5-ethyl-1,3-dioxane-5-methanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenicity of Cyclic Trimethylolpropane Formal (CTF) has been assessed in an appropriate battery of studies in vitro (Ames test, clastogenicity assay, mouse lymphoma assay).

CTF showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
It was concluded that CTF was not clastogenic when tested with Chinese hamster ovary cell in vitro.
It was concluded that CTF is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested to the predetermined maximum concentration of 1462 μg/mL (10 mM).

Link to relevant study records

Reference

Reference 1

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:

14 April 2008 - 02 September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No. 3777003; 99.9% purity

Target gene:

Histidine and Trytophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

In the first test, the following concentrations were used: 5, 15, 50, 150, 500, 1500 and 5000 µg.In the second test, the maximum concentration used was 5000 µg.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water- Justification for choice of solvent/vehicle: Test material was miscible with water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

-S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

+S9

Details on test system and experimental conditions:

In the first test, aliquots of 0.1ml of the test substance solutions, positive control or negative control were placed in glass vessels. The negative control was water, the vehicle. 0.5ml of S-9 mix or 0.1 M pH 7.4 phosphate buffer (0.5ml) was added, followed by 0.1 ml of a 10-hour bacterial culture and 2 ml of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes weer sued for each treatment. Plates were also prepared without the addition of bacteris to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at 37 °C for approximately 72 hours, after which the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter. In the second test, the pre-incubation assay was used in which the tubes containing mixtures of bacteria, buffer or S9 mix and test dilution were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was 5000 µg/plate, but only 5 concentration were used.

Rationale for test conditions:

The highest concentration of Cyclic Trimethylolpropane Formal tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with water to produce a series of lower concentrations, separated by approximately half-log10 intervals.

Evaluation criteria:

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E9/mL.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the first test, there was no evidence of toxicity was obtained following exposure to Cyclic Trimethylolpropane Formal. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Cyclic Trimethylolpropane Formal at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.In the second test, there was no evidence of toxicity was obtained following exposure to Cyclic Trimethylolpropane Formal. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Cyclic Trimethylolpropane Formal at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Mean revertant counts in the absence of metabolic activation

	Experiment 1					Experiment 2
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
0	29.7	172.3	33.0	12.3	130.3	31.7	164.0	21.0	12.0	165.7
5	31.0	165.0	30.3	14.7	126.7
15	37.0	167.7	30.7	20.7	107.7
50	35.3	160.0	30.0	18.7	133.0	29.3	148.7	18.7	10.7	154.3
150	36.3	146.7	22.7	15.7	137.7	37.3	146.3	16.0	8.3	185.0
500	27.3	143.3	23.7	14.0	152.3	31.3	136.3	18.7	12.0	158.0
1500	28.0	137.3	30.3	13.0	128.0	33.3	157.0	19.3	9.0	158.3
5000	22.3	124.0	29.3	11.7	119.7	29.7	135.0	17.3	8.7	143.3
2NF	193.7					317.3
NaN3		906.3	1150.0				910.3	994.0
AAC				558.0					326.0
NQO					1276.3					1758.3

Mean revertant counts in the presence of metabolic activation

	Experiment 1					Experiment 2
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
0	39.3	180.3	30.3	30.0	139.0	43.0	150.0	24.7	28.0	164.0
5	39.7	155.0	16.3	25.3	141.3
15	43.3	171.7	18.0	28.3	144.0
50	41.0	162.3	20.3	29.3	147.7	38.0	190.7	25.3	26.3	174.7
150	30.7	184.0	24.7	22.7	170.3	37.0	172.7	27.3	29.3	170.3
500	38.0	156.0	23.3	30.7	133.0	36.0	142.3	31.7	28.7	185.3
1500	29.3	158.7	20.3	28.0	120.7	34.3	129.7	23.7	25.7	175.3
5000	26.0	137.7	23.3	23.3	135.7	33.7	127.7	29.0	20.3	173.0
BaP	266.0			128.3		253.3			119.7
AAN		1982.7	429.7		545.3		1660.7	253.7		590.7

Conclusions:

Cyclic Trimethylolpropane Formal showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In the in vitro assessment of the mutagenic potential of Cyclic Trimethylolpropane Formal, conducted in accordance to GLP and OECD Guidelines for the Testing of Chemicals (1997). Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471, EC Commission Directive 2000/32/EC Annex 4D-B.13/14. Mutagenicity - Reverse mutation test in bacteria. No. L 136/57 and US EPA Health Effects Test Guidelines (1998). OPPTS 870.5100 Bacterial reverse mutation test. EPA 712-C-98-247, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli, strain WP2 uvrA (pKM101), were exposed to Cyclic Trimethylolpropane Formal diluted in water. Water was used as a negative control. Two independent mutation tests were performed in the presence and absence of S9 mix from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of Cyclic Trimethylolpropane Formal up to 5000 μg/plate were tested. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of Cyclic Trimethylolpropane Formal in either mutation test. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. It is concluded that Cyclic Trimethylolpropane Formal showed no evidence of mutagenic activity in this bacterial system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Sept 2009 - 01 March 2010

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

The test item, CTF (Cyclic Trimethylolpropane Formal), Batch No. 3977009 (purity 99.7%) was received from Perstorp Holding AB on 04 September 2009. The test item was supplied with an expiry date of 03 September 2010, which was one year from dispatch. The test item, a liquid, was stored at ambient temperature, protected from light and under Nitrogen headspace when not in use. A Certificate of Analysis was supplied with the test item.

Target gene:

Not applicable - chromosome aberration study

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line used was Chinese hamster ovary (CHO 10 B4) cells, obtained from the University of Leiden in 1987. The cell line is mycoplasma tested (in house) on a regular basis. The cells were grown as monolayers, and have a generation time of approximately 12 h. The modal chromosome number has been determined for these cells to be 21. The cells were incubated at ca 37°C.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colcemid was added to all cultures at a final concentration of 0.1 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

For the first experiment, 9 dose levels, covering a wide concentration range, were tested. The highest dose was the limit concentration of 1462 μg/mL (10 mM) and subsequent dose levels were halving dilutions.In the second experiment, the dose levels selected were: 183, 366, 731 and 1462 μg/mL

Vehicle / solvent:

The test item was freely soluble in Ham’s F-10 medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9; 20-50 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9; 10-40 µg/ml

Details on test system and experimental conditions:

The basic medium (Ham's F-10) containing HEPES buffer, was supplemented with the antibiotic minocycline. For cell growth and treatment in the absence of S9 mix, foetal bovine serum (10% v/v) was added. The medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment, was serum free.Cells were trypsinised from stock flasks at passage numbers 17 (Test 1) and 14 (Test 2), and resuspended in fresh culture medium at densities of 0.1 x 10exp6 or 0.05 x 10exp6 cells/mL. These cells, in 5 mL volumes, were dispensed into 25 cm² tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 h post treatment respectively. Test cultures were established from the stock flask about 20 h before testing.All experimental procedures, up to harvesting, were conducted using aseptic technique and under amber light.Tests were conducted both in the presence and absence of S9 mix. Treatments with test item or vehicle control substances were performed on du plicate cell cultures. Several concentrations of the positive controls were tested using single cultures. Cultures to be treated in the presence of S9 mix were washed before treatment with serum free medium. Exposure medium was prepared, immediately before dosing, in sterile containers. The final approximate volume per culture was 5 mL.Test 1 was conducted both with and without S9 mix, the treatment period was 0-6 h, with a recovery period of 6-22 h. Colcemid was added at 22-24 h, and the cells were harvested at 24 h.Test 2 was conducted with S9 mix in a identical manner to Test 1. Test 2 conducted without S9 mix employed either: no recovery period, colcemid was added at 22-24 h and cells harvested at 24 h; or a 22-46 h recovery period, colcemid added at 46-48 h and cells harvested at 48 h. After treatment, cells were washed twice with serum free medium, then full growth medium added, for the recovery period and colcemid treatment. The volume of medium for the recovery period was 5 mL.Harvesting of cultures: Colcemid was added to all cultures at a final concentration of 0.1 μg/mL. Culturing the cells in medium containing colcemid for 2 h accumulated cells in metaphase; the stage of cell division at which chromosomes can be examined using light microscopy. Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation (approximately 190 g), and treated with hypotonic solution (1% trisodium citrate) for 15 min at room temperature. The cells were then fixed (after sedimentation as before) using 4 mL of freshly prepared fixative (methanol:glacial acetic acid, 3:1). Two further changes (after sedimentation as before) of fixative were made. Monolayer cells were trypsinised, counted and discarded. This provided a quantitative measure of toxicity. For both experiments, 3 slides per culture were made. All slides were marked with the study number and assigned a unique, coded number from a computer generated sequence. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. The slides were stained with 5% Giemsa, then made permanent by mounting coverslips with DPX mountant. Living cultures were examined for evidence of changes to cell morphology, once at the end of the treatment period and again before harvesting of cultures. Slides were examined for evidence of metaphase cells and signs of cellular necrosis. Three concentration levels were selected for assessment of chromosomal aberrations. From 2 slides per culture, up to 50 metaphase cells per slide, a total of 100 metaphase cells per culture, were examined where possible. Slides were scored in order of coded number. A reduced number of metaphases were scored if a high proportion (≥40%) of metaphase cells were found to be damaged. A microscope was used for this assessment, the magnification used being x 1000 or x 1250, achieved with x 10 or x 12.5 eyepieces and x 100 objective. The number of chromosomes in each metaphase cell and all abnormalities, using the nomenclature of Gebhart (1970) was recorded. The types of structural and numerical aberrations recorded are listed in abbreviations page. The positions on the slides of any structurally aberrant cells were recorded using the Vernier scale on the microscope stage.As cultures harvested at both culture times were negative with regards to structural aberrations, a further assessment of polyploidy was made. The Study Director selected the later harvest time as being more appropriate for this analysis. This decision was based on results from the previous assessments. For this assessment, approximately 300 metaphase cells were cursorily examined at a magnification of x 400 or x 500 and deemed to be either diploid, polyploid or endoreduplicated. In this assessment no metaphase cell in a field was rejected. This assessment was considered more objective, avoiding scorer selection of either normal or polyploid cells. In addition, the larger sample size gave a more accurate frequency of such cells in the population.

Rationale for test conditions:

For the first experiment, 9 dose levels, covering a wide concentration range, were tested. The highest dose was the limit concentration of 1462 μg/mL (10 mM) and subsequent dose levels were halving dilutions. In the second experiment, the dose levels selected were: 183, 366, 731 and 1462 μg/mL

Evaluation criteria:

Toxicity: A dose level was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.Clastogenicity: The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historical negative control data. The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99% confidence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate. A test was positive if the response in at least one acceptable dose level was significant by the criterion described above. A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence limits). Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.

Statistics:

No statistical analysis was performed.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

No toxicity was noted in any of the cultures treated with CTF, when tested to a limit concentration of 1462 μg/mL.Cultures treated with the following dose levels of CTF were selected for assessment of chromosomal aberrations: 366, 731 and 1462 μg/mL.The vehicle control cultures had levels of structural and numerical aberrations within the 95% confidence limits of the historical negative control data. The positive control substances, cyclophosphamide in the presence and methyl methanesulphonate in the absence of S9 mix, induced positive frequencies of structural aberrations in at least one concentration level per section. These results demonstrated the sensitivity of the test system.All cultures treated with CTF had levels of structural aberrations within the 95% confidence limits for a negative response. An extra assessment of polyploidy was carried out on the cultures treated in the absence of S9 mix and harvested at 48 h. All the cultures treated with CTF had levels of polyploidy within the 95% confidence limits for a negative response.

Frequencies of aberrant cells (exluding gaps)

		Test 1		Test 2
		6h/24h		6h/24h	22h/24h	22h/48h
		+S9	-S9	+S9	-S9	-S9
CTF	0 µg/mL	0.0	0.0	0.0	0.0	0.0
	388 µg/mL	0.0	0.0	0.0	0.0	0.0
	731 µg/mL	0.0	0.5	0.0	0.5	0.0
	1462 µg/mL	0.0	0.0	0.5	0.0	0.0
CPA	20 µg/mL			12
	30 µg/mL	19		27
	40 µg/mL	40
MMS	10 µg/mL				1
	20 µg/mL				10	3
	30 µg/mL		6			10
	40 µg/mL		10

Conclusions:

It was concluded that CTF was not clastogenic when tested with Chinese hamster ovary cell in vitro.

Executive summary:

CTF (cyclic trimethylolpropane formal) was submitted for testing and evaluation of clastogenic potential. Chromosomal aberrations assays were performed with duplicate, Chinese hamster ovary cell cultures. This study was conducted incorporating 2 independent tests. Ham’s F-10 medium was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used in both tests. Both tests were conducted in the presence and absence of a post-mitochondrial supernatant fraction obtained from the livers of adult, male rats treated with Aroclor 1254 (S9) and a NADPH-generating system. Cultures, established approximately 20 h before testing, were treated for 6 h in the presence and 6 h or 22 h in the absence of S9 mix. Cultures were harvested at 24 h (Test 1 and 2) or 48 h (Test 2) post treatment. CTF was non toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix when tested to the limit concentration of 1462 μg/mL (10 mM). There was no evidence that CTF induced structural chromosomal aberrations in either the presence or absence of S9 mix. CTF induced an increase in polyploidy in both the presence and absence of S9 mix in cultures harvested 48 h post treatment. It was concluded that CTF was not clastogenic when tested with Chinese hamster ovary cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 November 2009 - 21 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

CTF (Batch 3977009), a clear, colourless liquid, was received from Perstorp Holding AB on 04 September 2009 and was stored protected from light and under a nitrogen headspace, at ambient room temperature. The purity was quoted as 99.7% and the Sponsor supplied an expiry date of 03 September 2010. A Certificate of Analysis was provided.

Target gene:

tk+tk- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

tk+tk- -3.72C mouse lymphoma L5178Y cell line, obtained from Dr Clive, Burroughs Wellcome and Company, Research Triangle Park USA (in Dec 1982). The cells grow in suspension culture, have a generation time of about 11 hours, have a stable, near-diploid chromosome number and have a high cloning efficiency in serum-enriched cloning medium. The basic cuture medium was RPMI 1640 medium, supplemented with penicillin, streptomycin, sodium bicarbonate and pluronic acid.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from the livers of Aroclor 1254 induced adult male Fischer rats

Test concentrations with justification for top dose:

Toxicity test: 0.15, 0.44, 1.46, 4.39, 14.6, 43.86, 146.2, 438.6 and 1462 μg/mLMutagenicity assay: 183, 366, 731 and 1462 µg/mL.

Vehicle / solvent:

Basic tissue culture medium.The CTF formulations were prepared within 1 h of dosing. Detailed records of preparation of the dosing solutions were maintained to allow checking of procedures. As the formulations were prepared within 1 h of dosing, and when added to the system were subject to metabolism by the cells and S9 mix, it was deemed unnecessary to assess the stability of CTF in the solvent used.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9; 250 µg/ml 4 h exposure, 150 µg/ml 24 h exposure

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 10 µg/ml 4 h exposure, 5 µg/ml 24 h exposure

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: with S9; 2.5 & 10 µg/ml

Details on test system and experimental conditions:

