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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-conducted study performed under current GLP with complete test material characterization available. Reliability was changed from "1" to "2" according to the ECHA guidance document "Practical guide 6: How to report read-across and categories".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): Erucamide
- Molecular formula (if other than submission substance): C22H43NO
- Molecular weight (if other than submission substance): 337.59

Method

Target gene:
None
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9, phenobarbital/ß-naphthoflavone induced
Test concentrations with justification for top dose:
4.9 to 1250 µg/ml; additional information below.
Vehicle / solvent:
acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9-mix

Migrated to IUCLID6: 1000 µg/ml (Experiment I); 900 µg/ml (Experiment II)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix

Migrated to IUCLID6: 1.4 µg/ml with activation
Details on test system and experimental conditions:
Large stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany) were stored in liquid nitrogen in the cell bank of Harlan CCR, which allows the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with Neomycin (5 µg/mL) and Amphotericin B (2.5 µg/mL). The cells were sub-cultured twice a week. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
The highest concentration used in the cytogenetic experiments was chosen considering the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible.
With respect to the solubility of the test item, 1250.00 µg/mL of Erucamide (approx. 3.7 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 4.9 and 1250.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item was observed at 312.5 µg/mL and above in the absence of S9 mix and at 625.0 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Dose selection of Experiment II was influenced by test item precipitation obtained in Experiment I and observations made during the experimental performance of Experiment II. Therefore, 450.0 µg/mL (without S9 mix) and 300.0 µg/mL (with S9 mix) were chosen as top treatment concentrations in Experiment II.
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium were added.
Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 14 hours.

Colcemid was added to the culture medium (0.2 µg/mL) 15.5 hours after the start of the treatment. The cells were treated, 2.5 hours later, on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" [5]) using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
The evaluation of cytotoxicity indicated by reduced cell numbers was made after the preparation of the cultures on spread slides. The cell numbers were determined microscopically by counting 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.
Statistics:
Statistical significance was confirmed by Fisher’s exact test (p < 0.05); however, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, classification with regard to historical data and biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
noted at 300 µg/ml after 18 hour incubation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation noted at 300 µg/ml after 4 hour incubation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In both experiments, in the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see Table). The aberration rates of the cells after treatment with the test item (0.0 - 3.8 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls.
In both experiments, either EMS (900.0 or 1000.0 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results of the chromosome aberration study with Erucamide

Exp.

Preparation

Test item

Cell numbers

Mitotic indices

Aberrant cells

 

 

interval

concentration

in %

in %

in %

 

 

 

in µg/mL

of control

of control

incl. gaps*

excl. gaps*

with exchanges

 

 

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

100.0

100.0

3.0

1.5

0.5

 

 

 

Positive control2

n.t.

104.9

22.0

21.0S

14.0

 

 

 

78.1

88.8

99.6

2.5

2.5

0.5

 

 

 

156.3

77.3

104.4

2.5

2.5

1.0

 

 

 

312.5P##

84.8

76.4

4.0

3.8

1.3

 

 

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

100.0

100.0

3.0

2.5

0.0

 

 

 

Positive control3#

n.t.

40.3

42.0

40.0S

13.0

 

 

 

150.0

67.3

97.8

3.0

1.5

0.0

 

 

 

300.0

51.9

57.5

2.5

2.5

0.0

 

 

 

450.0

60.9

68.4

1.0

1.0

0.5

 

 

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

100.0

100.0

1.5

1.5

0.5

 

 

 

Positive control4

n.t.

52.3

9.5

8.5S

2.5

 

 

 

156.3

96.3

118.2

3.5

2.5

0.0

 

 

 

312.5

82.9

107.7

0.5

0.5

0.0

 

 

 

625.0P

90.5

78.2

2.5

1.5

0.0

 

II

18 hrs

Solvent control1

100

100

0.5

0.5

0.0

 

 

 

Positive control4

n.t.

90.4

13.5

12.0S

5.0

 

 

 

75.0

60.0

102.9

0.0

0.0

0.0

 

 

 

150.0

84.2

106.4

1.5

0.5

0.0

 

 

 

300.0P

67.8

116.4

2.5

1.0

0.5

 

*     Inclusive cells carrying exchanges

#     Evaluation of 50 metaphases per culture

##    Evaluation of 200 metaphases per culture

n.t. Not tested

P     Precipitation occurred at the end of treatment

S     Aberration frequency statistically significant higher than corresponding control values

1     Acetone 0.5 % (v/v)

2           EMS    1000.0 µg/mL;3      EMS    900.0 µg/mL;4    CPA  1.4 µg/mL

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item Erucamide did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to precipitating or cytotoxic concentrations.
Executive summary:

The test item Erucamide, suspended in acetone, was assessed for its potential to induce structural chromosome aberrations inV79cells of the Chinese hamsterin vitroin two independent experiments. In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated. The highest applied concentration (1250.0 µg/mL; approx. 3.7 mM) was chosen with regard to the solubility properties of the test item in an appropriate solvent and with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In Experiment I in the absence and presence of S9 mix no clear cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred. In Experiment II in the absence of S9 mix clear cytotoxicity was observed at 300.0 and 450.0 µg/mL. Test item precipitation occurred in the presence of S9 mix at the highest evaluated concentration and the cell number was reduced to 60 % and 67.8 % of control after treatment with 75.0 and 300.0 µg/mL, respectively.

No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. Under the experimental conditions reported, the test item did not induce structural chromosome aberrations inV79cells (Chinese hamster cell line)in vitro. Therefore, erucamide is considered to be non-clastogenic in this chromosome aberration test in the presence and absence of metabolic activation when tested up to precipitating or cytotoxic concentrations.