Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study start date 20th November 2015 - Report date 18th August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

ANSC RDI 2860-477: development number for Elfamosit ACReaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

DNA polymerase A deficient

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA97a

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix activated with Aroclor 1254

Test concentrations with justification for top dose:

There was no diminution or clearing of the background lawn observed at any dose level, indicating that the test article was not cytotoxic to TA-100 at 0.001 to 5.0 µl/plate. The Study Director chose 5.0 µl/plate as the top test article concentration for the Main Assay.

Test concentrations 0.05, 0.1, 0.5, 1.0 and 5 μl/plate

Vehicle / solvent:

Identity : Tissue culture water (TCH2O), Lot #RNBD2215
Supplied by : Sigma
Date Received : 11 Sep 2014
Expiration Date : Apr 2016
Storage : Room temperature.
Description : Clear colorless liquid
Sample Preparation : Used as received





VEHICLES FOR POSITIVE CONTROLS
Identity : Tissue culture water (TCH2O), Lot #RNBD2215 (See above)



Identity : Dimethyl sulfoxide (DMSO), Lot #133317
(See Appendix B for Certificate of Analysis)
Supplied by : Fisher Scientific
Date Received : 18 Jul 2013
Expiration Date : May 2018
Storage : Room temperature.
Description : Clear colorless liquid
Sample Preparation : Used as received

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: acridine, 6-chloro-9-(3-((2-chloroethyl)amino) propyl)amino-2-methoxy, dihydrochloride (ICR- 191)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin hydrochloride

Details on test system and experimental conditions:

Preparation of the Tester Strains
Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. #26-555). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100.

The cultures were incubated at 37ºC ± 2ºC with agitation. The cultures were used after they reached the late exponential growth phase as determined by absorbance readings at 600 nm.

Exogenous Metabolic Activation
An activation buffer containing 10% S9 obtained from the livers of Aroclor 1254-treated adult Sprague Dawley® rats was prepared according to the manufacturer’s (Moltox) instructions. Each vial of Regensys B (Moltox cat# 60-201) was reconstituted with approximately 1 ml of Regensys A (Moltox cat# 60-200). The solution was transferred back into the Regensys A bottle. S9 (Moltox cat# 11-101) was then mixed with Regensys A+B to yield a 10% S9 buffer stock solution. The stock was separated into aliquots in sterile 15 ml conical tubes and refrigerated at 2-8ºC until used.


The exogenous metabolic activation mixture was added to one set of all doses – each test article concentration, vehicle control and positive control for each of the bacterial tester strains.


Treatment of the Test System
Top agar supplemented with appropriate amino acids were prepared, as 2 ml aliquots, and maintained at 45-50ºC in sterile culture tubes. Dulbecco’s Phosphate Buffered Saline (DPBS) was added to the tubes not undergoing S9 activation (i.e. without S9, or –S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, vehicle control or positive control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were gently vortexed and overlaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37ºC ± 2ºC for 48-72 hours. Plates that were not scored immediately following the incubation period were stored at 2-8ºC until scoring.



Screen
Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O). The test article was freely soluble at a concentration of 50 µl/ml. The Study Director chose TCH2O as the vehicle for the study. A cytotoxicity screen was conducted in the TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0 μl/plate) of the test article, two plates per concentration, with and without S9. A vehicle control (TCH2O) was run concurrently, with and without S9. The plates were incubated at 37ºC ± 2ºC for 48-72 hours.
Main Assay Five concentrations (0.05, 0.1, 0.5, 1.0 and 5.0 μl/plate) of the test article were tested in each of five bacterial tester strains (Escherichia coli WP2 uvrA, and Salmonella typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Two sets of culture plates were dosed per concentration (+S9 and –S9). A vehicle control and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37ºC ± 2ºC for 48-72 hours.

Revertant Colony Count
Counting of the revertants per plate was performed using an AlphaImager™ 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article. The plates were also examined visually for test article precipitate.

Independent Repeat Assay
The guidelines recommend that equivocal results be clarified by further testing, preferably using a modification of experimental conditions (e.g.concentrations tested), and that negative results need to be confirmed on a case-by-case basis. Justification should be provided when confirmation of negative results is not considered necessary. There is no recommendation of an independent repeat assay (confirmatory test) when the assay results are clearly positive.

There appeared to be a boarderline trend towards a poitive response with TA-97a. For this reason, an independent repeat assay (confirmatory test) was conducted in tester strain TA-97a, with and without S9, using test article concentrations of 0.05, 0.1, 0.5, 1.0 and 5.0 μl/plate

Rationale for test conditions:

The purpose of this study was to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 – Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 – Bacterial Reverse Mutation Test.

This assay is based on the methodology originally described by Ames, et al. (1975)1 and updated by Maron and Ames (1983)2.

