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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
Study period:
13 Jan - 23 Feb 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

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)
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Ländlichen Raum und Verbraucherschutz
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Erucamide
- Physical state: solid
- Storage condition of test material: room temperature
- Expiration date of the lot/batch: 02 Jul 2012
- Analytical purity: 99.2%
- Lot/batch No.: 0821205214

Method

Target gene:
Not applicable
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:
Experiment I:
- 4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250 μg/mL (with and without metabolic activation)
Experiment II:
- 2.3, 4.7, 9.4, 18.8, 37.5, 75, 150, 300, 450 μg/mL (without metabolic activation)
- 4.7, 9.4, 18.8, 37.5, 75, 150, 300 μg/mL (with metabolic activation)

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9-mix: Ethylmethanesulfonate: 1000 µg/mL (experiment I), 900 µg/mL (experiment II); +S9-mix: Cyclophosphamide: 1.4 µg/mL (experiment I + II)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 14 h; 18 h treatment: 18 h

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
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:
with and 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 and without
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 4, 5, 7 and 8, pages 29, 30, 32 and 33). 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 (see ANNEX III). 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

Table 2: Summary of results of the chromosome aberration study with Erucamide

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps*

without gaps*

Exposure period 4 h, fixation time 18 h, without S9-mix

Solvent control

-

100

3

1.5

EMS

1000

104.9

22

21S

 

Test substance

78.1

99.6

2.5

2.5

156.3

104.4

2.5

2.5

312.5 (P)##

76.4

4

3.8

Exposure period 4 h, fixation time 18 h, with S9-mix

Solvent control

-

100

1.5

1.5

CPA

1.4

52.3

9.5

8.5S

 

Test substance

156.3

118.2

3.5

2.5

312.5

107.7

0.5

0.5

625 (P)

78.2

2.5

1.5

Exposure period 18 h, fixation time 18 h, without S9-mix

Solvent control

-

100

3

2.5

EMS#

900

40.3

42

40S

 

Test substance

150

97.8

3

1.5

300

57.5

2.5

2.5

450

68.4

1

1

Exposure period 4 h, fixation time 18 h, with S9-mix

Solvent control

-

100

0.5

0.5

CPA

1.4

90.4

13.5

12S

 

Test substance

75

102.9

0

0

150

106.4

1.5

0.5

300 (P)

116.4

2.5

1

EMS: Ethylmethanesulfonate

CPA: Cyclophosphamide

*Inclusive cells carrying exchanges

# Evaluation of 50 metaphases per culture

##Evaluation of 200 metaphases per culture

P Precipitation oft he test substance

S Aberration frequency statistically significant higher than corresponding control values 

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.