1,1-dimethylheptanethiol

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (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:

Tert-Dodecanethiol (CAS # 25103-58-6)

IUPAC Name:

Tert-Dodecanethiol (CAS # 25103-58-6)

Details on test material:

- Supplier: Elf Aquitaine Production
- Name of test material (as cited in study report): t-dodecyl mercaptan
- Physical state: liquid
- Analytical purity: 98.58%
- Purity test date: April 1996
- Lot/batch No.: 42414
- Expiration date of the lot/batch: December 1997

Method

Species / strain

Species / strain / cell type:

other: Human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals

Test concentrations with justification for top dose:

16.89, 22.53, 30.03 µg/ml (20-h treatment, -S9)
53.39, 71.19, 94.92 µg/ml (3-h treatment, -S9)
16.89, 22.53, 30.03 (44-h treatment, -S9)
94.92 µg/ml (3-h treatment, +S9)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline 1-oxide (-S9) and cyclophosphamide (+S9)

Details on test system and experimental conditions:

t-Dodecyl mercaptan was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male human donor. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The highest dose level used, 300 µg/mL was just in excess of the limit of solubility in culture medium.

Treatment in the absence of S-9 was continuous for 20 or 44 hours (20+0, 44+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17 or 41 hour recovery period (3+17, 3+41). The test article dose levels for chromosome analysis were selected by evaluating the effect of t-dodecyl mercaptan on mitotic index. Following 20+0 hour treatments, -S-9 and 3+17 hour treatments, +S-9, chromosome aberrations were analysed at three consecutive dose levels. The highest concentrations chosen for analysis, 30.03 µg/mL and 94.92 µg/mL, induced approximately 48% and 70% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively.

The effects of these single concentrations only, (30.03 µg/mL, without and 94.92 µg/mL with S-9) were initially investigated at the delayed (44+0, 3+41) sampling time. Slides from cultures treated with 16.89 and 22.53 µg/mL in the absence of S-9 were subsequently examined to further investigate chromosome damage induced under these conditions.

Appropriate negative (solvent) control cultures and untreated cultures were included in the test system under each treatment condition.

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Where possible, 100 metaphases from each code were analysed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
Slides were examined, uncoded, for mitotic index (MI) or percentage of cells in mitosis. Slides from enough dose levels from each treatment regime were scored to determine if chemically induced mitotic inhibition had occurred. This is defined as a clear decrease in mitotic index compared with negative controls (based on at least 1000 cells counted), preferably dose-related.

OTHER EXAMINATIONS:
- Determination of polyploidy/ endoreplication: Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 46 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately.

Evaluation criteria:

The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and
2) the proportion of cells with structural aberrations at such doses exceeded the normal range.
A positive result only at the delayed harvest was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met. 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". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one structural aberration were to be considered of particular biological significance.

Statistics:

Fisher's exact test. Probability values of p <0.05 were accepted as significant

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

30.03 µg/mL and 94.92 µg/mL, induced approximately 48% and 70% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Treatment of cultures with t-dodecyl mercaptan in the absence and presence of S-9 resulted, at the 20 hour sampling time, in frequencies of cells with aberrations which were similar to those seen in concurrent negative controls. Frequencies of cells with aberrations (excluding gaps) fell just outside historical negative control ranges in one culture at the highest dose level analysed after treatment both with and without S-9, but insofar as the effect was small and not seen in both replicates it was considered unlikely to be biologically significant. Treatment of cultures with t-dodecyl mercaptan for 44 hours in the absence of S-9 resulted in numbers of cells with aberrations which were significantly higher than in concurrent negative controls at all dose levels analysed. The numbers of cells with aberrations (excluding gaps) observed exceeded the historical negative control range, but this effect was only seen in both replicates at the highest concentration tested (30.03 µg/mL) at which severe mitotic inhibition was apparent. At both 16.89 and 22.53 µg/mL numbers of aberrant cells exceeded the normal range in only one of the two replicate cultures analysed. No increase in aberrant cells was seen at the delayed sampling time after treatment in the presence of S-9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Human lymphocytes

Any other information on results incl. tables

t-Dodecyl Mercaptan: cells with structural aberrations

TABLE 1

20 hour treatment -S-9, 0 hour recovery (20+0) Donor sex: male

Treatment	Replicate	Cells	Cells with	Cells with         Significance	Mitotic
(µg/mL)		scored	aberrations including gaps	aberrations              § encluding gaps	index (mean)
Solvent	A	100	4	2	4.7
	B	100	4	4	4.1
	Totals	200	8	6	(4.4)
16.89	A	100	6	3	3.9
	B	100	7	3	4.3
	Totals	200	13	6                      NS	(4.1)
22.53	A	100	7	3	3.4
	B	100	6	3	4.3
	Totals	200	13	6                      NS	(3.9)
30.03	A	100	3	1	2.1
	B	100	10	6	2.5
	Totals	200	13	7                      NS	(2.3)
NQO, 2.5	A	25	S	S
	B	25	$	$
	Totals	50	16	16               p 0.001

TABLE 2

3 hour treatment +S-9, 17 hour recovery (3+17) Donor sex: male

Treatment	Replicate	Cells	Cells with	Cells with         Significance	Mitotic
(µg/mL)		scored	aberrations including gaps	aberrations               § excluding gaps	index (mean)
Solvent	A	100	1	1	5.5
	B	100	2	1	5.0
	Totals	200	3	2	(5.3)
53.39	A	100	5	3	3.2
	B	100	3	0	5.2
	Totals	200	8	3                      NS	(4.2)
71.19	A	100	$	4	4.3
	B	100	3	2	2.9
	Totals	200	11	6                      NS	(3.6)
94.92	A	100	0		0.7
	B	100	6	4	2.5
	Totals	200	16	11                  p <0.01	(1.6)
CPA, 12.5	A	25	19	i
	B	25	13
_	Totals	50	32	25                 p <0.001

TABLE 3

44 hour treatment -S-9, 0 hour recovery (44+0) Donor sex: male

Treatment	Replicate	Cells	Cells with	Cells with        Significance	Mitotic
(µg/mL)		scored	aberrations including gaps	aberrations               § encluding gaps	index (mean)
Solvent	A	100	1	0	2.8
	B	100	3	2	4.3
	Totals	200	4	2	(3.6)
16.89	A	100	9	2	3.3
	B	100	11		3.2
	Totals	200	20	8                  p <0.05	(3.3)
22.53	A	100	5	3	2.8
	B	100	9	7	2.8
	Totals	200	14	10                 p < 0.05	(2.8)
30.03	A	56	9	7	0.6
	B	100	24	14	0.7
_	Totals	156              _	33	21                 p <0.001	(0.7)

TABLE 4

3 hour treatment +S-9, 41 hour recovery (3+41) Donor sex: male

Treatment	Replicate	Cells	Cells with	Cells with        Significance	Mitotic
(µg/mL)		scored	aberrations including gaps	aberrations              § excluding gaps	index (mean)
Solvent	A	100	2	1	4.3
	B	100	2	2	3.6
	Totals	200	4	3	(4.0)
94.92	A	100	5	4	3.3
	B	100	4	2	1.8
	Totals	200	9	6                      NS	(2.6)

§ Statistical significance (Appendix 5b) NS = not significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation at cytotoxic concentrations
negative with metabolic activation

It is concluded that tert-dodecanethiol induced chromosome aberrations in cultured human peripheral blood lymphocytes. The effect, however, was restricted to prolonged, cytotoxic treatment in the absence of S-9.

Executive summary:

In a chromosomal aberration assay performed according to OECD guideline #473, human lymphocytes were exposed to tert-dodecanethiol concentrations up to 94 µg/ml, with and without metabolic activation. No effects were observed at the 20-hour sampling time. At 44 hours in the absence of S9, the number of cells with aberrations was significantly higher than in concurrent negative controls at all dose levels analyzed. The numbers of cells with aberrations observed exceeded the historical control range, but this effect was only seen in both replicates at the highest concentration tested (30.03 µg/mL) at which severe mitotic inhibition was apparent. It was concluded that tert-dodecanethiol induced chromosomal aberrations in cultured human peripheral blood lymphocytes; however, this effect was restricted to prolonged, cytotoxic treatment in the absence of S9, and is therefore, considered to be ambiguous.