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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
19 Nov 1992 - 27 Feb 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance Fatty acids, C18-unsatd., dimers (CAS No. 61788-89-4). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: UK Department of Health Report on Health and Social Subjects No. 35. Guidelines for the testing of chemicals for mutagenicity (1989)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dimer acid
- Physical state: viscous golden liquid
- Substance type: UVCB
- Analytical purity: dimer content 73%, trimer content 21%
- Expiration date of the lot/batch: April 1993
- Storage condition of test material: room temperature

Method

Target gene:
Not applicable
Species / strain
Species / strain:
lymphocytes: human blood (males)
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640 + 20% foetal calf serum + 175 µg/mL phytohaemagglutinin
- Properly maintained: 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:
Experiment 1:
0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75, 150, 300 µg/mL (18 h harvest, with and without metabolic activation)
Experiment 2:
37.5, 75, 150, 300 µg/mL (18 h harvest, without metabolic activation)
75, 150, 300 µg/mL (18 h harvest, with metabolic activation)
9.4, 18.8, 37.5, 75, 150, 300 µg/mL (32 h harvest, without metabolic activation)
18.8, 37.5, 75, 150, 300 µg/mL (32 h harvest, with metabolic activation)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on assessment of test substance solubility prior to commencing of testing.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO (10 µL/mL)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
500 and 750 µg/mL without metabolic activation
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO (10 µL/mL)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
10 and 15 µg/mL with metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 and 32 h (without metabolic activation) ; 3 h (with metabolic activation)
- Expression time (cells in growth medium): 15 and 29 h (after treatmet with metabolic activation)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 32 h

SPINDLE INHIBITOR: colchicine (0.25 µg/mL)
STAIN: Giemsa (1:9 dilution in buffered distilled water)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
A test result was considered positive, if a clear treatment-related and statistically significant increase (Fisher’s test) in the number of cells containing chromosome aberrations, not falling within the historical control of the testing facility (0-5.25%) and some evidence of a dose-relationship was observed.
Statistics:
The number of aberrant cells in each treatment group was compared with the solvent control value by means of the Fisher's test.

Results and discussion

Test results
Species / strain:
lymphocytes: human blood (males)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 300 µg/mL (18 h harvest ) and from 150 µg/mL (32 h harvest), without metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: prior to commencing testing, the solubility of the test substance was assessed. The test substance dissolved at 500 mg/mL in DMSO. However on dosing at 1% v/v into aqueous tissue culture medium, a precipitate was found. The final concentration used for subsequent testing was 300 µg/mL, at which a slight precipitate was observed.
- Precipitation: in the main tests, slight to heavy precipitate was observed at 150 and 300 µg/mL with and without metabolic activation for both the 18 and 32 h harvest.

COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent control values were within the historical control of the testing facility (0-5.25%).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1, at 300 µg/mL, the mitotic index was reduced to 65% of the solvent control value in the absence of S9 mix and no decrease was observed in its presence.
In Experiment 2, in the presence of S9 mix (18 and 32 h harvest) and in the absence of S9 mix (18 h harvest), no significant reductions in mitotic index were observed at the maximum achievable concentration of 300 µg/mL. In the absence of S9 mix at 32 h harvest, the highest analysable concentration of 150 µg/mL caused a decrease in mitotic index to 63% of the solvent control value. The highest concentration of 300 µg/mL was too toxic for analysis.

Any other information on results incl. tables

Table 1. Results of chromosome aberration test in human lymphocytes (duplicate cultures), Experiment 1.

Test item

Concentration

(µg/mL)

Relative mitotic index (%)

Mean No. of aberrant cells per 100 cells examined

Without gaps

With gaps

Exposure: without S9 mix, 18 h harvest

DMSO

10 µL/mL

100

0.25

0.25

Test substance

0.6

90

ND

ND

1.2

98

ND

ND

2.3

101

ND

ND

4.7

95

ND

ND

9.4

93

ND

ND

18.8

100

ND

ND

37.5

107

ND

ND

75

104

1.5

1.5

150

80

1.5

1.5

300 (Pa)

65

1.0

1.5

Ethylmethanesulphonate

500

ND

28.0*

28.0*

Exposure: with S9 mix, 18 h harvest (3 h + 15 h recovery)

DMSO

10 µL/mL

100

0.5

0.5

Test substance

0.6

102

ND

ND

1.2

107

ND

ND

2.3

111

ND

ND

4.7

112

ND

ND

9.4

112

ND

ND

18.8

127

ND

ND

37.5

110

ND

ND

75

114

0.0

0.0

150

91

0.5

0.5

300 (Pb)

111

0.0

0.0

Cyclophosphamide

10

ND

34.0*

34.0*

 

ND: not determined

Pa: Precipitate on dosing, still apparent when the culture was harvested.

Pb: Precipitate on dosing, still apparent at the end of treatment (3 h) and when the culture was harvested.

* P < 0.001

 

Table 2. Results of chromosome aberration test in human lymphocytes (duplicate cultures), Experiment 2.

Test item

Concentration

(µg/mL)

Relative mitotic index (%)

Mean No. of aberrant cells per 100 cells examined

Without gaps

With gaps

Exposure: without S9 mix, 18 h harvest

DMSO

10 µL/mL

100

0.25

0.25

Test substance

37.5

100

ND

ND

75

85

0.5

0.5

150 (Pc)

100

0.0

0.0

300 (Pd)

70

0.0

0.0

Ethylmethanesulphonate

500

ND

14.5*

15.0*

Exposure: with S9 mix, 18 h harvest (3 h + 15 h recovery)

DMSO

10 µL/mL

100

1.25

1.5

Test substance

75

94

0.5

1.0

150 (Pc)

100

1.0

1.0

300 (Pe)

113

0.0

0.0

Cyclophosphamide

10

ND

27.0*

28.0*

Exposure: without S9 mix, 32 h harvest

DMSO

10 µL/mL

100

1.25

2.0

Test substance

9.4

103

ND

ND

18.8

72

ND

ND

37.5

53

ND

ND

75

63

ND

ND

150 (Pc)

63

0.5

0.5

300 (Pd)

3

ND

ND

Exposure: with S9 mix, 32 h harvest (3 h + 29 h recovery)

DMSO

10 µL/mL

100

1.0

1.25

Test substance

18.8

98

ND

ND

37.5

103

ND

ND

75

103

ND

ND

150 (Pf)

118

ND

ND

300 (Pg)

89

1.0

1.0

 

ND: not determined

Pc: Slight precipitate on dosing, not apparent when the cultures were harvested.

Pd: Heavier precipitate on dosing, still apparent when the cultures were harvested.

Pe: Heavier precipitate on dosing, not apparent when the cultures were harvested.

Pf: Slight precipitate on dosing, not apparent at the end of treatment (3 h).

Pg: Heavier precipitate on dosing, still apparent at the end of treatment (3 h).

* P < 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative