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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was tested in three in-vitro genetic toxicology tests.  An in-vitro mammalian cell micronucleus test OECD 486 in human lymphocytes, an In-vitro mammalian cell gene mutation assay (L5178Y TK +/- Mouse lymphoma assay) OECD 490 and Bacterial reverse mutation assay (Ames Assay) OECD 471. All three test were negative. Therefore, there is no indication of any mutagenic or clastogenic or aneugenic activity for the test substance in-vitro. As the in-vitro studies were negative no further testing is required, and it can be concluded that the substance is not genotoxic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

DNA polymerase A deficient

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

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)

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.

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

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

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

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

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.

   

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

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 03 November 2016 Experimental completion date 09 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Nuclei of normal human lymphocytes

Species / strain / cell type:

mammalian cell line, other: Humna lymphocytes

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 27 years
Main Experiment: female, aged 24 years

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).

Test concentrations with justification for top dose:

The molecular weight of the test item was given as 147.17, therefore, the maximum dose level was 1472 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level.
0, 5.75, 11.5, 23, 46, 92, 184, 368, 736, 1472 µg/mL

Vehicle / solvent:

Not applicable

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Minus S9- mix: 0.2 µg/mL for 4-hour exposure

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Democolcine

Remarks:

Minus S9-mix: 0.075 µg/mL for 24-hour continuous exposure

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Plus S9- mix: 5 µg/mL for 4-hour exposure

Details on test system and experimental conditions:

The test item was soluble in Minimal Essential Medium (MEM) at 14.72 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, dissolved in MEM and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
 
4.3.2 Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

Rationale for test conditions:

4.5 Acceptability Criteria
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9 mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed

Evaluation criteria:

Please refer to "Any other information on materials and methods"

Statistics:

4.7 Statistical Analysis
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Key result

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 5.75, 11.5, 23, 46, 92, 184, 368, 736, and 1472 µg/mL. The maximum dose was the maximum recommended dose level, the 10 mM concentration. 

No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, in any of the three exposure groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 1472 µg/mL in the 4-hour exposure, both in the presence and absence of metabolic activation (S9) and in the 24-hour continuous exposure group.  The test item demonstrated no evidence of toxicity in any of the exposure groups.

The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level and was 1472 µg/mL for all three exposure groups. 

 Micronucleus Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Exposure Group	Final concentration of test itemElfaMoist AC - N-[2-(2-Hydroxyethoxy)ethyl]acetamide(µg/mL)
4-hour without S9	0*, 46, 92, 184*, 368*, 736*, 1472*, MMC0.2*
4-hour with S9 (2%)	0*, 46, 92, 184*, 368*, 736*, 1472*, CP5*
24-hour without S9	0*, 46, 92, 184*, 368*, 736*, 1472*, DC0.075*

 

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 1472 µg/mL in all three exposure groups.

Precipitate of test item was not seen in the blood cultures at the end of the exposure period in any of the exposure groups.

The CBPI data confirm the qualitative observations in that no dose-related inhibition of CBPI was observed, and that no marked inhibition of cell proliferation was observed in any of the three exposure groups. The maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level, 1472 µg/mL.

 The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

*              = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC = Mitomycin C

CP            = Cyclophosphamide

DC          = Demecolcine

Conclusions:

The test ite, did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

 Introduction

This report describes the results of anin vitrostudy for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that in the absence of precipitate or toxicity the maximum concentration should be the maximum recommended dose level. The dose levels selected for the Main Test were as follows:

Exposure Group	Final concentration of test itemElfaMoist AC - N-[2-(2-Hydroxyethoxy)ethyl]acetamide(µg/mL)
4-hour without S9	46, 92, 184, 368, 736, 1472
4-hour with S9 (2%)	46, 92, 184, 368, 736, 1472
24-hour without S9	46, 92, 184, 368, 736, 1472

Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level. 

Conclusion

The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 07 November 2016 Experimental Completion Date: 28 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines • ``Kanpoan No. 287 - - Environment Protection Agency`` • ``Eisei No. 127 - - Ministry of Health and Welfare`` • ``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian cell gene mutation assay

Specific details on test material used for the study:

I

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.


Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation (2% S9)

Test concentrations with justification for top dose:

Prliminary cytotoxicity test: 0, 5.75, 11.5, 23, 46, 92, 184, 368, 736 and 1472 µg/mL. Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was no evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was no evidence of marked reductions of %RSG values of cells treated with test item. A precipitate of the test item was not observed at any dose level. In the subsequent mutagenicity experiments the maximum dose was set at the maximum 10 mM recommended dose level of 1472 µg/mL.

Main test: 0, 46, 92, 184, 368, 736 and 1472 µg/mL

Vehicle / solvent:

Solvent R10 media

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and formulated in R0 media prior to serial dilutions being prepared. The test item had a molecular weight of 147.17 therefore the maximum dose level was set at the maximum recommended dose level(approximately 10 mM) of 1472 µg/mL, with a correction for purity (82%). There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). (The solubility data was shared with the corresponding In Vitro Micronucleus test Envigo project Number KR24BM and was considered to be appropriate for this study).


No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (R0 media) (Sigma batch 1791403 Expiry 08.2017*) exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBQ0451V Expiry 02.17*) at 400 µg/mL and 150 µg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros batch A0355340 Expiry 01.01.20 Purity 97%) at 1.5 µg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

Microsomal Enzyme Fraction
Lot No. PB/beta-NF S9 25/08/16 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a certificate of S9 efficacy is presented in Appendix 2.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test Procedure
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 5.75 to 1472 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 105 cells/mL.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:

i) Maximum recommended dose level, 5000 µg/mL or 10 mM.

ii) The presence of excessive precipitate where no test item-induced toxicity was observed.

iii) Test item-induced toxicity, where the maximum dose level used should produce approximately 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).


Mutagenicity Test
Main Experiment
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. For the 4-hour exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 106 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks The treatments were performed in duplicate (A + B), at 6 dose levels of the test item, 46 to 1472 µg/mL for all three exposure groups. To each universal was added 2 mL of S9-mix (2%) if required, 2 mL of the treatment dilutions, (0.15 or 0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group). The treatment vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Evaluation criteria:

Please refer to "Any other information on materials and methods" below

Statistics:

The Delta Building Monitoring System was used during the course of the study.

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

Key result

Species / strain:

mouse lymphoma L5178Y cells

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: No increase in mutant frequency

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 5.75 to 1472 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
5.75	95	97	110
11.5	103	93	108
23	118	103	110
46	106	95	101
92	94	88	94
184	105	109	83
368	106	98	86
736	102	96	92
1472	100	100	102

 

In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was no evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was no evidence of marked reductions of %RSG values of cells treated with test item. A precipitate of the test item was not observed at any dose level. In the subsequent mutagenicity experiments the maximum dose was set at the maximum 10 mM recommended dose level of 1472 µg/mL.

 

Mutagenicity TesT

4 -hour exposure in the absence and presence of metabolic activation

There was no evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of marked reductions in viability (%V), therefore indicating that residual toxicity had not occurred, in either the absence and presence of metabolic activation. The test item was tested up to the maximum recommended dose level (approximately 10 mM) of 1472 µg/mL. Acceptable levels of toxicity were seen with both positive control substances.

 

Neither of the vehicle control mutant frequency values were outside the normal in-house range of 50 to 170 x 10^-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

 

The test item did not induce any toxicologically increases in the mutant frequency x 10^-6per viable cell, at any dose level, in either the absence or presence of metabolic activation. 

 

24-hour exposure in the absence of metabolic activation

There was no evidence of marked toxicity following exposure to the test item as indicated by the %RSG and RTG values. There was no evidence of marked reductions in viability (%V), therefore indicating that residual toxicity had not occurred in this exposure group. The test item was tested up to the maximum recommended dose level (approximately 10mM) of 1472 µg/mL. Acceptable levels of toxicity were seen with the positive control substance.

 

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point did not have a marked effect on the toxicity of the test item. The vehicle control mutant frequency was within the normal range of 50 to 170 x 10^-6viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

 

The test item did not induce any toxicologically increases in the mutant frequency x 10^-6per viable cell, at any dose level, in either the absence or presence of metabolic activation.

