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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential for the substance ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol to induce genotoxic effects in vitro has been investigated in an Ames test, a study of chromosomal aberration in cultured primary human lymphocytes and in a mouse lymphoma assay. All studies report negative results, indicating that the substance does not have the potential to induce genotoxic effects.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

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

Target gene:

Reversion to histidine / tryptophan independence

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.

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.

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

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

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 July - 20 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July 2016).

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable: chromosomal aberration

Species / strain / cell type:

lymphocytes:

Remarks:

human, primary

Details on mammalian cell type (if applicable):

Blood samples obtained from two healthy female volunteer donors 35 years old, were pooled for use. The volunteers were non-smoker and were not receiving any medication or radiation exposure to the time of sampling.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colcemid was added (0.2 μg/mL final concentration) for the last three hours, leading up to harvesting.

Metabolic activation:

with and without

Metabolic activation system:

Male SD rat liver S9 fraction (phenobarbital / 5,6-benzoflavone-induced)

Test concentrations with justification for top dose:

Based on the preliminary solubility assay, concentrations of 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 and 0.0781, 0.0391 and 0.0178 μL/mL were used for all treatment series The highest concentration represents the limit concentration for a UVCB according to OECD 476.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Three treatment series were performed (short-term treatment with and without S9 metabolism and long-term treatment), including negative and positive controls. Two cultures were prepared at each test point. Lymphocyte cultures were treated fourty-eight hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.

For the short-term treatment, the treatment medium was added to the tubes and the cultures incubated for 3 hours at 37°C. At the end of treatment, cultures were centrifuged and washed twice with PBS. Fresh medium was added and the cultures incubated for a further 21 hours (Recovery Period) before harvesting.
For the long-term treatment, the treatment medium was added to the tubes and the cultures incubated for 24 hours. Colcemid was added (0.2 μg/mL final concentration) for the last three hours, leading up to harvesting.

Lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant removed. Cells were resuspended in hypotonic solution and centrifuged. The pellet was fixed in fresh methanol/acetic acid fixative and washed two times with fixative. A few drops of the cell suspension obtained in this way were dropped onto clean, wet, grease-free, glass slides and air-dried to produce metaphase chromosome spreads. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were stained in 3% Giemsa in tap water and rinsed twice in tap and distilled water.

The mitotic index (MI) was determined for each of the concentrations. This parameter is based on the number of metaphases observed per 1000 cells and is expressed as a percentage. The highest dose level for genotoxicity assessment was selected as a concentration which produced a substantial reduction in mitotic index compared with the concurrent solvent control. Ideally the reduction should be approximately to 45±5% of the concurrent negative control. Two lower concentrations were also selected for the scoring of chromosomal aberrations. Slides were independently coded before microscopic analysis for chromosomal aberrations. Metaphases that differed from the modal chromosomal complement by more than two centromeres were not scored. The number of chromosomes, the specific types and numbers of aberrations were recorded. Polyploid and endoreduplicated cells encountered were recorded, but not included in the count of eligible metaphases. For each culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells.

Rationale for test conditions:

Based on the preliminary solubility assay, concentrations of 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 and 0.0781, 0.0391 and 0.0178 μL/mL were used for all treatment series. In addition, for the continuous treatment in the absence of S9, an addtional concentration of 0.00977 μL/mL was included. Appropriate negative and positive control cultures were included in all treatment series. Using the short-term treatment, since tests with and without metabolic activation were done
concurrently, positive control cultures were treated only with cyclophosphamide (18.0 and 23.0 μg/mL). Using the long-term treatment, positive control cultures were treated with Mitomycin-C (0.300 and 0.450 μg/mL). Following treatment, the pH and osmolality of the treatment media were determined.

Evaluation criteria:

In this assay, the test item is considered as clearly positive if the following criteria are met:

– Any concentration shows a statistically significant increase in aberration-bearing cells (excluding gaps)
– The incidence of cells bearing aberrations is outside the normal distribution of historical control values
– The increase of cells bearing aberration is concentration-related when evaluated with an appropriate trend test

The test item is considered clearly negative in this assay, if none of the above criteria is met.

Statistics:

Fisher’s Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures.
Bonferroni’s corrections were applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps. Cochran-Armitage trend test (one-sided) was performed to aid determination of concentration-response relationship. The percentage of cells bearing aberrations excluding gaps was considered for the evaluation of the outcome of the study.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Results show that the proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range based on historical control data. Adequate numbers of cells (at least 300 at each test point) and test item concentrations were analysable. The positive control items,Mitomycin-C and Cyclophosphamide, induced statistically significant increases in the incidence of cells with structural aberrations compared with the concurrent negative control and the responses were compatible with the historical control range. The study was accepted as valid.

Remarks on result:

other: No indication of mutagenicity

The proportion of cells showing chromosomal aberrations was within the historical control range at all concentrations and treatment conditions. Test item treatment did not induce statistically significant increases in aberrant cells and no statistically significant concentration-response relationship was apparent. The incidences of cells with chromsomal aberrations were within the distribution of the historical data for negative controls. On the basis of these results and in accordance with the criteria for outcome of the study, the test item was not considered to induce chromosomal aberrations in human lymphocytes.

Summary of findings

Treatment	3h				24h
Sampling	24h
Metabolic activation	-S9		+S9		-S9
Treatment	CA	MI	CA	MI	CA	MI
DMSO	0.0	100%	0.0	100%	0.0	100%T
0.00977 µL/mL	-	-	-	-	-	81%
0.0195 µL/mL	-	93%	-	90%	-	96%
0.0391 µL/mL	-	86%	-	98%	-	81%
0.0781 µL/mL	-	86%	-	87%	-	84%
0.156 µL/mL	-	81%	-	109%	0.0	75%
0.313 µL/mL	-	81%	-	88%	0.0	56%
0.625 µL/mL	0.0	73%	0.0	66%	0.0	65%
1.25 µL/mL	0.0	63%	0.0	51%	-	18%
2.50 µL/mL	0.0	22%	0.0	43%	-	18%
5.00 µL/mL	-	0%	-	20%	-	0%
CPA 18.0 µg/mL	-	-	41	27%	-	-
CPA 23.0 µg/mL	-	-	-	19%	-	-
MMC 0.300 µg/mL	-	-	-	-	-	52%
MMC 0.450 µg/mL	-	-	-	-	55	44%

CA: chromosomal aberrations (/300 cells scored) excluding gaps

MI: relative mitotic index

Conclusions:

No evidence of clastogenicity was seen under the conditions of this study.

Executive summary:

The test material (caprolactone, oligomeric reaction products with 2,2’-oxydiethanol) was assayed for the ability to induce chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation. Three treatment series were included in the study. A short-term treatment was performed where cells were treated for 3 hours in the presence and absence of S9 metabolism. The harvest time of 24 hours corresponding to approximately 1.5 cell cycle was used. A long-term (continuous) treatment was also performed in the absence of S9 metabolism until harvest at 24 hours. Solutions of the test material were prepared in DMSO. Concentrations of 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 and 0.0781, 0.0391 and 0.0178 μL/mL were used for all treatment series. In addition, for the continuous treatment in the absence of S9, a concentration of 0.00977 μL/mL was included. Each treatment series included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. For all treatment series, concentrations were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity (as determined by the reduction in mitotic index). For each culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells. Following treatment, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any concentration and treatment condition. No concentration-related increase of the incidence of cells bearing aberrations was observed and all incidences were within the distribution of the historical data for negative controls. Statistically significant increases in the incidence of aberrant cells bearing aberrations (including and excluding gaps) were seen following positive control treatments with Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system. It is concluded that the test material (caprolactone, oligometric reaction products with 2,2’-oxydiethanol) does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under these experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 February - 24 April 2018

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)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (tk)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/− (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked. The cells are checked at regular intervals for the absence of mycoplasmal contamination.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / 5,6-Benzoflavone - induced rat liver S9 fraction

Test concentrations with justification for top dose:

A preliminary cytotoxicity test was performed in order to select appropriate concentrations for the mutation assays. In this test a wide range of concentrations was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours. A single culture was used at each test point. Based on the results of the cytotoxicity test, concentrations selected for the main assay were 1000, 800, 640, 512, 410 and 328 µg/mL (-S9); 4000, 3200, 2560, 2050, 1640 and 1310 µg/mL (-S9)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Duplicate cultures were exposed to concentrations of the test material, negative or positive controls for 3 hours. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities determined. The number of cells was adjusted to 2×10e5 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% relative humidity) to allow expression of the mutant phenotype. During the 2-day expression period, cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10e5 cells/mL. After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 ×10e3 cells were plated in each well of four 96-well plates. Plateswere incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies, as well as the number of those containing small colonies were scored. After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified and counted.

