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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Mar - 29 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 90%
- Batch No.: 0600016235
- Expiry date: 18 Apr 2017
- Appearance: Beige solid
- Storage conditions: At room temperature

Method

Species / strain
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors: 1 male donor, aged 21 years (Experiment I); 1 female donor, aged 33 years (Experiment II)
- Whole blood was used
- Methods for maintenance in cell culture: 3 µg/mL phytohemeagglutinine in medium; cells were cultured at 37 °C with 5.5% CO2 in humidified air

MEDIA USED
- Type and identity of media including CO2 concentration: DMEM/F12 (1:1) supplemented with penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively), FBS (10%), HEPES (10 mM) and heparin (125 U.S.P.-U/mL)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Experiment I:
4 h treatment (without metabolic activation): 69.63, 121.9 and 213.2 µg/mL
4 h treatment (with metabolic activation): 39.79, 69.63 and 121.9 µg/mL

Experiment II:
20 h treatment (without metabolic activation): 79.01, 118.5 and 177.8 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen due 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:
cyclophosphamide
mitomycin C
other: demecolcin (125 ng/mL in deionized water (- S9 mix, 4 h, continuous treatment))
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation method: 48 h
- Exposure duration: 4 and 20 h
- Fixation time: 40 h

STAIN: Giemsa

NUMBER OF REPLICATIONS: Duplicates each in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a cleas microscope slide and thereafter stained with Giemsa.

NUMBER OF CELLS EVALUATED: 500 binucleated cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: 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. According to the study director, the criteria of Countryman and Heddle (1976) were taken for the evaluation of micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index was determined and cytotoxicity was expressed as %cytostasis.

OTHER EXAMINATIONS:
- Other: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

[Reference: Countryman P.I. and Heddle J.A. (1976): The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.]
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% confidence interval).
Statistics:
Statistical significance will be confirmed by the Chi square 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 resultsopen allclose all
Key result
Species / strain:
lymphocytes: lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. I (4 h): ≥ 373.2 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. I (4 h): ≥ 373.2 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Exp. II (20 h): at 177.8 µg/mL
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. II (20 h): ≥ 400.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the presence of S9 mix and a 4-h exposure period, a statistical significant increase (1.05%) in micronucleated cells was observed at the lowest evaluated concentration (39.79 μg/mL). In the absence of S9 mix and a 4-h exposure period, a statistical significant increase (1.10%) in micronucleated cells was observed at the highest evaluated concentration (213.2 μg/mL). However, both values can be declared as biologically irrelevant, because they are well within the range of the historical laboratory control data (0.02 – 1.15% in the absence and 0.08 – 1.20% in the presence of S9 mix).

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH determined in the maximum concentration without metabolic activation was within the same range as in the solvent control.
- Effects of osmolality: The osmolality of the solvent control did not differ from the osmolality of the maximum concentration without metabolic activation.
- Precipitation: The test substance precipitated at the end of the treatment at 213.2 µg/mL and above with and without metabolic activation in Experiment I. In Experiment II precipitation occured at 400.0 µg/mL at the end of treatment without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, blood cultures were exposed to the test substance at 12.99, 22.74, 39.79, 69.63, 121.9, 213.2, 373.2, 653.1, 1143 and 2000 µg/mL culture medium with and without metabolic activation for 4 h exposure time. Strong cytotoxic effects were determined at concentrations of 373.2 µg/mL and above with and without metabolic activation. Precipitation was observed in the culture medium at concentrations of 213.2 µg/mL and above with and without metabolic activation. Since the pre-test fulfilled the requirements for cytogenetic evaluation, it was designated Experiment I. The concentration levels for Experiment II were selected based on the results of the range-finding test (Experiment 1).

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: the number of mono-, bi- and multi-nucleated cells were determined.

NUMBER OF CELLS WITH MICRONUCLEI
- Binucleated cells per culture were scored for cytogenetic damage.

HISTORICAL CONTROL DATA
- Positive historical control data: The values of the positive control were within the range of the historical control data [ranges: 2.10 - 6.40 (-S9, continuous treatment); 4.15 - 24.00 (-S9; pulse treatment); 2.25 - 11.30 (+S9, pulse treatment)].
- Negative (solvent/vehicle) historical control data: The values of the negative control were within the range of the historical control data [ranges: 0.05 - 1.43 (-S9, continuous treatment); 0.15 - 1.25 (-S9; pulse treatment); 0.15 - 1.35 (+S9, pulse treatment)].

