ε-Caprolactone, oligomeric reaction products with propylidynetrimethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6th to 15th January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

CAPA 3050

IUPAC Name:

CAPA 3050

Constituent 2

Reference substance name:

ε-Caprolactone, oligomeric reaction products with propylidynetrimethanol

EC Number:

500-099-5

EC Name:

ε-Caprolactone, oligomeric reaction products with propylidynetrimethanol

Cas Number:

37625-56-2

Molecular formula:

(C6H14O3)x.C6H10O2 (x=0-6)

IUPAC Name:

2-Oxepanone, polymer with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol

Constituent 3

Reference substance name:

2-Oxepanone, polymer with 2-ethyl-2-(hydroxymethyl)-1, 3-propanediol

IUPAC Name:

2-Oxepanone, polymer with 2-ethyl-2-(hydroxymethyl)-1, 3-propanediol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): CAPA 3050
- Substance type: Oligomer
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: WCB000999
- Expiration date of the lot/batch: 23th August 2015
- Stability under test conditions: Stable for the duration of the study
- Storage condition of test material: Ambient

Method

Target gene:

Histidine or tryptophan loci

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the liver of Aroclor 1254-induced adult male Fischer rats

Test concentrations with justification for top dose:

Toxicity test: 17, 50, 167, 500, 1667 and 5000 µg/plate.Mutation experiment: 17, 50, 167, 500, 1667 and 5000 µg/plate.

Vehicle / solvent:

Initial solubility tests showed that dimethysulphoxide (DMSO) was a suitable solvent, and it was used to dissolve and dilute the test item throughout the study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; all strains with S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: without S9; TA 1535 and TA 100

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: without S9; TA 1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: without S9; TA 98

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: without S9; WP2uvrB

Details on test system and experimental conditions:

Samples of each bacterial strain were grown up by culturing for 16 h at 37°C in nutrient broth (25 g Oxoid Nutrient Broth No. 2 per litre). Fresh overnight cultures were used in the tests for this study. Samples from cultures were kept for up to 7 days at 4°C to allow relevant checks to be performed.Two experiments were conducted in both the absence and presence of S9 mix. The first test was performed using the Direct Plate Incorporation Method. If the first test was negative, the repeat test was performed using the Pre-Incubation Method. If the the first test was positive, the second test was also performed using the Direct Plate Incorporation Method to confirm the response. Triplicate plates were poured for each exposure level (n=6) and bacterial strain (n=5) in the absence or presence of S9. Aseptic techniquies, conducted under amber light, were used throughout the study. All water used in the preparation of reagents wa sproduced (in-house) by reverse osmosis followed by mixed-bed deionisation and sterilisation by autoclaving.Toxicity tests: S. typhimurium strain TA 100 was used for the toxicity tests, according to the direct plate mutation assay (see below). Plates were incubated at 37°C for 2-3 days. The number of revertant colonies were noted, and the paltes examined microscopically for thinning of the background lawn or microcolonies. Mutation testsDiluted agar (0.6% Difco Bacto-agar, 0.6% NaCl) was autoclaved, and just before use supplemented as follows: For S. typhimurium, sterile 1.0 mM L-histidine.HCl, 1.0 mM biotin solution was added at 50 mL per litre of soft agar. For E. coli, sterile 1.35 mM L-tryptophan solution was added at 10 mL per litre of soft agar. These soft agars were thoroughly mixed and kept in a water bath at 45°C.Direct plate method: 2 mL of soft agar were dispensed into a small, plastic, sterile tube. S9 mix or 0.05 M phosphate buffer, pH 7.4 (0.5 mL) was added, followed by 0.1 mL of bacteria and, finally, the test item solution (0.1 mL). The tube contents (which were continually cooling) were mixed, then poured on to minimal medium plates prepared in-house. The minimal medium plates contained 20 mL of 1.5% BBL Purified Agar in Vogel-Bonner Medium E (Vogel and Bonner, 1956) with 2% glucose.Pre-incubation method: For this assay method, 0.5 mL of S9 mix or 0.05 M phosphate buffer, pH 7.4 was dispensed into a small, plastic, sterile tube followed by 0.1 mL of bacteria and, finally, the test solution (0.1 mL). The tubes were then placed in a shaking incubator at ca 37°C for 20 min. After incubation, 2 mL of soft agar was added to each tube. The tube contents were then mixed and poured onto minimal medium plates (preparation described above).When the soft agar had set, the plates were inverted and incubated at ca 37°C for 2 or 3 days and then examined. The numbers of mutant colonies on each plate were determined using a Sorcerer Colony Counter and captured electronically in a validated software system Ames Study Manager (both from Perceptive Instruments). The plates were also examined microscopically for precipitates and for microcolony growth.

Evaluation criteria:

Toxicity test: number of revertant colonies and thinning of the background lawn.Mutation tests: For S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli WP2uvrA, at least a doubling of the mean concurrent vehicle control value was required before mutagenic activity was suspected. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value was considered indicative of a mutagenic effect.If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 (representing a 2-fold increase over 10) was required before a response was registered. A concentration-related response was also required for identification of a mutagenic effect. At high concentrations this relationship may be reversed because of, for example, toxicity of the test item to the bacteria, specific toxicity of the test item to the mutants, or inhibition of S9 enzymes (where a mutagen requires metabolic activation by the S9 mix). A response should be reproducible in an independent test.

Statistics:

The mean number of mutant colonies, plus standard deviation, was calculated for each set of 3 plates. In addition, the fold-increase over the vehicle control was calculated for all test item and positive control treatments. No statistical analysis was performed in this study.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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 examined

Positive controls validity:

valid

Test results 2

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:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity test: No toxicity to TA 100 was evident. CAPA 3050 precipitated at 1667 and 5000 μg per plate.Mutation tests: The vehicle control values were within the normal/historical ranges recorded in the test laboratory and reported in the literature with these strains of S. typhimurium and E. coli. The positive control values were within the normal/historical ranges recorded in the test laboratory for each bacterial strain and activation condition. No evidence of mutagenic activity was obtained with CAPA 3050 in any strain in either test.CAPA 3050 was not toxic to any strain in either test. CAPA 3050 precipitated at 1667 and 5000 μg per plate in both tests.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No evidence of mutagenic activity was obtained with CAPA 3050 in any strain in either test. CAPA 3050 was not toxic to any strain in either test. CAPA 3050 precipitated at 1667 and 5000 μg per plate in both tests.

Summary of findings

µg/plate	Experiment 1
	TA1535		TA1537		TA98		TA100		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
-control	15.0	12.0	15.3	12.0	27.7	30.0	95.7	102.0	4.7	12.0
17	14.3	11.7	9.3	14.3	27.0	26.3	112.7	108.7	6.7	10.7
50	8.0	5.7	11.3	13.7	30.7	29.3	106.7	109.3	6.7	8.3
167	12.3	11.7	10.0	14.0	16.3	34.0	93.0	104.3	9.3	10.3
500	15.7	16.7	12.7	13.0	25.0	33.7	115.7	104.0	4.7	10.3
1667	13.3	11.3	11.3	12.0	26.3	36.7	109.0	89.7	5.7	11.7
5000	12.0	13.0	12.3	13.7	31.0	30.3	120.7	95.7	8.7	8.0
+control	508	759	3135	756	712	1296	1095	1928	72	493
µg/plate	Experiment 2
	TA1535		TA1537		TA98		TA100		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
-control	11.0	12.3	11.7	10.3	25.7	36.0	86.3	96.7	6.0	8.0
17	14.7	13.3	16.3	11.7	29.0	29.7	56.0	86.7	4.3	12.3
50	12.3	12.0	8.7	13.3	19.7	33.3	80.0	94.0	3.7	10.7
167	11.7	15.7	15.0	14.0	17.7	27.0	59.3	84.0	2.3	10.3
500	10.7	9.3	13.3	13.0	13.0	34.3	110.3	84.7	2.0	15.0
1667	11.3	11.7	12.0	13.0	20.0	33.3	110.7	104.0	3.0	14.3
5000	11.3	11.7	7.0	10.3	20.7	41.7	102.3	93.0	3.0	8.3
+control	141	274	1050	264	733	815	1041	938	95.7	343

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negative with and without metabolic activationNo evidence of mutagenicity was seen under the conditions of this study.

Executive summary:

CAPA 3050 was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2uvrA. CAPA 3050 was dissolved and diluted in dimethylsulphoxide. Two independent tests were conducted on agar plates in triplicate in the absence and presence of exogenous metabolic activaiton (S9 mix). The first test was conducted by the direct plate incorporation method, while the second test was conducted by the pre-incubation method. CAPA 3050 was dosed at concentrations ranging from 17 to 5000 μg per plate (the predetermined maximum). Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. No evidence of mutagenic activity was obtained with any strain in either test. CAPA 3050 was not toxic to the bacteria. CAPA 3050 precipitated at concentrations of 1667 and 5000 μg per plate. It was concluded that CAPA 3050 is not mutagenic in strains of Salmonella typhimurium and Escherichia coli when tested in dimethylsulphoxide in the absence and presence of metabolic activation at concentrations up to the predetermined maximum of 5000 μg per plate. The concentration of 5000 μg per plate also exceeded the limit of solubility of CAPA 3050 in the test system