Didodecyl 3,3'-thiodipropionate

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:

1992-08-05, 1992-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, performed under GLP (exposure period of 2 hours)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(exposure period of 2 hours)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Didodecyl 3,3'-thiodipropionate

EC Number:

204-614-1

EC Name:

Didodecyl 3,3'-thiodipropionate

Cas Number:

123-28-4

Molecular formula:

C30H58O4S

IUPAC Name:

didodecyl 3,3'-sulfanediyldipropanoate

Details on test material:

- Name of test material (as cited in study report): Dilauryl thiodipropionate
- Physical state: solid, white powder
- Analytical purity: 96.1 %
- Purity test date: 1992-06-01, 1992-06-11
- Lot/batch No.: Reagens 05/92
- Storage condition of test material: room temperature in the dark

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium, 10% (v/v) foetal calf serum (FCS), and 100 µg/mL gentamycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

29.4, 42, and 60 µg/mL (experiment I) and 60, 92.3, and 142 µg/mL (experiment II)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 h (with metabolic activation, cells were then washed twice with sterile saline and re-fed with fresh medium containing foetal calf serum and gentamycin, cultures were then incubated for a further 18 or 42 hours before harvesting), 20 and 44 hours (without metabolic activation)
- Expression time (cells in growth medium): 18 and 42 hours (with S9-mix), 20 and 44 hours (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 and 44 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine (one and a half hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 (4, only for the negative control)
NUMBER OF CELLS EVALUATED: 100 metaphases of each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Determination of hyperdiploid cells: yes

Evaluation criteria:

The CHO assay was to be considered valid if the following criteria were met:
1) the binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.
2) the proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range and the percentage of polyploid/ endoreduplicated/ hyperdiploid cells was <10%
3) at least 160 cells out of an intended 200 were analysable at each treatment level.
4) the positive control chemicals induced statistically significant increases in the number of cells with structural aberrations.

The test chemical was to be considered as clearly positive in this assay if:
1) statistically significant increases in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentration
2) the proportion of aberrant cells at such data points exceeded the normal range.
3) the results were confirmed in the second experiment.

Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations not exceeding the normal range or occurring only at very high or very toxic concentrations were likely to be concluded as "equivocal". Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance. A positive result only at the delayed harvest in Experiment II was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met.

Statistics:

Aberrant cells in each culture were categorized as follows:
1. cells with structural aberrations including gaps
2. cells with structural aberrations excluding gaps
3. polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures was used to determine whether the assay was acceptable or not.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of cells in category 2 for each test treatment condition, were compared with the proportion in negative controls using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to historical control (normal) ranges.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: low
- Precipitation: at 39 µg/mL and above

COMPARISON WITH HISTORICAL CONTROL DATA: Comparable. Few values (mostly positive controls, one test substance, and two solvent controls) exceeded historical negative control ranges.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table: Chromosomal aberrations in CHO cells after treatment with the test substance:

Experiment I
Test item	Concentration	Mitotic Index	Cells with aberrations
	in µg/mL	mean	including gaps	excluding gaps
Exposure period 2h, fixation time 18h, with S9 mix
Solvent	0	10.6	8	5
CPA	12.5	-	24	21
Test substance	29.4	14.5	6	6
	42	11.9	7	5
	60	12.8	4	3
Exposure period 20h, without S9 mix
Solvent	0	5	4	2
NQO	0.0625	-	24	23
Test substance	29.4	3.9	8	4
	42	5.1	8	4
	60	3.7	4	2
Experiment II
Exposure period 2h, fixation time 18h, with S9 mix
Solvent	0	7.3	2	2
CPA	25	-	47	46
Test substance	60	6.3	4	2
	92.3	7.3	6	4
	142	6.5	7	5
Exposure period 20h, without S9 mix
Solvent	0	4.6	5	1
NQO	0.125	-	22	22
Test substance	60	4.3	6	3
	92.3	2.6	5	3
	142	3	4	1
Exposure period 2h, fixation time 42h, with S9 mix
Solvent	0	1.8	11	6
Test substance	92.3	1.1	9	7
Exposure period 44h, without S9 mix
Solvent	0	5.2	8	2
Test substance	142	7.8	7	2

CPA: cyclophosphamide

NQO: 4-nitroquinoline-N-oxide

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation