Reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-d

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:

July 28, 2016 - December 9, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(ethylenedioxy)diethanol

EC Number:

203-953-2

EC Name:

2,2'-(ethylenedioxy)diethanol

Cas Number:

112-27-6

Molecular formula:

C6H14O4

IUPAC Name:

2,2'-[ethane-1,2-diylbis(oxy)]diethanol

Constituent 2

Reference substance name:

3,6,9-trioxaundecane-1,11-diol

EC Number:

203-989-9

EC Name:

3,6,9-trioxaundecane-1,11-diol

Cas Number:

112-60-7

Molecular formula:

C8H18O5

IUPAC Name:

2,2'-[oxybis(ethane-2,1-diyloxy)]diethanol

Constituent 3

Reference substance name:

3,6,9,12-tetraoxatetradecane-1,14-diol

EC Number:

225-341-4

EC Name:

3,6,9,12-tetraoxatetradecane-1,14-diol

Cas Number:

4792-15-8

Molecular formula:

C10H22O6

IUPAC Name:

3,6,9,12-tetraoxatetradecane-1,14-diol

Method

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strains).

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fractions

Test concentrations with justification for top dose:

156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate
Top dose was selected because recommended top dose in the guidelines for testing in the Confirmatory Mutation Assay.vehicle

Vehicle / solvent:

Distilled water (Pusher/Pentaethylene glycol mixture and positive control material sodium azide). DMSO for positive controls 4-nitroquinoline 1-oxide, 0-aminoacridine hydrochloride hydrate, 2-nitrofluorene, and 2-aminoanthracene.

Controls

Untreated negative controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Initial Toxicity Mutation Assay
Before commencing the Confirmatory Mutation Assay, Pentaethylene Glycol Mixture was tested for cytotoxicity to all Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101). The experiment was conducted both in the absence and presence of metabolic activation (5% v/v S9 mix).
Tubes containing 2 mL of molten top agar 0.5 mM histidine/biotin were maintained at 45 ± 2 °C for Salmonella typhimurium tester strains. Tubes containing 2 mL of plain top agar were maintained at 45 ± 2 ºC for Escherichia coli WP2 uvrA (pKM101) tester strain and prior to the addition of the test item and controls, 100 µL of tryptophan solution (100 µg/mL) was added to the top agar tubes. Volumes of 100 µL of the relevant stock solution of test item and distilled water were used for treatment and negative control, respectively.
A volume of 500 µL of 5% v/v S9 mix was added in the presence of metabolic activation and 500 µL of 0.2 M phosphate buffer was added in the absence of metabolic activation. Finally 100 µL of bacterial culture was added to the tubes and mixed. Cultures used were checked for cell viability prior to testing. This treatment mixture was incubated in shaking water bath for
20 ± 2 minutes at 37 ± 1 °C. After incubation, 2 mL molten top agar was added to this treatment mixture and poured on MGA plates and allowed to solidify. Duplicate sets were maintained for each concentration of Pentaethylene Glycol Mixture, negative control and positive control. The petri plates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the background bacterial lawn pattern and reduction in number of colonies.

Confirmatory Mutation Assay
The Confirmatory Mutation Assay was conducted as an independent experiment. In the trial, the treatment was performed both in the absence and presence of metabolic activation (10% v/v S9 mix). The treatment was performed by the preincubation technique as described in the Initial Toxicity Mutation Assay. Triplicate sets of plates were treated for each test concentration of Pentaethylene Glycol Mixture, negative and positive controls.
The tester strains were exposed at the concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 µg Pentaethylene Glycol Mixture/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation for Confirmatory Mutation Assay, respectively.
Volumes of 100 µL of relevant stock solutions A – F were used to obtain the required test concentrations for treatment both in the absence and presence (10% v/v S9 mix) of the metabolic activation system.
Samples were collected and sent for analysis.
The numbers of revertant colonies were recorded approximately after the 48 h incubation period.
Treatment with 2-aminoanthracene in the absence of metabolic activation was performed for tester strain TA100 in both the trials to demonstrate the efficiency of the S9 fraction used in the study.

Analytical Verification and Stability of Test Item
Dose formulations of Pentaethylene Glycol Mixture were prepared in distilled water by the study director during the study. Dose formulations for the Initial Toxicity Mutation Assay and the Confirmatory Mutation Assay were not corrected for purity. Samples of dose formulations and the vehicle (distilled water) were sent to the Department of Chemistry, JRF for active ingredient concentration analysis for Confirmatory Mutation Assay.

Sampling
Samples were collected from all six prepared dose formulations along with vehicle (distilled water) for active ingredient concentration and lowest and highest dose formulation for stability analysis during the confirmatory mutation assay following the detailed procedures below:
Two sets of single replicates of 3 mL each of concentration (1562.5, 3125, 6250, 12500, 25000, and 50000 µg/mL) and vehicle (distilled water) were taken from middle portion for active ingredient concentration analysis at 0 hour. Two sets of single replicates of 3 mL each of highest concentration (50000 µg/mL) and lowest concentration (1562.5 µg/mL) were taken from middle portion for stability analysis at 4 hour of dose formulation preparation i.e. at the end of plate exposure. All the aliquots were stabilized by immediate freezing (-70 ± 10 °C) to quench any degradation of test item. The first sets of replicates were sent to Department of Chemistry (JRF) for analysis. Second set of replicates were stored in deep freezer (-70 ± 10 ºC) as backup. Due to extraction problem, back up samples were reanalysed using same analytical parameter.

Stability
Stability of the test item in distilled water was tested concurrently by test samples before the initiation of exposure and after completion of plate exposure (4 hours) by using the validated analytical method provided by the sponsor. All prepared concentrations were sampled before the initiation of plate exposure, while only the lowest and the highest test concentrations were sampled at 4 hours post dose formulation preparation for stability analysis.

Analysis
The samples collected during the confirmatory mutation study as described above were analysed for active ingredient concentration and stability.

Evaluation criteria:

Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.

Strains TA98, TA1535, and TA1537
Data 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 negative control value.

Strains TA100 and Escherichia coli WP2 uvrA (pKM101)
Data 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 negative control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be non mutagenic.

Results and discussion

Test results

Species / strain:

other: S.typhimuriam TA1537, TA1535, TA98, TA100, and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative Controls
The results of the study indicate that the values for the negative controls (Distilled Water) in all strains were within limits of historical range.

Positive Controls
2-Aminoanthracene was used as the positive control in the presence of a metabolic activation for all the tester strains during the Initial Toxicity Mutation Assay and Confirmatory Mutation Assay. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation.
Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges.This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.
An increase in the mean number of revertants was not observed in Salmonella typhimurium tester strain TA100 (Initial Toxicity Mutation Assay and Confirmatory Mutation Assay) treated with 2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity Mutation Assay
Bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate (two plates/concentration). Normal growth was observed up to the tested concentration of 5000 µg/plate both in the absence and presence of metabolic activation system (5% v/v S9 mix). No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control. Hence, 5000 µg Pentaethylene Glycol Mixture/plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation system (10% v/v S9 mix), respectively, for Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101) tester strain.

Confirmatory Mutation Assay
From the results of the Initial Toxicity Mutation Assay, 5000 µg Pentaethylene Glycol Mixture/plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation system (10% v/v S9 mix), for Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101) tester strain.
In the confirmatory mutation assay, bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate (three plates/concentration).
Normal growth was observed up to the tested concentration of 5000 µg/plate in the absence and presence of the metabolic activation system (10% v/v S9 mix) in all the Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101). No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

Dose Formulation Analysis
Pentaethylene Glycol Mixture was found to be within acceptable range of ± 15% of nominal (% RSD < 10%) in distilled water at all the tested concentrations during confirmatory mutation assay. The doses complied with the presence of test item for its claimed concentration (± 15% of nominal) of active ingredient. Average recoveries were 97.2%, 98.7%, 95.5%, 103%, 104%, and 103% at test concentrations of 1562.5, 3125, 6250, 12500, 25000, and 50000 µg/mL, respectively. Results of stability analysis showed that the test item was stable at the end of treatment (4 hours) and recovery of the test concentrations 1562.5 and 50000 µg/mL (lowest and highest concentration) was 104% and 104% of the 0 hour concentration, respectively.