Single cultures were treated for each concentration in the initial toxicity tests. In the mutation assays, duplicate cultures were treated for all CTF concentrations. All experimental procedures were conducted using aseptic technique and under amber light.Toxicity test: The toxicity test was performed using the standard 4 h exposure period in the absence and presence of S9 mix. An additional toxicity test was performed in the absence of S9 mix with 24 h exposure to CTF, as a contingency against the later requirement for a full experiment using this extended exposure period. Observations on the precipitation of CTF were made after dosing and at the end of the exposure period. Observations of pH change (colour change in indicator in RPMI medium) were made and if any change was noted, pH measurements were made.Mutation test 4 h exposure: On the day of test, samples of cell culture were dispensed to sterile tubes and freshly prepared 1 mL S9 mix was added, followed by 4 mL test solution (or appropriate control). The final reaction mixture in all cultures contained 10 m cells, at a population density of ca 6x10E5 cells/mL. All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 4 h. After this, the cells were gently sedimented by centrifugation at ca 200 g for 5 min and resuspended in 10% horse-serum growth medium (20 mL). This step was repeated to give a cell density of ca 3x10E5/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3x10E5 cells/mL on Day 1.Mutation test 24 h exposure: On the day of test, samples of cell culture were dispensed to sterile tubes. 2 mL growth medium:serum (50:50) was added to each tube followed by 8 mL of test solution (or appropriate controls). The final reaction mixture in all cultures contained 20 mL cells, at a population desnsity of ca 3x10E5 cells/mL in growth medium + 10% serum. All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 24 h. After this (on Day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in growth medium + 10% serum (20 mL). This step was repeated. Cell counts were made and the densities adjusted (where higher) to give ca 3x10E5 cells/mL. The cells were returned to the rotatingdrum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3x10E5 cells/mL on Day 2.On Day 2 (4 h exposure) or Day 3 (24 h exposure), cell counts were determined. The cell counts over the 2 or 3 days of the experiments provided a measure of suspension growth. All treated cultures were selected for assessment. The cultures were then assessed for expression of genetic damage. This was determined by performing two parallel cloning assays: the cloning efficiency assay and the mutant selection assay.For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well. For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1x10E4 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well. All dishes were incubated at ca 37°C in an atmosphere of 5% CO2:95% air (v/v) until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay).The plates were scored using a dissecting microscope. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies. Large colonies are defined as covering greater than ¼ of the floor of the well, while small colonies cover less than ¼ of the well. Relative suspension growth was used to assess toxicity, the total suspension growths were expressed as percentages of the vehicle control mean value. Relative total growth was used to assess cytotoxicity, combining suspension growth with the cloning efficiency of the non-mutants. The number of empty wells from the non-mutant cloning efficiency assay and the number of empty wells from the TFT-resistance assay were used to calculate the mutant fraction. Each mutant fraction was expressed per 10E6 viable cells.

Rationale for test conditions:

It was necessary to perform initial toxicity tests in the absence and presence of S9 mix.
The final concentrations of CTF used in the toxicity test were as follows: 0.15, 0.44, 1.46, 4.39, 14.6, 43.86, 146.2, 438.6 and 1462 μg/mL The highest concentration represents the predetermined maximum of 10 mM, as recommended by relevant guidelines (being lower than the alternative, 5000 μg/mL). Subsequently, four mutation assays were performed, as detailed below. The concentrations of CTF tested were as follows (μg/mL): Assay 1 (in the absence of S9 mix*): 183, 366, 731 and 1462 Assay 2 (in the presence of S9 mix*): 183, 366, 731 and 1462 Assay 3 (in the absence of S9 mix**): 183, 366, 731 and 1462 Assay 4 (in the presence of S9 mix*): 183, 366, 731 and 1462. (* Experiment using a 4 h exposure period. ** Experiment using a 24 h exposure period )

Evaluation criteria:

An experiment was considered positive if one or more concentrations were biologically significant and there was a significant linear trend.

Statistics:

The results for each experiment were subjected to statistical analysis by the recommended UKEMS method.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results of the toxicity tests show that CTF did not cause an appreciable reduction in cell growth at the predetermined maximum concentration of 1462 μg/mL (10 mM) in either the 4 h exposure system (absence and presence of S9 mix) or the 24 h exposure system.The solvent control mean mutant fractions were within the normal ranges experienced in the test laboratory and reported in the literature. All positive control log mutant fractions were significantly higher than the vehicle controls at P<0.05.Assays 1 and 2: 4 h exposure with and without S9None of the assessed concentrations tested significant for increase in log mutant fraction in either experiment. In the absence of S9 mix, the test for linear trend was not significant (P = 0.72), while in the presence of S9 mix the test for linear trend was not reported as the slope was negative. There was no toxicity at any concentration of CTF in either experiment. Both experiments were classed as negative.Assays 3 and 4: 24 h exposure without S9, 4 h exposure with S9In the absence of S9 mix, the overall mutant (Hm) heterogeneity factor was statistically significantly higher than the historical control and therefore no statistical analysis was reported for this assay. The mutant fractions obtained with CTF were all very close to the vehicle control value and it can be assumed that no mutagenic activity was present in these cultures. In the presence of S9 mix, none of the assessed concentrations tested significant for increase in log mutant fraction and the test for linear trend was not significant (P = 0.70). CTF was not toxic in either experiment.Both experiments were classed negative.

Assay 1 (-S9) 4 hour exposure		RTG (%)	MF (/10^6)	IMF (/10^6)	Small:large colonies
Vehicle	-	100	57	-	0.73
EMS	250 µg/mL	58	551	493	0.57
MMS	10 µg/mL	36	1015	958	3.79
CTF	183 µg/mL	97	53	-	1.07
	366 µg/mL	114	71	14	1.37
	731 µg/mL	102	72	15	1.65
	1462 µg/mL	92	59	2	2.30

RTG: relative total growth

MF: mutant frequency

IMF: induced mutant frequency

Assay 2 (+S9) 4 hour exposure		RTG (%)	MF (/10^6)	IMF (/10^6)	Small:large colonies
Vehicle	-	100	64	-	1.30
3MC	2.5 µg/mL	79	600	535	1.40
	10 µg/mL	63	986	921	1.41
CTF	183 µg/mL	78	81	17	0.66
	366 µg/mL	99	71	6	1.69
	731 µg/mL	93	57	-	1.90
	1462 µg/mL	88	59	-	0.91

RTG: relative total growth

MF: mutant frequency

IMF: induced mutant frequency

Assay 3 (-S9) 24 hour exposure		RTG (%)	MF (/10^6)	IMF (/10^6)	Small:large colonies
Vehicle	-	100	62	-	2.20
EMS	150 µg/mL	32	1726	1664	0.47
MMS	5 µg/mL	22	1963	1901	1.77
CTF	183 µg/mL	100	71	9	0.59
	366 µg/mL	96	53	-	1.45
	731 µg/mL	101	56	-	1.45
	1462 µg/mL	91	36	-	1.70

RTG: relative total growth

MF: mutant frequency

IMF: induced mutant frequency

Assay 4 (+S9) 4 hour exposure		RTG (%)	MF (/10^6)	IMF (/10^6)	Small:large colonies
Vehicle	-	100	64	-	1.76
3MC	2.5 µg/mL	90	814	750	1.43
	10 µg/mL	45	989	925	1.29
CTF	183 µg/mL	93	91	27	2.11
	366 µg/mL	98	70	6	1.48
	731 µg/mL	89	87	23	1.70
	1462 µg/mL	86	71	7	1.50

RTG: relative total growth

MF: mutant frequency

IMF: induced mutant frequency

Conclusions:

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

Executive summary:

CTF was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine kinase locus: tk+tk- to tk-tk-, according to OECD guideline 476. CTF was dissolved and diluted in tissue culture medium. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). In preliminary cytotoxicity tests, CTF was shown was shown to be non-toxic at the predetermined maximum concentration of 1462 μg/mL (10 mM). Four independent mutation assays were conducted with final test concentrations of 183, 366, 731 and 1462 µg/mL, employing exposure times of 4 h (with and without S9) and 24 h (without S9). Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of ‘small’ type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate.

No evidence of mutagenic activity was obtained with CTF in any of the 4 assays. It was concluded that CTF is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested to the predetermined maximum concentration of 1462 μg/mL (10 mM).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP/OECD Guideline 471 study to determine the mutagenic potential of 5 -ethyl-1,3 -dioxane-5 -methanol (CTF) in bacterial cells in vitro (May, 2008), negative results were obtained using the bacterial reverse mutation assay (Ames test) in Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and in Escherichia coli,strain WP2 uvrA (pKM101) in the presence and in the absence of metabolic activation (S9 mix). In an OECD Test Guideline 473 study to determine the clastogenicity of 5 -ethyl-1,3 -dioxane-5 -methanol in Chinese Hamster Ovary cells in vitro (Murie, 2010), no clastogenic potential was found in the presence or in the absence of metabolic activation. Negative results were also reported in an OECD Test Guideline 476 study to determine the mutagenic potential of the substance using the mouse lymphoma assay (L5178Y cell line), in the presence and the absence of metabolic activation using S9 mix (Riach, 2010). Both studies were compliant with GLP and involved testing up to the respective limit concentrations.

Justification for classification or non-classification

5 -ethyl-1,3 -dioxane-5 -methanol is not mutagenic in bacterial cells or in mammalian cells in vitro. The substance does not meet the criteria for classification for genetic toxicity according to the CLP Regulation.