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Toxicological information

Genetic toxicity: in vitro

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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
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:
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

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.

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 t-dodecyl mercaptan 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 t-dodecyl 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 t-dodecyl mercaptan 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.