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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Two in vitro studies are available. Ames test: Negative result (Verspeek-Rip, 2014); Chromosome aberration: Negative result (Buskens, 2014).
Link to relevant study records
Reference
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:
From 12 Jun to 20 Aug 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Cytogenetic assay 1A:
Without and with S9-mix: 10, 50, 70, 90, 110 and 130 μg/mL culture medium
Second cytogenetic assay:
Without S9-mix : 5, 15, 25, 35, 45, 55 and 65 μg/mL culture medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility in vehicle: >90 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9 mix
Details on test system and experimental conditions:
Test system
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (OECD, EC).
Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2013) are presented below:
Dose range finding test / first cytogenetic assay: age 31, AGT = 13.5 h
Cytogenetic assay 1A: age 35, AGT = 13.2 h
Second cytogenetic assay: age 26, AGT = 13.1 h

Dose range finding test / First cytogenetic assay
Lymphocytes (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of test substance for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.

Cytogenetic assay 1A
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of test substance for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS (Hanks’ Balanced Salt Solution). After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.

Second cytogenetic assay
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of test substance for 24 h and 48 h in the absence of S9-mix.
The cells were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

SPINDLE INHIBITOR (cytogenetic assays): colchicine

NUMBER OF CELLS EVALUATED: at least 1000 cells (with a maximum deviation of 5%)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Analysis of slides for chromosome aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with WIL Research Europe study identification number and code was placed over the marked slide. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.
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.
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:
X2=(N-1) (ad-bc)2/((a+b) (c+d) (a+c) (b+d))
where b = the total number of aberrant cells in the control cultures.
d = the total number of non aberrant cells in the control cultures.
n0 = the total number of cells scored in the control cultures.
a = the total number of aberrant cells in treated cultures to be compared with the control.
c = the total number of non aberrant cells in treated cultures to be compared with the
control.
n1 = the total number of cells scored in the treated cultures.
N = sum of n0 and n1
If P [X2 >(N-1) (ad-bc)2/ ((a+b) (c+d) (a+c) (b+d))](one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose range finding test / first cytogenetic assay:
No dose levels could be selected for scoring of chromosome aberrations since the highest tested concentration was too toxic for scoring. The experiment was repeated in cytogenetic assay 1A.

Cytogenetic assay 1A:
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.
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:
Test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
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. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Finally, it is concluded that this test is valid and that the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Two in vitro studies are available.

Ames test conducted according to OECD Guideline 471 under GLP (Verspeek-Rip, 2014).

The test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration test conducted according to OECD Guideline 473 under GLP (Buskens, 2014).

It is concluded that this test is valid and that the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.


Justification for selection of genetic toxicity endpoint
In vitro Mammalian Chromosome aberration test selected as higher level study.

Justification for classification or non-classification

In vitro studies gave negative result.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, this substance should not be classified for this endpoint.