Evaluation criteria:

Quality Check of the Assay
Sterility Test:
Sterile technique is required throughout the course of the study. To ascertain each component’s sterility, a small amount (1 ml) was placed on agar plates and incubated to encourage growth of any contaminating microorganisms. The plates were incubated for 48-72 hours at 37º± 2ºC and visually
observed for microbial growth.



Vehicle Control:
The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain
of bacteria was calculated and compared to in-house historical ranges.

Positive Control:
Positive control treatment for each tester strain of bacteria must result in at least a 2-fold increase of revertants over the mean vehicle control value. The effectiveness of the exogenous metabolic activation mixture was demonstrated by the positive response of the control.

Statistics:

Analysis of Data
There is not specific statistical analysis. Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality
checks of the assay were taken into account.



In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose-
related increases approaching a 2-fold increase are deemed equivocal.



A negative result is determined by the absence of a dose-related increase in all five tester strains, again
taking into account cytotoxicity of the test article as well as the quality checks of the assay.



Positive results from the bacterial reverse mutation test indicate that the substance induces point
mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or
Escherichia coli. Negative results indicate that under the test conditions, the test substance is not
mutagenic in the tested strains.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

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:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 97

Remarks:

TA-97a

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:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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:

not applicable

Positive controls validity:

valid

Test results 4

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:

not applicable

Positive controls validity:

valid

Test results 5

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:

not applicable

Positive controls validity:

valid

Additional information on results:

Criteria:
The spontaneous reversion rate should be comparable to both in-house historical data and ranges provided by the supplier.
Positive control must result in at least a 2-fold increase of revertants over vehicle control value.

TA does have mutagenecity potential if:
TA results in a concentration related increase of the number of revertants over the range tested and/or a reproducible increase at one or more concentrations in the number of revertants (at least 2-fold increase of revertants over vehicle control value) in at least one strain with or without metabolic activation.

Any other information on results incl. tables

   

Main Study Quality Control

   

Strain	Treatment (μl/plate)	S9	Mean		SD	SEM	Fold Increase over Vehicle
WP2	vehicle	+	43.5		6.8	2.8	2.3	×
WP2	10 μg 2aa	+	101.2		8.8	3.6
WP2	vehicle	–	29.3		7.7	3.1	24.3	×
WP2	2.5 μl MMS	–	710.8		22.9	9.4
TA-97a	vehicle	+	56.5		9.4	3.8	11.2	×
TA-97a	10 μg 2aa	+	633.7		48.9	20.0
TA-97a	vehicle	–	53.7		4.1	1.7	13.8	×
TA-97a	1 μg ICR191	–	738.5		98.5	40.2
TA-1535	vehicle	+	12.2		2.3	0.9	15.4	×
TA-1535	10 μg 2aa	+	187.8		15.5	6.3
TA-1535	vehicle	–	13.8		5.2	2.1	33.9	×
TA-1535	1.5 μg sodium azide	–	467.2		21.3	8.7
TA-98	vehicle	+	17.8		4.6	1.9	164.2	×
TA-98	10 μg 2aa	+	2923.0		77.1	31.5
TA-98	vehicle	–	15.0		3.5	1.4	53.2	×
TA-98	6 μg daunomycin	–	797.7		38.1	15.6
TA-100	vehicle	+	106.5		8.1	3.3	23.1	×
TA-100	10 μg 2aa	+	2464.2		149.1	60.9
TA-100	vehicle	–	95.8		15.9	6.5	5.0	×
TA-100	1.5 μg sodium azide	–	474.3		15.1	6.1

 

* = >2       + = presence           - = absence

 

Main study testing of 2680 -47

 

		Revertant Colonies
	Conc (μg/plate)	Mean		SD		Fold Increase over Vehicle
Strain		+ S9	-S9	+ S9	-S9	+ S9		-S9
Veh	vehicle	43.5	29.3	6.8	7.7
WP2	0.05	47	31	12.5	3.5	1.1		1.1	
	0.1	48.3	29.3	2.1	6.7	1.1		1.0	
	0.5	38	33.7	7.0	1.2	0.9		1.2	
	1	41.7	23.7	5.0	5.8	1.0		0.8	
	5	44.7	35	12.9	4.4	1.0		1.2	
Veh	vehicle	56.5	53.7	9.4	4.1
TA-97a	0.05	91.7	38	7.8	7.8	1.6		0.7	
	0.1	91.3	57.7	9.3	5.7	1.6		1.1	
	0.5	83	60	14.0	1.7	1.5		1.1	
	1	103	63.3	8.9	1.2	1.8		1.2	
	5	94.7	60.3	14.6	11.1	1.7		1.1	
Veh	vehicle	12.2	13.8	2.3	5.2
TA-1535	0.05	11	12.7	2.6	4.7	0.9		0.9	
	0.1	14.3	16.7	6.7	5.7	1.2		1.2	
	0.5	10	17.7	2.0	1.5	0.8		1.3	
	1	9.7	16	2.9	7.5	0.8		1.2	
	5	11.7	13	5.1	1.7	1.0		0.9	
Veh	vehicle	17.8	15	4.6	3.5
TA-98	0.05	18.7	11	0.6	1.0	1.1		0.7	
	0.1	18.7	13	2.3	2.6	1.1		0.9	
	0.5	22.7	19	4.2	8.0	1.3		1.3	
	1	15	17	8.5	3.5	0.8		1.1	
	5	20	14.7	1.0	4.0	1.1		1.0	
Veh	vehicle	106.5	95.8	8.1	15.9
TA-100	0.05	102.3	90.3	20.3	4.9	1.0		0.9	
	0.1	111.3	100.7	10.4	8.0	1.0		1.1	
	0.5	111	94.7	8.7	4.5	1.0		1.0	
	1	113.7	93	16.3	8.9	1.1		1.0	
	5	113.7	85.7	15.3	6.0	1.1		0.9	