                  Summary of Results

 

Treatment (µg/ml)		4-hours-S-9				Treatment (µg/ml)		4-hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	83.55		0		100	1.00	122.91
46		97	0.99	86.76		46		105	1.00	109.87
92		95	0.99	90.37		92		101	1.20	100.88
184		96	0.95	89.16		184		98	1.25	86.01
368		97	0.82	95.28		368		93	1.18	95.01
736		91	0.86	107.44		736		90	0.92	137.82
1472		86	0.87	90.48		1472		99	1.62	62.76
MF positive response threshold = 209.55						MF positive response threshold = 248.91
EMS						CP
400		70	0.84	458.34		1.5		73	0.58	1015.38

               

Treatment (µg/ml)		24-hours-S-9
		%RSG	RTG	MF§
0		100	1.00	137.68
46		92	1.05	110.68
92		98	1.06	126.20
184		93	0.99	116.23
368		94	1.14	108.88
736		97	0.90	126.07
1472		80	0.78	113.31
MF positive response threshold = 263.68
EMS
150		38	0.36	1755.05

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells at all dose levels, either with or without metabolic activation.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

 

Methods…….

One main experiment was performed. In the main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 6 dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24-hour exposure in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for expression of mutant colonies were as follows:

 

Main Experiment

Group	Concentration of substance: (µg/mL)  plated for mutant frequency
4-hour without S9	46, 92, 184, 368, 736, 1472
4-hour with S9 (2%)
24-hour without S9

 

 

Results……..

The maximum dose level used was the maximum recommended dose level of 1472 (µg/mL). A precipitate of the test item was not observed at any dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation.

 

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells at all dose levels, either with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The in vitro study results are conclusive. There is no need to perform in vivo studies.

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Profiling and QSAR results indicate no interaction to DNA, no mutagenicity, no carcinogenicity and an overall low toxicity.

 

For each endpoint, bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity, a recent guideline and GLP compliant study is available which all indicate that there is no concern for genotoxicity.

 

Bacterial mutagenicity

Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol has been tested for bacterial mutagenicity in the Salmonella typhimurium (S.typhimurium; TA-97a, TA-1535, TA-98, and TA-100) and Escherichia coli (WP2 uvrA) reverse gene mutation assay by direct plate incorporation method followed by an independent repeat. 

Tissue culture water was used as vehicle. Five concentrations (0.05, 0.1, 0.5, 1.0 and 5.0 μl/plate) were tested. There was no cytotoxicity observed either with or without S9. Vehicle controls and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations.

Results: In the Main Assay, 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 any bacterial tester 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 in TA-97a, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

 

Genotoxicity in mammalian cells:

Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerolwas investigated for clastogenic and aneugenic effects on the nuclei of normal human lymphocytesin vitroin the absence and presence of metabolic activation with S9 homogenate. The was performed under GLP according to OECD 487 guideline.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels selected for the Main Test were as follows:

- 4-hour without S9: 46, 92, 184, 368, 736, 1472 µg/mL

- 4-hour with S9 (2%): 46, 92, 184, 368, 736, 1472 µg/mL

- 24-hour without S9: 46, 92, 184, 368, 736, 1472 µg/mL

Results: All vehicle (Minimal Essential Medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level.

 

 

Mutagenicity in mammalian cells:

Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerolwas investigated for its potential to induce mutations on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted under GLP according to OECD 490 guideline.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for expression of mutant colonies were 46, 92, 184, 368, 736, and 1472 (recommended max.10 mM) µg/mL for all exposure conditions: 4-hour without S9, 4-hour with S9 (2%), and 24-hour without S9.

Results: No precipitation was noted at these evaluated concentrations. The vehicle (solvent) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency.

The test item did not induce any toxicologically significant increases in the mutant frequency at

any of the dose levels, either with or without metabolic activation.

Justification for classification or non-classification

Reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide (DGA) and Glycerolwas shown not to be mutagenic in the Salmonella typhimurium reverse mutation assay, not clastogenic or aneugenic in human lymphocytes and not mutagenic in the mouse lymphoma L5178Y test system.

Consequently, no classification for genotoxic effects is required.