ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 June 2017 to 17 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Described in Council Regulation (EC) No. 440/2008 (as amended).

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-oxydiethanol

EC Number:

203-872-2

EC Name:

2,2'-oxydiethanol

Cas Number:

111-46-6

Molecular formula:

C4H10O3

IUPAC Name:

(2-hydroxyethoxy) ethan-2-ol

Constituent 2

Reference substance name:

Diethylene glycol 6-hydroxyhexanoate

Molecular formula:

C10H20O5

IUPAC Name:

Diethylene glycol 6-hydroxyhexanoate

Constituent 3

Reference substance name:

Diethylene glycol di-6-hydroxyhexanoate

Molecular formula:

C16H30O7

IUPAC Name:

Diethylene glycol di-6-hydroxyhexanoate

Constituent 4

Reference substance name:

Diethylene glycol tri-6-hydroxyhexanoate

Molecular formula:

C22H40O9

IUPAC Name:

Diethylene glycol tri-6-hydroxyhexanoate

Constituent 5

Reference substance name:

Diethylene glycol tetra-6-hydroxyhexanoate

Molecular formula:

C28H50O11

IUPAC Name:

Diethylene glycol tetra-6-hydroxyhexanoate

Constituent 6

Reference substance name:

Diethylene glycol penta-6-hydroxyhexanoat

Molecular formula:

C34H60O13

IUPAC Name:

Diethylene glycol penta-6-hydroxyhexanoat

Constituent 7

Reference substance name:

Diethylene glycol hexa-6-hydroxyhexanoate

Molecular formula:

C40H70O15

IUPAC Name:

Diethylene glycol hexa-6-hydroxyhexanoate

Test material form:

liquid: viscous

Details on test material:

Appearance viscous, colorless liquid
CAS No. 36890-68-3
EINECS-No. 500-092-7

Method

Target gene:

Reversion to histidine / tryptophan independence

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:

Rat liver S9 fraction

Test concentrations with justification for top dose:

Toxicity test
The test item ϵ-Caprolactone, oligometric reaction products with 2,2’-oxydiethanol was assayed in the toxicity test at a maximum concentration of 5.00 μL/plate and at four lower concentrations spaced at approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 μL/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Neither increases in revertant numbers, nor toxic effects were observed at any dose level, with any tester strain in the absence or presence of S9 metabolism.
Main Assays
On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5.00, 2.50, 1.25, 0.625 and 0.313 μL/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; water
- Justification for choice of solvent/vehicle: This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity.

Controls

Untreated negative controls:

yes

Remarks:

untreated plate

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: The prepared plates were inverted and incubated for approximately 72 hours at 37°C.
NUMBER OF REPLICATIONS:
Three replicate plates were used at each test point.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight thinning of background lawn observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight thinning of background lawn observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity test
The test item ϵ-Caprolactone, oligometric reaction products with 2,2’-oxydiethanol was assayed in the toxicity test at a maximum concentration of 5.00 μL/plate and at four lower concentrations spaced at approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 μL/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Neither increases in revertant numbers, nor toxic effects were observed at any dose level, with any tester strain in the absence or presence of S9 metabolism.

Main Assays
On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5.00, 2.50, 1.25, 0.625 and 0.313 μL/plate. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments and using the same concentrations of Main Assay I. Slight toxicity, as indicated by thinning of the background lawn, was observed with TA1535 and TA100 tester strains at the highest dose level in the absence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Any other information on results incl. tables

Toxicity Results

 

Toxicity test without metabolic activation

Solvent: DMSO

 

Dose level (μL/plate)	TA1535 Rev/pl.	TA1537 Rev/pl.	TA98 Rev/pl.	TA100 Rev/pl.	WP2uvrA Rev/pl.
Untreated	22	15	25	163	28
0.000	20	20	28	168	29
0.050	17	13	25	138	28
0.158	17	13	31	123	30
0.500	17	14	30	138	29
1.580	24	15	21	133	26
5.000	18	14	24	141	26

  

Toxicity test with metabolic activation

Solvent: DMSO

 

Dose level (μL/plate)	TA1535 Rev/pl.	TA1537 Rev/pl.	TA98 Rev/pl.	TA100 Rev/pl.	WP2uvrA Rev/pl.
Untreated	20	20	35	169	35
0.000	18	18	34	155	34
0.050	16	18	28	168	35
0.158	14	14	28	149	31
0.500	18	15	34	135	39
1.580	16	14	34	136	33
5.000	14	18	34	146	28

 

Main Study Results

Treatment (µl/plate)			Mean revertants/plate ± standard deviation
			-S9 mix				+S9 mix
			Exp 1		Exp 2		Exp 1		Exp 2
Strain TA98
DMSO	Untreated		27 ± 1.2		27 ± 1.9		38 ± 0.9		34 ± 0.0
ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol	0		29 ± 1.2		31 ± 0.9		38 ± 1.2		35 ± 2.3
	0.313		26 ± 1.3		31 ± 2.3		33 ± 2.0		43 ± 0.6
	0.625		31 ± 2.2		30 ± 0.3		30 ± 1.5		38 ± 1.9
	1.25		26 ± 1.2		32 ± 1.9		37 ± 0.3		36 ± 1.2
	2.50		30 ± 1.5		33 ± 2.1		28 ± 1.5		34 ± 0.9
	5.00		26 ± 1.5		31 ± 1.8		30 ± 2.5		23 ± 1.5
Controls
DMSO	100 μL/pl		29 ± 0.7		31 ± 0.9		-		-
2-Nitrofluorene	2 μg/pl		142 ± 4.8		117 ± 1.2		-		-
DMSO	100 μL/pl		-		-		38 ± 1.2		35 ± 2.3
2-Aminoanthracene	1 μg/pl		-		-		813 ± 10.8		923 ± 19.5
Strain TA100
DMSO	Untreated		134 ± 1.2		136 ± 3.8		155 ± 2.3		157 ± 2.1
ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol	0		138 ± 1.9		128 ± 3.1		135 ± 2.9		133 ± 5.1
	0.313		142 ± 1.8		146 ± 2.7		152 ± 4.6		123 ± 5.2
	0.625		131 ± 1.0		140 ± 2.0		154 ± 2.3		146 ± 0.3
	1.25		134 ± 3.8		143 ± 2.5		165 ± 6.2		126 ± 5.0
	2.50		165 ± 2.9		123 ± 2.1		168 ± 4.2		119 ± 4.1
	5.00		149 ± 2.0		*132 ± 5.5		165 ± 3.7		119 ± 3.8
Controls
Untreated	0		134 ± 1.2		136 ± 3.8		-		-
Sodium Azide	1 μg/pl		433 ± 10.2		474 ± 16.6		-		-
DMSO	100 μL/pl		-		-		135 ± 2.9		133 ± 5.1
2-Aminoanthracene	1 μg/pl		-		-		1443 ± 70.7		1389 ± 67.5
Strain TA1535
DMSO		Untreated		18 ± 1.0		16 ± 0.6		19 ± 1.2		15 ± 1.5
ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol		0		17 ± 1.3		19 ± 2.5		15 ± 1.9		18 ± 1.5
		0.313		19 ± 0.9		17 ± 0.9		19 ± 2.4		20 ± 0.9
		0.625		21 ± 1.5		19 ± 1.2		17 ± 1.2		17 ± 2.4
		1.25		15 ± 0.9		16 ± 2.3		21 ± 1.8		16 ± 1.5
		2.50		17 ± 1.7		16 ± 1.5		20 ± 0.6		17 ± 1.2
		5.00		18 ± 0.9		*15 ± 1.5		22 ± 2.5		19 ± 1.8
Controls
Untreated		0		18 ± 1.0		16 ± 0.6		-		-
Sodium Azide		1 μg/pl		429 ± 17.7		440 ± 18.2		-		-
DMSO		100 μL/pl		-		-		15 ± 1.9		18 ± 1.5
2-Aminoanthracene		1 μg/pl		-		-		160 ± 14.4		122 ± 1.7
Strain TA1537
DMSO	Untreated			15 ± 1.3		20 ± 0.7		20 ± 0.9		17 ± 1.5
ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol	0			14 ± 0.7		19 ± 0.6		18 ± 0.9		16 ± 0.7
	0.313			17 ± 1.5		16 ± 0.9		17 ± 0.9		18 ± 1.2
	0.625			18 ± 0.6		17 ± 2.0		15 ± 1.3		19 ± 1.5
	1.25			19 ± 3.2		17 ± 2.6		14 ± 0.6		16 ± 2.1
	2.50			15 ± 0.7		16 ± 2.3		19 ± 0.7		16 ± 1.5
	5.00			21 ± 0.6		15 ± 1.5		18 ± 1.0		16 ± 0.6
Controls
DMSO	100 μL/pl			14 ± 0.7		19 ± 0.6		-		-
9-Aminoacridine	50 μg/pl			111 ± 3.7		196 ± 66.0		-		-
DMSO	100 μL/pl			-		-		18 ± 0.9		16 ± 0.7
2-Aminoanthracene	1 μg/pl			-		-		117 ± 4.4		118 ± 1.2
Strain WP2 uvrA
DMSO	Untreated		30 ± 1.5		26 ± 1.3		35 ± 0.9		28 ± 1.2
ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol	0		31 ± 0.9		24 ± 0.7		39 ± 1.3		27 ± 0.3
	0.313		28 ± 0.3		27 ± 1.2		28 ± 2.6		31 ± 0.7
	0.625		30 ± 1.2		27 ± 0.3		35 ± 1.7		29 ± 1.5
	1.25		29 ± 2.0		26 ± 0.9		35 ± 1.5		35 ± 1.2
	2.50		33 ± 1.3		28 ± 1.2		33 ± 2.6		33 ± 2.1
	5.00		28 ± 0.3		28 ± 0.9		32 ± 0.9		33 ± 0.6
Controls
Untreated	0		30 ± 1.5		26 ± 1.3		-		-
MMS	500 μg/pl		164 ± 7.2		136 ± 0.7		-		-
DMSO	100 μL/pl		-		-		39 ± 1.3		27 ± 0.3
2 -AA	10 μg/pl		-		-		233 ± 10.4		185 ± 5.0

*: Slight thinning of the background lawn

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item ϵ-Caprolactone, oligometric reaction products with 2,2’-oxydiethanol does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

ϵ-Caprolactone, oligometric reaction products with 2,2’-oxydiethanol was examined for the ability to induce gene mutations in strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The test item was used as a solution in dimethylsulfoxide (DMSO). On the basis of toxicity test results, Main Assay I (plate incorporation method) used concentrations of 5.00, 2.50, 1.25, 0.625 and 0.313 μL/plate. No toxicity was observed with any tester strain at any concentration in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed including a pre-incubation step for all treatments and using the same concentrations as Main Assay I. Slight toxicity, as indicated by thinning of the background lawn, was observed in strains TA1535 and TA100 at the highest concentration in the absence of metabolic activation. No precipitation of the test item was observed at the end of the incubation period at any concentration. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of metabolic activation. It is concluded that ϵ-Caprolactone, oligometric reaction products with 2,2’- oxydiethanol does not induce reverse mutation in Salmonella typhimurium or Escherichia coli strains in the absence or presence of S9 metabolism, under the conditions of this study.