Rationale for test conditions:

Standard conditions according to the test guideline and taking into account the solubility and toxicity of the test material.

Evaluation criteria:

The assay was considered valid if the following criteria were met:
(1) The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
(2) The untreated/solvent control suspension growth over 2 days fell within the range 8-32
(3) The mutant frequencies in the untreated/solvent control cultures fell within the range 50−170×10e−6 viable cells.

Every assay was also evaluated as to whether the positive control met at least one of the following two acceptance criteria:
(1) The positive control induced a clear increase above the spontaneous background (induced mutant frequency = IMF) of at least 300×10e−6. At least 40% of the IMF was reflected in the small colony MF.
(2) The positive control induced a clear increase in the small colony IMF of at least 150×10e−6.

For a test material to be considered mutagenic in this assay, it is required that:
(1) The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10−6) at one or more concnetrations.
(2) There is a significant dose-response relationship as indicated by the linear trend analysis.

Results which only partially satisfied these criteria are dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Mutant frequencies (MF) for each treatment were calculated using Poisson statistics. Results of individual plates within a replicate treatment were checked for consistency by calculation of the term χ2. For negative control and test item treatments, the consistency between replicate cultures was evaluated by calculation of the heterogeneity factor (H). The heterogeneity factors (H) were calculated for viability (Hv) and mutation (Hm). Values obtained should not exceed 10.8 times the current heterogeneity factor where 10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinite degrees of freedom. The overall consistency was evaluated using the mean values for Hv and Hm. The estimated H experiment values were combined with the current heterogeneity factors to define the updated estimate factors. The evaluation of a linear trend in mutant frequency with treatment dose was performed using weighted regression.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the absence of S9 metabolic activation, no cell survived after treatment at the highest concentration, severe toxicity reducing relative total growth (RTG) to 5% of the concurrent negative control was noted at 800 µg/mL, moderate to slight toxicity was observed between 640-410 µg/mL, while no relevant toxicity was noted at the lowest concentration tested. In the presence of S9, treatment with at 4000 µg/mL yielded marked toxicity reducing RTG to 13% of the concurrent negative control value, while no relevant toxicity was observed over the remaining concentrations tested.

In the absence of S9 metabolism, no increase in mutant frequency was noted at any concentration tested. In the presence of S9 metabolism, statistically significant increases in mutant frequency were observed at the highest and at an intermediate concentration (2560 µg/mL) and a linear trend was indicated. However, the observed increases were lower than the Global Evaluation Factor and mutation frequencies were within the range defined for the untreated/solvent control cultures; they were therefore considered to be of no biological relevance. For the negative and positive controls, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.

Summary of results in the absence of metabolic activation

-S9
Concentration	RTG	Precipitation	MF	IMF	Proportion small colonies
0 µg/mL	100%	-	91.6	-	0.34
328 µg/mL	72%	-	70.6	-	-
410 µg/mL	58%	-	62.1	-	-
512 µg/mL	34%	-	78.2	-	-
640 µg/mL	23%	+	71.0	-	-
800 µg/mL	5%	+	110.2	18.53	-
1000 µg/mL	0%	+
MMS	74%	-	424.7	333#	0.41

RTG: relative total growth (%)

MF: Mutant Frequency (/10^-6cells)

IMF: Induced Mutant Frequency

#: IMF > Global Evaluation Factor

Summary of results in the presence of metabolic activation

+S9
Concentration	RTG	Precipitation	MF	IMF	Proportion small colonies
0 µg/mL	100%	-	75.0	-	0.18
1310 µg/mL	137%	-	84.1	9.08	-
1640 µg/mL	133%	-	73.6	-	-
2050 µg/mL	111%	-	102.3	27.29	-
2560 µg/mL	86%	+	137.2*	62.19	-
3200 µg/mL	75%	+	99.0	24.02	-
4000 µg/mL	13%	+	161.7**	86.70	-
B(a)P	44%	-	624.8	549.8#	0.37

RTG: relative total growth (%)

MF: Mutant Frequency (/10^-6cells)

IMF: Induced Mutant Frequency

#: IMF > Global Evaluation Factor

Conclusions:

It is concluded that ϵ-Caprolactone, oligomeric reaction products with 2,2’-oxydiethanol does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the conditions of this study.

Executive summary:

The potential mutagenicity of ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol was investigated in a mouse lymphoma assay (OECD 490) in the absence and presence of exogenous metabolic activation (phenobarbital / 5,6 -Benzoflavone-induced SD rat liver S9 fraction). Duplicate cell cultures were exposed for three hours to ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol(dissolved in DMSO) at concenterations of up to 1000 µg/mL (-S9) or 4000 µg/mL (+S9). Concentrations were based on the results of a preliminary cytotoxicity assay performed at up to the limit concentration for UVCB substances of 5000 µg/mL. After an expression period of 2 days, cells were cultured in selective medium containing trifluorothymidine for 14 days. Precipitation of the test material was seen at the three highest tested concentrations in the absence and presence of S9. The highest analysable concentrations of 800 µg/mL (-S9) and 4000 µg/mL (+S9) showed high levels of cytotoxicity. In the absence of S9, no increase in mutant frequency was noted at any concentration tested. In the presence of S9, statistically significant increases in mutant frequency were observed at the highest and at an intermediate concentration (2560 µg/mL) and a linear trend was indicated. However, the observed increases were lower than the Global Evaluation Factor and mutation frequencies were within the range defined for the untreated/solvent control cultures; they were therefore considered to be of no biological relevance. It is therefore concluded that ϵ-Caprolactone, oligomeric reaction products with 2,2’-oxydiethanol does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of metabolic activation, under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

ϵ-Caprolactone, oligometric reaction products with 2,2’-oxydiethanol has been investigated to determine 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 under the conditions of this study.

Chromosomal aberration study

The test material (caprolactone, oligomeric reaction products with 2,2’-oxydiethanol) was assayed for the ability to induce chromosomal damage in cultured human lymphocytes, following in vitrotreatment in the absence and presence of S9 metabolic activation. Three treatment series were included in the study. A short-term treatment was performed where cells were treated for 3 hours in the presence and absence of S9 metabolism. The harvest time of 24 hours corresponding to approximately 1.5 cell cycle was used. A long-term (continuous) treatment was also performed in the absence of S9 metabolism until harvest at 24 hours. Solutions of the test material were prepared in DMSO. Concentrations of 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 and 0.0781, 0.0391 and 0.0178 μL/mL were used for all treatment series. In addition, for the continuous treatment in the absence of S9, a concentration of 0.00977 μL/mL was included. Each treatment series included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. For all treatment series, concentrations were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity (as determined by the reduction in mitotic index). For each culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells. Following treatment, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any concentration and treatment condition. No concentration-related increase of the incidence of cells bearing aberrations was observed and all incidences were within the distribution of the historical data for negative controls. Statistically significant increases in the incidence of aberrant cells bearing aberrations (including and excluding gaps) were seen following positive control treatments with Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system. It is concluded that the test material (caprolactone, oligometric reaction products with 2,2’-oxydiethanol) does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under these experimental conditions.

Mouse lymphoma assay

The potential mutagenicity of ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol was investigated in a mouse lymphoma assay (OECD 490) in the absence and presence of exogenous metabolic activation (phenobarbital / 5,6 -Benzoflavone-induced SD rat liver S9 fraction). Duplicate cell cultures were exposed for three hours to ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol (dissolved in DMSO) at concenterations of up to 1000 µg/mL (-S9) or 4000 µg/mL (+S9). Concentrations were based on the results of a preliminary cytotoxicity assay performed at up to the limit concentration for UVCB substances of 5000 µg/mL. After an expression period of 2 days, cells were cultured in selective media containing trifluorothymidine for 14 days. Precipitation of the test material was seen at the three highest tested concentrations in the absence and presence of S9. The highest analysable concentrations of 800 µg/mL (-S9) and 4000 µg/mL (+S9) showed high levels of cytotoxicity. In the absence of S9, no increase in mutant frequency was noted at any concentration tested. In the presence of S9, statistically significant increases in mutant frequency were observed at the highest and at an intermediate concentration (2560 µg/mL) and a linear trend was indicated. However, the observed increases were lower than the Global Evaluation Factor and mutation frequencies were within the range defined for the untreated/solvent control cultures; they were therefore considered to be of no biological relevance. It is therefore concluded that ϵ-Caprolactone, oligomeric reaction products with 2,2’-oxydiethanol does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the conditions of this study.

Justification for classification or non-classification

No classification is proposed for germ cell mutagenicity on the basis of the negative results obtained in the Ames test, chromosomal aberration assay and mouse lymphoma assay with the substance ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol.