Reaction mass of 1,3-dioxane-5,5-dimethanol and 1,3-Propanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]-

Genetic toxicity: in vitro

Administrative data

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:

2 September 2009 to 6 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and Guideline compliant study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Acetaldehyde, reaction products with formaldehyde, by-products from

EC Number:

270-480-6

EC Name:

Acetaldehyde, reaction products with formaldehyde, by-products from

Cas Number:

68442-60-4

IUPAC Name:

68442-60-4

Constituent 2

Reference substance name:

Polyol PX

IUPAC Name:

Polyol PX

Test material form:

other: solid

Details on test material:

- Physical state: amber waxy solid
- Lot/batch No.: 391191549 (B)
- Expiration date of the lot/batch: 15 May 2014
- Storage condition of test material: in the dark at ambient temperature

Method

Target gene:

Not applicable (clastogenicity study)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Basic medium (Ham's F-10 medium containing HEPES buffer and supplemented with minocycline) for each treatment condition at approximately 5x10^5 cells/25 cm2 flask.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 (Aroclor 1254-induced rat liver), mixed with 25 mM glucose-6-phosphate, 4mM nicotinamide adenine dinucleotide phosphate (NADP) and disodium salt

Test concentrations with justification for top dose:

The following test concentrations were : 156, 313, 625, 1250 in ug/mL active ingredient.
There were also positive, negative and untreated controls (consisting of cells in complete medium or S-9 reaction mixture) used.
Polyol PX did not change the colour of the culture medium, therefore, no pH measurements were made.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

cyclophosphamide with activation

Positive control substance:

cyclophosphamide

Controls 2

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

methylmethanesulfonate without activation

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

In the definitive assay CHO cells were seeded at 5 x 10000 cells per 25 cm2 flask about 20 hours before exposure. Flasks were re-fed with medium, S9 where appropriate and the cells were exposed to various test substance concentrations for 6 hours, without metabolic activation or with S9 activation. For the non-activated system, two hours prior to scheduled harvest the cells were treated with Colcemid. For the activated system, after six hours exposure the treatment medium was removed, cells washed twice with serum free medium, then full growth medium was added for the recovery period, two hours prior to scheduled harvest the cells were treated with Colcemid. Cells were harvested by trypsinisation approximately 12 hours after initiation of treatment. Cells were collected by centrifugation 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. Slides were prepared, the cells were stained with Giemsa and permanently mounted.

METHOD OF APPLICATION: in medium - treatment was carried out by refeeding the flasks with basic medium or S-9
reaction mixture and into this the dosing solution was added.
DURATION- Preincubation period: of the CHO cells was 20 hours
- Exposure duration: 6 hours for the non-activated study and 6 hours for the activated S9 study
Non-activated study: 2 hours prior to the scheduled cell harvest, Colcemid was added to the treatment flasks at a final
concentration of 0.1 ug/mL and the cells were returned to the incubator until cell collection.
Activated S-9 study: After exposure, the treatment medium was removed, the cells were washed in serum free medium, refed with
full growth medium for the recovery period. 2 hours prior to the scheduled cell harvest, colcemid was added to each treatment flask at a
final concentration of 0.1 ug/mL and the flasks were incubated for 2 more hours.
- Harvesting of cells: occurred 12 hours after treatment initiation
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: minimum of 100 metaphase spreads were examined and scored (50 per duplicate
flask)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

The test material was considered to give a negative result if the responses were within the 95% confidence limit of 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.
The test material was considered to give a positive result if the response in at least one acceptable dose level was significant by the criterion described above.

Statistics:

None reported.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

: toxicity at 313-5000 µg/mL with metabolic activation; cytotoxicity at 625-5000 µg/mL without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9 mix, a positive response was noted in the lesions per cell parameter in both cultures treated with 625 μg/mL. In one of the cultures treated with this concentration, a positive response was also noted in the aberrant cells excluding gaps parameter, with a suspicious response in the aberrant cells including gaps parameter. The duplicate culture had levels of aberrations within the 95% confidence limits of the historical negative control data in these two parameters. All the concentrations where the clastogenic responses were observed were deemed toxic to the cells. Due to the positive responses noted in Test 1, it was not deemed necessary to conduct the second test.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Concentration	Aberrant cells excluding gaps (%)
	Test 1
	+S9	-S9
	6h / 24h	6h / 24h
-control	0,0	0,0
156 µg/mL	0,0	0,0
625 µg/mL	3,1	0,0
1250 µg/mL	16,13	2,5
+control	13	7
+control	21	11

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
positive with metabolic activation
positive without metabolic activation

Evidence of clastogenicity was seen under the conditions of this assay.

Executive summary:

Polyol PX was submitted for testing and evaluation of clastogenic potential. A chromosomal aberration assay was performed with duplicate, Chinese Hamster Ovary cell cultures. Ham’s F-10 medium was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used. The test was 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 absence of S9 mix. Cultures were harvested at 24 h post treatment. Polyol PX was toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix (Appendices 1 and 2). It was tested to the maximum permitted concentration of 5000 μg/mL. Toxicity was noted at 313-5000 μg/mL in the presence of S9 mix and at 625-5000 μg/mL in the absence of S9 mix. The concentrations assessed for aberrations were 156, 625 and 1250 μg/mL (presence of S9 mix) and 156, 313 and 625 μg/mL (absence of S9 mix). There was evidence that Polyol PX induced structural chromosomal aberrations in both the presence and absence of S9 mix. It was concluded that Polyol PX was clastogenic when tested with Chinese hamster ovary cells in vitro, in the presence and absence of S9 mix, at concentrations deemed toxic to the cells.