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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 487 (in vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
EXPERIMENT I
- without S9 mix: 13.7, 24.1, 42.1, 73.7, 128.9, 225.7, 394.9, 691.1, 1209.4, 2116.4 µg/mL
- with S9 mix: 13.7, 24.1, 42.1, 73.7, 128.9, 225.7, 394.9, 691.1, 1209.4, 2116.4 µg/mL

EXPERIMENT II
- without S9 mix: 13.7, 24.1, 42.1, 73.7, 128.9, 225.7, 394.9, 691.1, 1209.4, 2116.4 µg/mL
Vehicle / solvent:
Deionised water
Controlsopen allclose all
Untreated negative controls:
other: solvent control served as negative control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 - pulse treatment
Untreated negative controls:
other: solvent control served as negative control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: demecolcin
Remarks:
without S9 - continuous treatment
Untreated negative controls:
other: solvent control served as negative control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
HUMAN LYMPHOCYTES
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (29 years old) for Experiment I and from a male donor (31 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

CULTURE CONDITIONS
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Evaluation criteria:
Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culturewere scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI is determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CBPI=(MONCx1)+(BINCx2)+(MUNCx3)/n

CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BINC Binucleate cells
MUNC Multinucleate cells

Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]

T Test item
C Solvent control
Statistics:
Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

In both experiments no precipitation of the test item in the culture medium was observed.

No relevant influence on osmolarity was observed. The pH was adjusted to physiological values.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

No relevant increase in micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, at a concentration of 691.1 μg/mL a statistically significant increase in the number of micronucleated cells (0.75 %) was observed after treatment with the test item. The value lies within the range of the laboratory historical control data (0.15 – 1.70 % micronucleated cells) and can be estimated as biologically irrelevant.

In both experiments, either Demecolcin (100.0 ng/mL), MMC (2.0 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

 Experiment  Exposure  Conc. [µg/mL]  CBPI  Cytostasis [%]  Micronucleated cells [%]
 I  4 h -S9  SC  1.88    0.30
 I  4 h -S9  PC  1.14  84.7  10.30
 I  4 h -S9  691.1  1.67  24.4  0.10
 I  4 h -S9  1209.4  1.79  10.0  0.50
 I  4 h -S9  2116.4  1.75  14.7  0.35
 II  20 h -S9  SC  1.51    1.35
 II  20 h -S9  PC  1.44  12.5  5.45
 II 20 h -S9  394.9  1.79  nc  0.55
 II  20 h -S9  691.1  1.60  nc  0.60
 II  20 h -S9  1209.4  1.53  nc  0.20
 I  4 h +S9  SC  1.92    0.30
 I  4 h +S9  PC  1.46  50.3  4.85
 I  4 h +S9  691.1  1.95  nc  0.75
 I  4 h +S9  1209.4  1.91  1.2  0.70
 I  4 h +S9  2116.4  1.91  0.7  0.30

SC = Solvent control

PC = Positive control

CBPI = Proliferation index

nc = not calculated (CBPI equal or higher than solvent control value)

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, SAT 140001 is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required or evaluable concentrations.
Executive summary:

The test item SAT 140001, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

Without S9 

 With S9

 

 Exp. I

 Exp. II

 Exp. I

 Stimulation period

 48 h

 48 h

 48 h

 Exposure period

 4 h

 20 h

 4 h

 Recovery

 16 h

 -

 16 h

 Cytochalasin B exposure

 20 h

 20 h

 20 h

 Total culture period

 88 h

 88 h

 88 h

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2116.4 μg/mL of the test item) was chosen with regard to the purity (94.5%) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

No relevant increase in micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, at a concentration of 691.1 μg/mL a statistically significant increase in the number of micronucleated cells (0.75 %) was observed after treatment with the test item. The value lies within the range of the laboratory historical control data (0.15 – 1.70 % micronucleated cells) and can be estimated as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, SAT 140001 is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required or evaluable concentrations.