 10 mL/kg. 24 h after the second administration of the test item, the bone marrow cells were collected for micronuclei analysis. Six males per test group were evaluated for the occurrence of micronuclei. Per animal, 4000 polychromatic erythrocytes were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item, the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated: 500, 1000, and 2000 mg/kg bw.


The highest dose (maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. The animals treated with the test item and the vehicle control as well as with the test item did not exhibit any clinical symptoms. Accuracy of dose formulations was confirmed within phase number 20I13137-01-RATX. Recoveries were between 90 and 95% of nominal concentrations. Furthermore, within the same phase, bioavailability was confirmed by analytical detection of the test item in plasma (namely, the analytes 2,2-dimethyl-3-oxopropyl acetate (CAS 16184-79-5) and 3-aminomethyl-3,5,5-trimethylcyclohexylamine (CAS 2855-13-2)). 2,2-dimethyl-3-oxopropyl acetate could not be detected in plasma at any time point, whereas 3-aminomethyl-3,5,5-trimethylcyclohexylamine was present in plasma in test item treated animals and could be quantified in two of three animals at the 4 h after the second application time point (9.02 and 14.5 mg/L, respectively). In the third animal with blood withdrawal 4 h after the second application, 3-aminomethyl-3,5,5-trimethylcyclohexylamine could also be detected, but at a level which was below the Limit of Quantification (LOQ, <8 mg/L) but above the Limit of Detection (LOD, >2.4 mg/L). In conclusion, exposure of the blood and consequently also the bone marrow could be demonstrated. At 1 h after the second application, 3-aminomethyl-3,5,5-trimethylcyclohexylamine concentration in plasma was below LOQ in the three animals sacrificed at that time point.


After treatment with the test item, the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item not exert any cytotoxic effects in the bone marrow (see table below).


Table 1: Summary of micronucleus test results











































































Test
Group



Dose
mg/kg
b.w.



Sampling
time
(after 2nd application)



Mean MN/4000 PCE



SD  MN/4000 PCE



Range



Ratio
 PCE /total Ery



% ratio
Vehicle



min



max



Vehicle



0



24



7.0



3.1



2



11



0.732



100.00



Dose 1



500



24



4.5



2.3



1



8



0.750



102.46



Dose 2



1000



24



5.7



1.8



3



8



0.714



97.54



Dose 3



2000



24



5.7



2.4



3



10



0.716



97.81



Positive



40



24



115.7



32.9



61



144



0.729



99.59



 


In comparison to the corresponding vehicle controls, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei after administration of the test item with any dose level used. The concurrent vehicle control range (2-11 MN per 4000 PCE) was well within the range of the historical control data (0-18 MN per 4000 PCE). 40 mg/kg b.w. orally administered cyclosphosphamide was used as positive control which induced a substantial and statistically significant increase in cells with micronuclei (range: 61-144 MN per 4000 PCE), which was well within the range of the historical control data (range: 36-249 MN per 4000 PCE).


In conclusion, in can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-clastogenic in this in vivo micronucleus assay.


 


Endpoint conclusion


The test item did not indicate mutagenic potential in both in vitro bacterial reverse mutation assay and mammalian cell gene mutation assay. However, clastogenic potential was observed in an in vitro chromosome aberration assay in mammalian cells. To further elucidate the clastogenic potential of the substance, an in vivo Mammalian Erythrocyte Micronucleus Test was performed. The test was clearly negative showing that the test substance is non-clastogenic.


 


This result is also supported by physicochemical data of the test substance. The test item is known to undergo hydrolysis quickly in contact with water (please refer to IUCLID section 5.1.2). Therefore, it is likely that the hydrolysis products, 3-aminomethyl-3,5,5-trimethylcyclohexylamine and 2,2-dimethyl-3-oxopropyl acetate are mainly present in the actual assay. 3-aminomethyl-3,5,5-trimethylcyclohexylamine was demonstrated to be not clastogenic in a corresponding chromosome aberration assay in vitro as well as in a micronucleus assay in vivo. For more details please refer to REACH registration dossier of isophorone diamine.


With regard to 2,2-dimethyl-3-oxopropyl acetate, no data on the clastogenic potential is available. However, results of an available in vivo comet assay according to OECD 489 revealed a non-mutagenic activity of this hydrolysis product.


Thus, available data demonstrate that both hydrolysis products of the registered substance are of no concern in regards to genotoxicity.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for genotoxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for seventeenth time in Regulation (EU) No 2021/849.