Any other information on results incl. tables

TABLE1:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Initial Toxicity Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate (Absence of Metabolic Activation)
	Salmonella typhimurium Tester Strains																			E.coli WP2 uvrA (pKM101)
	TA1537				TA1535					TA98					TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean		SD	Ratio	Lawn Pattern	Mean		SD	Ratio	Lawn Pattern	Mean		SD	Ratio	Lawn Pattern
NC(distilled water)	7.00	4.24	1.00	NI	19.00	7.07	1.00	NI	20.50		2.12	1.00	NI	123.00		16.97	1.00	NI	117.00		11.31	1.00	NI
1.5	5.00	1.41	0.71	NI	18.50	3.54	0.97	NI	18.50		3.54	0.90	NI	126.50		9.19	1.03	NI	110.00		2.83	0.94	NI
5	8.50	0.71	1.21	NI	20.00	2.83	1.05	NI	16.00		1.41	0.78	NI	115.00		8.49	0.93	NI	115.00		7.07	0.98	NI
15	8.00	1.41	1.14	NI	16.50	6.36	0.87	NI	19.50		3.54	0.95	NI	121.00		7.07	0.98	NI	111.00		4.24	0.95	NI
50	7.50	3.54	1.07	NI	18.00	2.83	0.95	NI	20.50		7.78	1.00	NI	121.50		10.61	0.99	NI	119.50		3.54	1.02	NI
150	7.00	2.83	1.00	NI	19.00	5.66	1.00	NI	18.00		2.83	0.88	NI	121.00		4.24	0.98	NI	117.50		7.78	1.00	NI
500	8.50	3.54	1.21	NI	18.00	2.83	0.95	NI	19.00		1.41	0.93	NI	128.00		4.24	1.04	NI	118.50		0.71	1.01	NI
1500	8.50	0.71	1.21	NI	17.50	0.71	0.92	NI	20.00		1.41	0.98	NI	119.50		6.36	0.97	NI	109.50		3.54	0.94	NI
5000	6.50	0.71	0.93	NI	16.00	5.66	0.84	NI	17.50		2.12	0.85	NI	126.50		2.12	1.03	NI	118.00		4.24	1.01	NI
PC	301.50	26.16	43.07	NI	322.50	33.23	16.97	NI	582.50		58.69	28.41	NI	893.50		21.92	7.26	NI	1092.50		24.75	9.34	NI
2-Aa	NA	NA	NA	NA	NA	NA	NA	NA	NA		NA	NA	NA	133.50		6.36	1.09	NI	NA		NA	NA	NA

Key:        SD = Standard deviation, NC = Negative control, PC = Positive control {for TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), for TA1535 = Sodium azide (0.5 µg/plate), for TA98 = 2-Nitrofluorene (7.5 µg/plate), for TA100 = Sodium azide (5 µg/plate), forE. coli WP2 uvrA (pKM101) =4-Nitroquinoline 1-oxide(0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),Test item = Pentaethylene Glycol Mixture, NI = No Inhibition

TABLE2:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Initial Toxicity Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	Salmonella typhimurium Tester Strains															E.coli WP2 uvrA (pKM101)
	TA1537				TA1535				TA98				TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern
NC (Distilled water)	9.00	4.24	1.00	NI	20.50	6.36	1.00	NI	29.00	9.90	1.00	NI	129.00	12.73	1.00	NI	118.50	9.19	1.00	NI
1.5	6.50	2.12	0.72	NI	16.50	2.12	0.80	NI	25.50	10.61	0.88	NI	123.50	6.36	0.96	NI	119.00	7.07	1.00	NI
5	5.00	2.83	0.56	NI	21.00	7.07	1.02	NI	25.50	4.95	0.88	NI	125.50	6.36	0.97	NI	116.00	7.07	0.98	NI
15	6.00	2.83	0.67	NI	19.50	2.12	0.95	NI	25.50	6.36	0.88	NI	125.50	7.78	0.97	NI	114.50	6.36	0.97	NI
50	8.50	2.12	0.94	NI	21.00	1.41	1.02	NI	21.50	2.12	0.74	NI	123.50	13.44	0.96	NI	116.00	4.24	0.98	NI
150	8.50	0.71	0.94	NI	20.50	3.54	1.00	NI	25.50	3.54	0.88	NI	125.00	4.24	0.97	NI	120.50	6.36	1.02	NI
500	4.50	0.71	0.50	NI	18.50	4.95	0.90	NI	25.50	4.95	0.88	NI	129.50	13.44	1.00	NI	118.00	5.66	1.00	NI
1500	9.50	2.12	1.06	NI	19.00	2.83	0.93	NI	23.00	7.07	0.79	NI	127.50	3.54	0.99	NI	119.50	4.95	1.01	NI
5000	5.50	0.71	0.61	NI	16.00	1.41	0.78	NI	21.50	2.12	0.74	NI	125.50	3.54	0.97	NI	117.50	3.54	0.99	NI
PC(2-Aa)	281.50	43.13	31.28	NI	354.50	47.38	17.29	NI	564.50	60.10	19.47	NI	981.00	48.08	7.60	NI	1161.50	178.90	9.80	NI

Key:    SD = Standard deviation, NC = Negative control, PC = Positive control, 2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535,E. coli WP2 uvrA(pKM101) and 5 µg/plate for TA98 and TA100),Test item = Pentaethylene Glycol Mixture, NI = No Inhibition

TABLE3:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Confirmatory Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate (Absence of Metabolic Activation)
	Salmonella typhimurium Tester Strains																E.coli WP2 uvrA (pKM101)
	TA1537				TA1535				TA98				TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern
NC (Distilled water)	8.00	4.00	1.00	NI	17.00	5.29	1.00	NI	21.67	7.37	1.00	NI	127.00	13.53	1.00	NI	124.67	9.07	1.00	NI
156.25	7.33	3.51	0.92	NI	16.00	5.29	0.94	NI	20.67	7.64	0.95	NI	129.33	16.56	1.02	NI	123.33	8.50	0.99	NI
312.5	8.67	3.51	1.08	NI	16.00	4.58	0.94	NI	22.67	7.37	1.05	NI	127.67	10.07	1.01	NI	126.67	7.09	1.02	NI
625	7.67	1.53	0.96	NI	17.67	6.03	1.04	NI	21.00	8.72	0.97	NI	131.00	10.15	1.03	NI	122.00	7.55	0.98	NI
1250	8.67	1.53	1.08	NI	16.33	4.16	0.96	NI	22.67	4.16	1.05	NI	125.00	7.00	0.98	NI	127.33	8.62	1.02	NI
2500	7.67	3.51	0.96	NI	18.00	5.29	1.06	NI	22.00	6.24	1.02	NI	133.33	9.50	1.05	NI	122.67	5.86	0.98	NI
5000	7.33	2.52	0.92	NI	16.67	4.73	0.98	NI	21.00	5.29	0.97	NI	126.33	11.06	0.99	NI	121.33	12.06	0.97	NI
PC	419.33	38.03	52.42	NI	542.33	43.32	31.90	NI	765.67	55.05	35.33	NI	1073.67	14.19	8.45	NI	870.00	115.01	6.98	NI
2-Aa	-	-	-	-	-	-	-	-	-	-	-	-	128.67	6.66	1.01	NI	-	-	-	-

Key:    SD = Standard deviation, NC = Negative control, PC = Positive control {for TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), for TA1535 = Sodium azide (0.5 µg/plate), for TA98 = 2-Nitrofluorene (7.5 µg/plate), for TA100 = Sodium azide (5 µg/plate), for E. Coli WP2 uvrA (pKM101) =4-Nitroquinoline 1-oxide(0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),Test item = Pentaethylene Glycol Mixture, - = Not applicable, NI = No Inhibition

TABLE4:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Confirmatory Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	Salmonella typhimurium Tester Strains																E.coli WP2 uvrA (pKM101)
	TA1537				TA1535				TA98				TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern
NC (Distilled water)	9.33	4.51	1.00	NI	20.00	7.00	1.00	NI	23.33	6.81	1.00	NI	131.33	14.05	1.00	NI	131.00	10.82	1.00	NI
156.25	9.00	3.61	0.96	NI	20.33	4.04	1.02	NI	23.67	5.69	1.01	NI	134.00	9.17	1.02	NI	128.00	12.53	0.98	NI
312.5	9.33	3.21	1.00	NI	19.00	6.24	0.95	NI	22.67	6.51	0.97	NI	126.67	11.59	0.96	NI	132.33	9.02	1.01	NI
625	10.00	3.61	1.07	NI	19.33	5.13	0.97	NI	23.67	5.51	1.01	NI	137.00	10.82	1.04	NI	125.33	12.86	0.96	NI
1250	9.00	3.00	0.96	NI	20.67	5.69	1.03	NI	23.00	5.20	0.99	NI	125.00	10.82	0.95	NI	130.33	8.50	0.99	NI
2500	9.00	4.36	0.96	NI	20.33	6.81	1.02	NI	24.33	7.09	1.04	NI	133.00	11.14	1.01	NI	131.00	9.85	1.00	NI
5000	9.67	3.51	1.04	NI	19.00	7.21	0.95	NI	22.67	5.69	0.97	NI	130.00	14.53	0.99	NI	127.33	11.02	0.97	NI
PC	429.33	33.13	46.02	NI	629.00	56.56	31.45	NI	1005.67	61.91	43.11	NI	1175.00	87.61	8.95	NI	962.33	136.14	7.35	NI

Key:        SD = Standard deviation, NC = Negative control,PC = Positive control, 2Aa = 2-Aminoanthracene(10 µg/plate for TA1537, TA1535, E. coli WP2 uvrA (pKM101)and 5 µg/plate for TA98 and TA100), Test item = Pentaethylene Glycol Mixture, NI = No Inhibition

Applicant's summary and conclusion

Conclusions:

From the results of this study, under the specified experimental conditions, Pentaethylene Glycol Mixture was concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Executive summary:

The potential of Pentaethylene Glycol Mixture to induce reverse mutations in Salmonella typhimurium (strains: TA1537, TA1535, TA98, and TA100) and a tryptophan deficient strain, Escherichia coli WP2 uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method. 

Pentaethylene Glycol Mixture was tested in the absence and presence of metabolic activation using distilled water (DW) as the solvent. In the Initial Toxicity-Mutation Assay, bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (two plates/concentration). No cytotoxicity was observed up to the tested concentration of 5000 µg/plate, with and without metabolic activation, in any of the five strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

From the results of the Initial Toxicity-Mutation Assay, 5000 µg/plate was selected, being the recommended top dose indicated in the guidelines for testing in the Confirmatory Mutation Assay. In the Confirmatory Mutation Assay, bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate (three plates/concentration). No cytotoxicity was observed at all concentrations tested, with and without metabolic activation, in any of the five tester strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

Pentaethylene Glycol Mixture was found to be within acceptable range of ± 15% of nominal concentrations at the tested stock concentrations of 1562.5, 3125, 6250, 12500, 25000, and 50000 µg/mL during the Confirmatory Mutation Assay. Recoveries ranged between 95.5% to 104% at the above tested concentrations. Results of stability analysis showed that the test item was stable at the end of treatment (4 hours) and recovery of the test concentrations at 1562.5 and 50000 µg/mL (lowest and highest concentration) was 104% and 104% of the 0 hour concentrations, respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, Pentaethylene Glycol Mixture is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).