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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:
26 Mar to 09 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

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:
EU Method B.10 (Mutagenicity - 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
Details on test material:
- Name of test material (as cited in study report): sorbitan laurate
- Physical state: yellow turbid viscous liquid
- Analytical purity: >80%
- Lot/batch No.: 0000357933
- Expiration date of the lot/batch: 27 October 2011
- Storage condition of test material: stable at room temperature in the dark

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium supplemented with 20% (v/v) FCS, 2 mM L-glutamine, penicillin/streptomycin (50 U/mL and 50 mG/mL respectively) and 30 U/mL heparin
- 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 a combination of phenobarbital and beta-naphtoflavone
Test concentrations with justification for top dose:
3 h exposure time: 33, 100 and 333 µg/mL
24 h exposure time: 50, 100 and 600 µg/mL
48 h exposure time: 100, 450 and 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with rat liver S9-mix

Migrated to IUCLID6: 10 µg/mL for a 3 h exposure period (24 h fixation time)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without rat liver S9-mix

Migrated to IUCLID6: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 24 and 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 24 and 48 h; 24 h treatment: 24 h; 48 h treatment: 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 5% (v/v) in tap water

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Chi-square test, one-sided, p < 0.05

Results and discussion

Test results
Species / strain:
lymphocytes: cultured human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 333 µg/mL and higher sorbitan laurate precipitated in the culture medium

RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h and 24 h continuous exposure) or on toxicity inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).

Any other information on results incl. tables

  Table 1: Maximum number of cells with aberrations (with gaps)

 

Maximum number of cells with aberrations [%](with gaps)

solvent control

positive control

treatment (conc. µg/mL)

treatment [exposure time/fixation time]

with S9-mix

without S9-mix

with S9-mix

without S9-mix

with S9-mix

without S9-mix

3h/24h

2

7

31

24

3 (100)

5 (100)

3h/48h

3

-

29

-

2 (33)

-

24h/24h

-

1

-

32

-

3 (600)

48h/48h

-

1

-

26

-

4 (450)

Table 2: Maximum number of cells with aberrations without gaps

 

Maximum number of cells with aberrations [%](without gaps)

solvent control

positive control

treatment (conc. µg/mL)

treatment [exposure time/fixation time]

with S9-mix

without S9-mix

with S9-mix

without S9-mix

with S9-mix

without S9-mix

3h/24h

1

5

30

24

2 (333)

4 (333)

3h/48h

3

-

28

-

1 (33)

-

24h/24h

-

1

-

32

-

2 (100)

48h/48h

-

1

-

26

-

4 (450)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative