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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:
2008-07-16 to 2008-09-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
published June 8, 2000
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
other: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
Whole blood (0.4 ml) treated with heparin was added to culture medium. Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.
Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) 01 the cells and the age of the donor at the time the AGT was determined (December 2007):
Dose range finding study: age 42, AGT =14.5 h
First cytogenetic assay: age 29, AGT =16.3 h
Second cytogenetic assay: age 31, AGT =14.5 h
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fractions obtained from livers of adult male Wistar rats induced with Phenobarbital (80 mg/kg bw) and beta-Naphtoflavone (100 mg/kg bw) in corn oil.
Test concentrations with justification for top dose:
Dose range finding test:
- at 3 h exposure time: 3, 10, 33, 100, 333 µg/ml culture medium with and without S9-mix;
- at 24 h and 48 h exposure time: 3, 10, 33, 100, 333, 1000 µg/ml without S9-mix.
First cytogenetic assay:
- without and with S9-mix: 33, 100 and 333 µg/ml culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay:
- without S9-mix: 100, 300, 400, 500, 600, 700 and 800 µg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time)
- with S9-mix: 33, 100 and 300 µg/ml culture medium (3 h exposure time, 48 h fixation time).
Scoring of chromosome aberrations:
- without S9-mix: 100, 500 and 600 µg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time)
- with S9-mix: 33, 100 and 300 µg/ml culture medium (3 h exposure time, 48 h fixation time).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Stability of test substance in vehicle: unknown
- Solubilily in vehicle: not indicated
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Solvent positive control: Hanks' Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without calcium and magnesium

Migrated to IUCLID6: CP (CAS no. 50-18-0. Endoxan-Asta, Asta-Werke, Germany) was used as an indirect acting mut
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Solvent positive control: Hanks' Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without calcium and magnesium

Migrated to IUCLID6: MMC-C (CAS no. 50-07-7, Sigma, Zwijndrecht, NL) was used as a direct acting mutagen (-S9-m
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Cell culture:
- Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection Iymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
- Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ud., United Kingdom) was added.

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


SPINDLE INHIBITOR (cytogenetic assays):
- During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindel inhibitor colchicine (0.5 µg/ml medium) (Acros Organics, Belgium)

NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS SCORED: approx. 200


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells.
- At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells were scored in presence and absence of S9-mix.
- Determination of endoreplication: the number of endoreduplicated chromosomes were observed in presence and absence of S9-mix


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.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results
Species / strain:
other: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 600 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

DOSE-FINDING STUDY:
- At a concentration of 333 µg/ml the test substance precipitated in the culture medium.

FIRST CYTOGENETIC ASSAY
- Both in the absence and presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberratlons
- Both in the absence and presence of S9-mix, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
- Both in the absence and presence ot s9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations:
- Both in the absence and presence of ss-mb, the test substance did not increase the number of
polyploid cells and cells with endoreduplicated chromosomes.
COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range:
- The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range .

POSITIVE CONTROL CHEMICALS:
The positive control ehemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosome aberrations:

- Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

- No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion Isostearic acid, esters with methyl α-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report.
Executive summary:

In a mammalian cell cytogenetics assay, peripheral human lymphocyte cultures were exposed to Isostearic acid, esters with methyl α-D-glucoside (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl Glucoside), in dimethyl sulfoxide at concentrations of 0, 33, 100 and 333 µg/ml culture medium (3 h exposure time , 24 h fixation time with and without metabolic activation in the first cytogenetic assay. In the second cytotgenetic assay concentrations of 0, 100, 300, 400, 500, 600, 700 and 800 µg/ml (24 h and 48 h exposure time and fixation time) without S9-mix and 0, 33, 100 and 300 µg/ml (3 h exposure time, 48 h fixation time) with S9-mix were tested respectively.

Isostearic acid, esters with methyl α-D-glucoside was tested up to cytotoxic and precipitating concentration. In the first cytogenetic assay, the test substance was tested up to 333 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Isostearic acid, esters with methyl α-D-glucoside precipitated in the culture medium at this dose level. In the second cytogenetic assay, Isostearic acid, esters with methyl α-D-glucoside was tested up to 800 µg/ml for a 24 h and 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at 600 µg/ml. In the presence of S9-mix Isostearic acid, esters with methyl α-D-glucoside was tested up to 300 µg/ml for a 3 h exposure time with a 48 h fixation time. The test substancce precipitated in the culture medium at this dose level.

Isostearic acid, esters with methyl α-D-glucoside did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No biologically relevant effects of Isostearic acid, esters with methyl α-D-glucoside on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Isostearic acid, esters with methyl α-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report.

Positive controls induced the appropriate response. There was no evidence of Chromosome aberration induced over background. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

This study is classified as acceptable. This study satisfies the requirement for OECD Test Guideline 473.