2-Pentanone, 4-methyl-, reaction products with 2-(2-aminoethoxy)ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

To fulfil the genetic toxicology requirements three in-vitro tests were conducted in accordance with OECD Test Guidelines.

The results of the Bacterial Reverse Mutation Assay (OECD 471) indicated that, under the conditions of this study, the test material did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

The test substance, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells (OECD 473) in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. Under the conditions of the assay described in this report, the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

In-vitro mammalian cell gene mutation test (OECD 476) the experimental test results indicated that ZK Ketimin was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures, under the conditions and according to the criteria of the test protocol.

Overall, ZK Ketimin is considered to be negative for genotoxicity

Link to relevant study records

Reference

Reference 1

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:

Experimental starting date (first day of data collection): 26 June 2015; Experimental completion date: 24 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9.

Test concentrations with justification for top dose:

- In the initial toxicity-mutation assay:1.50/5.00/15.0/50.0/150/500/1500 and 5000 μg per plate.- In the confirmatory mutagenicity assay: 15.0/50.0/150/500/1500 and 5000 μg per plate.

Vehicle / solvent:

The vehicle used was :- Vehicle: DMSO- CAS number: 67-68-5- Supplier: Sigma-Aldrich- Lot number: BCBJ4366V- Purity: 99.99%- Expiration date: February 2018

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

diluted is DMSO

Positive control substance:

2-nitrofluorene

Remarks:

Positive control for TA 98 without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

diluted is sterile water

Positive control substance:

sodium azide

Remarks:

Positive control for TA100 and TA 1535 without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

diluted is DMSO

Positive control substance:

other: 2-aminoanthracene

Remarks:

2-Aminoanthracene is the positive control for TA98, TA100, TA1535, TA1537 and WP2 uvra with metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

diluted is DMSO

Positive control substance:

9-aminoacridine

Remarks:

Positive control for TA 1537 without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

diluted is DMSO

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control for WP2 uvra without metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).NUMBER OF REPLICATIONS:- Initial toxicity-mutation assay: In duplicate- Confirmatiry mutagenicity assay: in triplicateTreatment of Test SystemOn the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of sterile water for each 100 mL of minimal top agar. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottomagar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in each assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50.0 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance aliquot was replaced by a 50 μL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Evaluation criteria:

Evaluation of Test ResultsFor each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:Strains TA1535 and TA1537Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.Strains TA98, TA100 and WP2 uvrAData sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

Electronic Data Collection SystemsThe primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:- LIMS Labware System: Test Substance Tracking- Excel 2007 (Microsoft Corporation): Calculations- Sorcerer Colony Counter and Ames Study Manager (Perceptive Instruments): Data Collection/Table Creation- Kaye Lab Watch Monitoring system (Kaye GE): Environmental Monitoring- BRIQS: Deviation and audit reporting

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Solubility Test

DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Tester strain titer results:

Experiment	Tester strain
	TA98	TA100	TA1535	TA1537	WP2 uvra
	Titer value (x10E9 cells per ml)
B1	1.9	1.6	1.7	6.1	11.4
B2	1.4	1.2	1.1	1.6	2.2

Initial toxicity-mutation assay:

In Experiment B1 (Initial Toxicity-Mutation Assay), the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50.0 μL plating aliquot. The dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Confirmatory mutagenicity assay:

In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed.

CONCLUSION

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test material did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Historical negative and positive control values, 2014, Revertants per plate

Historical Negative and Positive Control Values 2014 Revertants per plate
Strain	Control	Activation
		None					Rat liver
TA98	Neg	16	5	5	42	6-26	24	7	5	53	10-38
	Pos	232	258	57	2691		400	165	109	1382
TA100	Neg	94	14	66	152	66-122	102	18	63	164	66-138
	Pos	681	176	213	1767		681	259	186	2793
TA1535	Neg	11	4	2	31	3-19	13	5	2	36	3-23
	Pos	586	226	16	2509		117	99	23	1060
TA1538	Neg	7	3	1	19	1-13	9	4	1	23	1-17
	Pos	411	355	32	2921		72	52	10	562
WP2 uvra	Neg	25	7	7	62	11-39	28	8	10	55	12-44
	Pos	376	123	99	1026		302	102	91	687
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Conclusions:

Interpretation of results (migrated information):negative with metabolic activationnegative without metabolic activationAll criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test material did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50.0 μL plating aliquot. The dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed.

Under the conditions of this study, the test material, was concluded to be negative in the Bacterial Reverse Mutation Assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2015 to 28 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Please see "principles of method if other than guideline" part.

Principles of method if other than guideline:

Deviation: The following deviation from the protocol occurred during the conduct of this study:Event No. 222395: The test article was evaluated in the definitive mutagenicity assay on14 August 2015 at concentrations of 296, 593, 790, 1054, 1405 and 1873 μg/mL with and without S9 based on an email directive from the Study Director on 13 August 2015. However, a protocol amendment was generated for dose selection on 14 August 2015 indicating that the second highest concentration should be 1402 μg/mL rather than 1405 μg/mL. In this assay, the top dose of 1873 μg/mL was dosed correctly. The assay needs to show maximum to little cytotoxicity, and the second highest concentration evaluated (1405 μg/mL), which was higher than the concentration selected by protocol amendment by 3 μg/mL, already showed substantial recovery both with and without S9. The results of the assay showed decreasing cytotoxicity with decreasing concentrations. Therefore, this deviation is not considered to have had an adverse impact on the integrity of the study or the conclusions derived from it.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

The test substance, ZK Ketimin, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80% (Li et al., 1987). The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN).Frozen stock cultures were tested to confirm the absence of mycoplasma contamination and for karyotpye stability.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

- Preliminary toxicity assay: 3.66/7.32/14.6/29.3/58.5/117/234/468/937 and 1873 μg/mL with and without S9.- Definitive mutagenoicity assay: 296/593/790/1054/1405 and 1873 μg/mL with and without S9.Test substance dilutions were prepared immediately before use and delivered to the test system.

Vehicle / solvent:

The vehicle used to prepare the test substance dose formulations, and also used as the vehicle control was DMSO, as indicated below:- Vehicle: DMSO- CAS number: 67-68-5- Supplier: Sigma-Aldrich- Lot number: SHBF1863V- Purity/Grade: 99,97%- Expiration date: 31 December 2017

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Remarks:

EMS is the positive control for the mutagenicity assay without S9. Benzo(a)pyrene is the positive control for themutagenicity assay with S9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumCells were treated for 5 ± 0.5 hours in the presence and absence of S9, by addition of the test and control substance formulations to the treatment medium (with or without S9, as appropriate). This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981; Li et al., 1987).Preliminary Toxicity Test for Selection of Dose LevelsCells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated (1873 μg/mL) approximated the 10 mM limit dose for this assay. Lower concentrations were prepared by 2-fold dilutions. Precipitation was assessed at the beginning and end of treatment. The pH of the cultures was measured using pH indicator strips, and was adjusted to within pH 7 ± 0.5 using 1N hydrochloric acid (HCl; Lot No. RNBD4214, Exp. Date: 31 March 2018, Supplier: Sigma-Aldrich) at concentrations ≥468 μg/mL. No pH adjustment was necessary to maintain neutral pH in the treatment medium at the remaining concentrations tested. Osmolality of the vehicle control and the highest concentration also was measured at the beginning of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival, calculated as described below.NUMBER OF REPLICATIONS: Duplicate

Evaluation criteria:

Please see any other information on materials and methods section

Statistics:

Please see any other information on materials and methods section

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Visible precipitate was observed at a concentration of 1873 μg/mL by the end of treatment only

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SOLUBILITY TEST:DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of ~500 mg/mL in the solubility test conducted at BioReliance.RANGE-FINDING/SCREENING STUDIES: ZK Ketimin was prepared in DMSO and evaluated at concentrations of 3.66, 7.32, 14.6, 29.3, 58.5, 117, 234, 468, 937 and 1873 μg/mL with and without S9. The maximum dose evaluated approximated the 10 mM limit dose for this assay. Visible precipitate was observed at a concentration of 1873 μg/mL by the end of treatment only. The pH of the cultures was adjusted at concentrations ≥468 μg/mL to maintain neutral pH, and the test substance had no adverse impact on the osmolality of the cultures [407 and 389 mmol/kg for the vehicle control and the highest concentration (1873 μg/mL), respectively]. Adjusted relative survival was 159.33 and 64.04% at concentrations of 1873 μg/mL with S9 and 937 μg/mL without S9, respectively. Adjusted relative survival approximated 0% at a concentration of 1873 μg/mL without S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results:

Definitive mutagenicity assay:

Based on the results of the preliminary toxicity assay, ZK Ketimin was evaluated in the definitive mutagenicity assay at concentrations of 296, 593, 790, 1054, 1405 and 1873 μg/mL with and without S9 (see Deviations). Visible precipitate was observed at a concentration of 1873 μg/mL by the end of treatment only. The pH of the cultures again was adjusted at concentrations ≥296 μg/mL to maintain neutral pH. The average adjusted relative survival was 87.55 and 79.66% at concentrations of 1873 μg/mL with S9 and 1405 μg/mL without S9, respectively. Cultures treated at concentrations of 296, 790, 1054, 1405 and 1873 μg/mL (with S9) and 296, 593, 790, 1054 and 1405 μg/mL (without S9) were chosen for mutant selection (cultures treated at other concentrations were excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p> 0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p< 0.01).

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

CHO/HPRT Assay Historical Control Data (2011 -2013)

	Non-activated		S9 -activated
	Solvent control	0.2 microL/mL EMS	Solvent control	4.0 microL/mL B(a)P
Mean MF	4.2	239.2	4.4	143.1
SD	3.6	144	3.9	76.2
Maximum	15.7	764.2	16.9	314.5
Minimum	0.0	12	0.0	5.8

Solvent control (culture medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor). It has been demonstrated that all of the above solvents exhibit the same mutant frequency range.

EMS Ethyl methanesulfonate

B(a)P Benzo(a)pyrene MF Mutant frequency per 106 clonable cells

SD Standard deviation

Conclusions:

Interpretation of results (migrated information):negativeThese results indicate ZK Ketimin was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures, under the conditions and according to the criteria of the test protocol.

Executive summary:

The test substance, ZK Ketimin, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

ZK Ketimin was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 3.66, 7.32, 14.6, 29.3, 58.5, 117, 234, 468, 937 and 1873 μg/mL with and without S9. The maximum dose evaluated approximated the 10 mM limit dose for this assay. Visible precipitate was observed at a concentration of 1873 μg/mL by the end of treatment only. The pH of the cultures was adjusted at concentrations ≥468 μg/mL to maintain neutral pH, and the test substance had no adverse impact on the osmolality of the cultures. Adjusted relative survival was 159.33 and 64.04% at concentrations of 1873 μg/mL with S9 and 937 μg/mL without S9, respectively. Adjusted relative survival approximated 0% at a concentration of 1873 μg/mL without S9.

Based on these results, ZK Ketimin was evaluated in the definitive mutagenicity assay at concentrations of 296, 593, 790, 1054, 1405 and 1873 μg/mL with and without S9. Visible precipitate was observed at a concentration of 1873 μg/mL by the end of treatment only. The pH of the cultures again was adjusted at concentrations ≥296 μg/mL to maintain neutral pH. The average adjusted relative survival was 87.55 and 79.66% at concentrations of 1873 μg/mL with S9 and 1405 μg/mL without S9, respectively. Cultures treated at concentrations of 296, 790, 1054, 1405 and 1873 μg/mL (with S9) and 296, 593, 790, 1054 and 1405 μg/mL (without S9) were chosen for mutant selection (cultures treated at other concentrations were excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p> 0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p< 0.01).

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

These results indicate ZK Ketimin was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures, under the conditions and according to the criteria of the test protocol.

Reference 3

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:

Experimental starting date: 26 June 2015; Experimental completion date: 22 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

See "principles of method if other than guideline"

Principles of method if other than guideline:

The following deviation from the assay-method SOPs occurred during the conduct of this study:Event 214545: During seeding of cells in the preliminary toxicity assay, the Coulter Counter used to take cell counts was still under monthly maintenance. During normal operations, the coulter counter is filled with Isoton Diluent; however, at the time of use, the counter filled with Coulter Klenz, as is the procedure during routine maintenance (monthly cleaning). The liquid (Isoton diluent or Coulter Klenz) that the counter was filled with, did not appear to have an impact on the equipment calibration. When the error was discovered a day later, the equipment was brought out monthly maintenance (i.e., filled with Isoton diluent) and calibrated. The calibration values were comparable. In addition, the baseline counts for the assay were within normal range (~5000). This suggests that the equipment functioned normally, despite being used for counts while filled with the cleaning solution. Therefore, the Study Director has concluded that this deviation did not adversely impact the integrity of the data or the validity of the study conclusion.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 metabolic activation system

Test concentrations with justification for top dose:

- Preliminary toxicity assay without exogenous metabolic activation (4-hrs treatment, 16 hrs recovery period):0,187 / 0,561 / 1,87 / 5,61 / 18,7 / 56,1 / 187 / 561 / 1870 μg/mL.- Preliminary toxicity assay with exogenous metabolic activation (4-hrs treatment, 16 hrs recovery period):0,187 / 0,561 / 1,87 / 5,61 / 18,7 / 56,1 / 187 / 561 / 1870 μg/mL.- Preliminary toxicity assay without exogenous metabolic activation (20-hrs continuous treatment):0,187 / 0,561 / 1,87 / 5,61 / 18,7 / 56,1 / 187 / 561 / 1870 μg/mL.- Chromosome aberration assay without metabolic activation, 4 hr treatment and a recovery period of 16 hrs:100 / 250 / 500 / 700 / 850 / 1000 / 1200 / 1350 / 1500 / 1700 μg/mL.- Chromosome aberration assay without metabolic activation, 20 hr treatment and a recovery period of 0 hrs:100 / 250 / 500 / 700 / 850 / 1000 / 1200 / 1350 / 1500 / 1700 μg/mL.Chromosome aberration assay with metabolic activation, 4 hr treatment and a recovery period of 16 hrs:250 / 500 / 1000 / 1500 / 1870 μg/mL.

Vehicle / solvent:

The vehicle used to was:- Vehicle: DMSO- Supplier: Sigma-Aldrich- CAS N°: 67-68-5- LotN°: BCBJ4366V- Exp. Date: Feb 2018Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO as it is used as vehicle.

True negative controls:

no

Positive controls:

yes

Remarks:

CP and MMC were dissolved in sterile water.

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin (MMC): Used as the positive control substance in the non-activated test system and Cyclophosphamide (CP): Used as positive control substance in the S9-activated test system.

Details on test system and experimental conditions:

Number of replication for preliminary assay: single culturesNumber of replication for chromosome aberration assay: duplicateNUMBER OF CELLS EVALUATED: The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990).Cytotoxicity was determined in the preliminary test for selection of dose levels (reduction in cell growth index relative to the vehicle control).

Evaluation criteria:

The test substance was considered to have induced a positive response if:• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and• the increase is concentration-related (p ≤ 0.05), and• results are outside the 95% control limit of the historical negative control data.The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Negative with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Assay

The osmolality in treatment medium was measured as follows:

Dose tested	Dose levels (μg/mL)	Osmolality (mmol/kg)
Vehicle	0	431
Highest	1870	412

The osmolality of the test substance dose level in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%. The pH of the highest dose level of test substance in treatment medium was 9.0. Therefore, in order to maintain a neutral pH in the treatment medium, pH was adjusted using 1N HCl as follows:

Dose level (μg/mL)	Original pH	Final pH	Volume of 1N HCl added (drops)
1870	9.0	7.0	7
561	8.0	7.0	2
187	7.5	-	-

Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 1870 μg/mL in the non-activated 4 and 20-hour exposure groups. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

Treatment conditions	Treatment time	Recovery time	Dose level (μg/mL)
Non-activated	4 hr	16 hr	100/250/500/700/850/1000/1200/1350/1500/1700
Non-activated	20 hr	0 hr	100/250/500/700/850/1000/1200/1350/1500/1700
S9 -activated	4 hr	16 hr	250/500/1000/1500/1870

Chromosome Aberration Assay

In the chromosome aberration assay, the test substance was soluble in DMSO and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The pH of the highest dose level of test substance in treatment medium was 9.5. Therefore, in order to maintain a neutral pH in the treatment medium, pH was adjusted using 1N HCl as follows:

Dose level (μg/mL)	Original pH	Final pH	Volume of 1N HCl added (drops)
1870	9.5	7.0	8
1700	9.0	7.0	4
1500	8.5	7.0	3
1350	8.5	7.5	2
1200	8.5	7.0	2
1000	8.0	7.5	1
850	8.0	7.0	1
700	8.0	7.0	1
500	7.5	-	-
250	7.5	-	-
100	7.0	-	-

Toxicity of the test item (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 51% at 1000 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 1000 μg/mL, was 36% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 500, 850, and 1000 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

Toxicity (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 37% at 1870 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 1870 μg/mL, was 12% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 500, 1000, and 1870 μg/mL. The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (3.7%) relative to vehicle control at 500 μg/mL (p ≤ 0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose-response (p > 0.05). In addition, the percentage of cells with structural aberrations was in line with the historical control data of 0.0% to 3.5%. Therefore, the statistically significant increase in structural aberrations was considered to be biologically irrelevant. The percentage of structurally aberrant cells in the CP (positive control) treatment group (26.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

The toxicity (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was 53% at 1000 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 1000 μg/mL, was 52% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 250, 700, and 1000 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (26.7%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

CONCLUSION

The positive and vehicle controls fulfilled the requirements for a valid test.

Under the conditions of the assay described in this report, the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Conclusions:

Interpretation of results (migrated information):negative with metabolic activationnegative without metabolic activationUnder the conditions of the assay described in this report, the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

The test substance, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dimethylsulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.187 to 1870 μg/mL. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 1870 μg/mL in the non-activated 4 and 20-hour exposure groups. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 100 to 1700 μg/mL for the non-activated 4 and 20-hour exposure groups, and from 250 to 1870 μg/mL for the S9-activated 4-hour exposure group.

In the chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 1000 μg/mL in the non-activated 4 and 20-hour exposure groups. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group. The highest dose analyzed under each treatment condition produced 55 ± 5% reduction in cell growth index relative to the vehicle control or was the highest dose tested in the chromosome aberration assay, which met the dose limit as recommended by testing guidelines for this assay.

The percentage of cells with structural aberrations in the non-activated 4 and 20-hour exposure groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (3.7%) relative to vehicle control at 500 μg/mL (p ≤ 0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose-response (p > 0.05). In addition, the percentage of cells with structural aberrations was in line with the historical control data of 0.0% to 3.5%. Therefore, the statistically significant increase in structural aberrations was considered to be biologically irrelevant.

The percentage of cells with numerical aberrations in the test substance-treated groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

Under the conditions of the assay described in this report, the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In-vivo testing not required

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).