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Any other information on results incl. tables

Table 1: Cytogenicity in Experiment I and II

  Concentration
[µg/mL]
mono-
nucleated cells
bi-nucleated cells multi-
nucleated
cells
proliferation index
CBPI
mono-
nucleated cells
bi-nucleated cells multi-
nucleated
cells
proliferation index
CBPI
Mean proliferation index
CBPI
Cytostasis [%] *
Experiment I: 4 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 94 329 77 1.97 107 326 67 1.92 1.94  
Positive control (MMC) 1.0 168 304 28 1.72 144 304 52 1.82 1.77 18.6
Test substance 69.63 160 304 36 1.75 83 350 67 1.97 1.86 8.8
Test substance 121.9 203 278 19 1.63 246 236 18 1.54 1.59 37.6
Test substance 213.2 P 190 286 24 1.67 211 272 17 1.61 1.64 32.1
Experiment I: 4 h with S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 146 286 68 1.84 162 263 75 1.83 1.82  
Positive control (CPA) 15.0 177 276 47 1.74 201 259 40 1.68 1.53 15.1
Test substance 39.79 106 300 94 1.98 99 307 94 1.99 1.88 NC
Test substance 69.63 117 313 70 1.91 147 282 71 1.85 1.86 NC
Test substance 121.9 167 287 46 1.76 145 295 60 1.79 1.68 4.9
Experiment II: 20 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 113 370 17 1.81 116 354 30 1.83 1.84  
Positive control (Demecolcin) 15.0 224 276 0 1.55 247 249 4 1.51 1.71 15.1
Test substance 79.01 97 374 29 1.86 94 366 40 1.89 1.98 NC
Test substance 118.5 108 355 37 1.86 107 355 38 1.86 1.88 NC
Test substance 177.8 178 308 14 1.67 169 316 15 1.69 1.79 4.9

P: Precipitation occurred at the end of treatment

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

NC: Not calculated as the CBPI is equal or higher than the solvent control value

Table 2: Number of micronucleated cells

  Concentration
[µg/mL]
binucleated cells with n micronuclei culture 1 sum binucleated cells with n micronuclei culture 2 sum sum in 2000 binucleated cells Micronucleated cells [%] **
    1 2 >2   1 2 >2      
Experiment I: 4 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 2 0 0 2 2 0 0 2 4 0.2
Positive control (MMC) 1.0 94 10 3 107 100 8 1 109 216 10.80 S
Test substance 69.63 2 1 0 3 6 0 0 6 9 0.45
Test substance 121.9 1 0 0 1 3 0 0 3 4 0.20
Test substance 213.2 P 12 2 0 14 8 0 0 8 22 1.10 S
Experiment I: 4 h with S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 2 0 0 2 6 0 0 6 8 0.40
Positive control (CPA) 15.0 34 4 0 38 33 3 0 36 74 3.70 S
Test substance 39.79 15 0 0 15 6 0 0 6 21 1.05 S
Test substance 69.63 4 0 1 5 5 0 0 5 10 0.50
Test substance 121.9 3 0 0 3 11 0 0 11 14 0.70
Experiment II: 20 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 9 0 0 9 5 1 0 6 15 0.75
Positive control (Demecolcin) 15.0 20 4 1 25 27 4 2 33 58 2.90 S
Test substance 79.01 9 1 0 10 9 0 0 9 19 0.95
Test substance 118.5 9 0 0 9 6 1 0 7 16 0.80
Test substance 177.8 # 32 2 1 35 29 1 1 311 66 1.65 S

P: Precipitation occurred at the end of treatment

S: The number of micronucleated cells is statistically significantly higher than corresponding control values

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

Table 3: Historical Control Data

Solvent Control without S9
Micronucleated cells in %
  Pulse treatment (4/40) Continuous treatment (20/40)
No. of experiments 50* 54**
Mean 0.61 0.55
95 % Ctrl limit 0.02 – 1.15 0.05 – 1.05
     
1x SD 0.27 0.25
2x SD 0.54 0.50
Min 0.15 0.05
Max 1.25 1.43
Solvent Control with S9
Micronucleated cells in %
  Pulse treatment (4/40)
No. of experiments 67***
Mean 0.64
95 % Ctrl limit 0.08 – 1.20
   
1x SD 0.28
2x SD 0.56
Min 0.15
Max 1.35

* Aqueous solvents – 23 Experiments; Organic solvents – 27 Experiments
** Aqueous solvents – 24 Experiments; Organic solvents – 30 Experiments

*** Aqueous solvents – 24 Experiments; Organic solvents – 43 Experiments

Applicant's summary and conclusion

Conclusions:
In the in vitro micronucleus test conducted with human lymphocytes according to OECD 487, clastogenicity could be evidenced in the absence of metabolic activation after a 20-h exposure period.