 

   * =>2       + = presence           - = absence

 

Confirmatory Assay

Quality Control

Strain	Treatment (μg/plate)	S9	Mean		SD	SEM	Fold Increase over Vehicle
TA-97a	vehicle	+	36.7		9.9	4.0	17.0	×
TA-97a	10 μg 2aa	+	623.0		59.1	24.1
TA-97a	vehicle	–	49.2		7.5	3.1	14.3	×
TA-97a	1 μg ICR191	–	701.2		38.3	15.6

        * = >2       + = presence       - = absence

Confirmatory Assay for 2680 -47 with TA-97a

TA :2680-47           Vehicle:TCH2O

		Revertant Colonies
	Conc (μg/plate)	Mean		SD		Fold Increase over Vehicle
Strain		+ S9	-S9	+ S9	-S9	+ S9		-S9
Veh	vehicle	36.7	49.2	9.9	7.5
TA-97a	0.05	59	46.3	6.1	15.5	1.6		0.9	
	0.1	57.3	52.7	8.1	4.2	1.6		1.1	
	0.5	58.7	44.7	7.2	3.1	1.6		0.9	
	1	55	53.7	7.0	7.6	1.5		1.1	
	5	62	48.3	7.5	5.5	1.7		1.0	

            * = >2          + = presence           - = absence

 

Applicant's summary and conclusion

Conclusions:

Under test conditions, test article 2680-47 did not have mutagenicity potential in the Bacterial Reverse Mutation Test. ANSC RDI 2860-47 was a development number for Elfamosit AC which is being registered as the Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol.

Executive summary:

Objective:  The purpose of this study was to evaluate the mutagenic potential of a test article 2860 -47 based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation.  This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 – Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 – Bacterial Reverse Mutation Test.

Method Synopsis:  Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O).  The test article was freely soluble at a concentration of 50 µl/ml.  The Study Director chose TCH2O as the vehicle for the study.  A cytotoxicity screen was conducted in the Salmonella typhimurium TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0 μl/plate) of the test article, two plates per dose.  The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation mixture (S9) and overlaid onto minimal glucose agar plates.  A TCH2O vehicle control was run concurrently, with and without S9.

Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1.0 and 5.0 μl/plate) of the test article were tested in each of five bacterial tester strains (E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).  Vehicle controls and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations.  The plates were incubated at 37ºC ± 2ºC

for 48-72 hours.  Revertant colony growth was determined by counting the colonies per plate using an AlphaImager™ imaging system.  The number of revertants of the test article treatment plates and positive control plates was divided by the number of revertants of the vehicle plates.  In general, a positive result is determined by a 2-fold increase above the vehicle control.

Due to a borderline trend towards a positive response for TA-97a in the presence of S9,but without a 2 fold increase at any concentration an independent repeat assay (confirmatory test) was conducted in tester strain TA-97a, with and without S9, using test article concentrations of 0.05, 0.1, 0.5, 1.0 and 5.0 μl/plate.

Summary:  Test article 2680 -47, in the vehicle, tissue culture water, was tested in a Bacterial Reverse Mutation Assay.  In the screen, the test article did not show obvious cytotoxicity to tester strain TA-100 at a dose range of 0.001 to 5.0 µl/plate, with or without S9.  In the Main Assay, there was a boarderline trend towards a positive response for TA-97a but a 2 fold incresae was not seen. There was no diminution or clearing of the background lawn observed at any dose level or with any strain.  In the independent repeat assay (confirmatory test), the test article at

0.05 to 5.0 µl/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of TA-97a, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

Conclusion: Under test conditions, test article 2680-47 did not have mutagenicity potential in the Bacterial Reverse Mutation Test.