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

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

615-768-8

Cas Number:

72480-17-2

Molecular formula:

C10 H21 N O2

IUPAC Name:

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

Test material form:

other: liquid

Method

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

Species / strain 1

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 2

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

Controls

Controls 1

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.

Controls 2

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.

Controls 3

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.

Controls 4

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.

Controls 5

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

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.