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Genetic toxicity: in vitro

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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:
16 Oct - 18 Apr 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance glycerides, castor-oil, mono, hydrogenated, acetates (CAS 736150-63-3). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

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

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
primary culture, other: human lymphocytes
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 (500 mg/kg bw)
Test concentrations with justification for top dose:
Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL

First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Only slides from cultures of the following dose groups were selected for metaphase analyses:

First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases
Evaluation criteria:
EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations

For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.

The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.

The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.

Results which failed to meet the above mentioned criteria were considered as equivocal.

Statistics:
When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.

Results and discussion

Test results
Key result
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.

Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).

Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.

In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.

Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of the preliminary toxicity test

 

Treatment (µg/mL)

Mitotic index (MI)

Without S9

With S9

Individual values

Relative mean MI (%)

Individual values

Relative mean MI (%)

0

4.8

5.3

100

3.8

2.9

100

313

4.8

4.3

90

3.1

3.5

99

625

4.3

4.1

83

2.9

3.5

96

1250

3.4

3.7

70

3.4

2.8

93

2500

3.7

3.3

69

2.1

1.8

58

5000

3.6

2.9

64

0.4

0.3

10

Mitotic index: percentage of cells at metaphase

Individual values: values for each of the duplicate     

Relative mean MI: relative mean mitotic index for the duplicates

 

 

Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)

Treatment (µg/mL)

S9 mix

Relative mean MI (%)

No. aberrant metaphases

Number and types of aberrations

Number of polyploid metaphases

Gaps

Breaks

Exchanges

 

0

-

100

1 / 0

2 / 1

1 / 0

 

1 / 0

1250

-

110

1 / 1

0 / 2

2 / 1

 

 

2500

-

75

1 / 4

1 / 3

1 / 4

 

 

5000

-

70

2 / 3

2 / 1

2 / 4

 

 

Daunomycin (0.015 µg/mL)

-

91

17 / 14 **

9 / 5

20 / 14

2 / 1

 

0

2%

100

0 / 0

1 / 2

0 / 0

 

 

625

2%

67

0 / 1

0 / 0

0 / 1

 

 

1250

2%

51

0 / 0

2 / 1

0 / 0

 

 

2500

2%

42

0 / 1

1 / 1

0 / 1

 

 

Cyclophosphamide (6 µg/mL)

2%

20

33a /19b **

16 / 3

36 / 20

15 / 9

 

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 3. Test results of the second main test (repeat test)

Treatment (µg/mL)

S9 mix

Relative mean MI (%)

No. aberrant metaphases

Number and types of aberrations

Number of polyploid metaphases

Gaps

Breaks

Exchanges

 

0

-

100

0 / 3

1 / 5

0 / 3

 

 

40

-

84

1 / 4

3 / 6

1 / 4

 

1 / 0

80

-

59

1 / 2

0 / 2

1 / 2

 

 

160

-

49

0 / 2

3 / 1

0 / 2

 

0 / 1

Daunomycin (0.015 µg/mL)

-

102

13 / 14 **

6 / 9

13 / 13

1 / 1

 

0

4%

100

2 / 1

3 / 2

1 / 1

1 / 0

 

625

4%

66

2 / 4

3 / 1

5 / 1

 

0 / 1

1250

4%

61

1 / 1

3 / 4

1 / 1

 

 

2500

4%

33

5 / 1

3 / 0

3 / 4

 

 

Cyclophosphamide (6 µg/mL)

4%

77

36 / 33 **

8 / 8

37 / 33

11 / 11

 

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 4. Historical Data (n = 9 previous study)

Treatment (µg/mL)

S9 mix

Frequency of metaphases with aberrant chromosomes excluding gaps (%)

Number of cultures

Mean

SD

Mimimum

Maximum

 

Negative control

-

0.8

0.8

0

3

32

Daunomycin (0.015 µg/mL)

-

15.1

7.4

7

34

32

Negative control

+

0.7

0.9

0

3

32

Cyclophosphamide (6 µg/mL)

+

43.0

13.6

23

70

32

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative