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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 January 2020 to 16 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
please see "Principles of method if other than guideline"
Principles of method if other than guideline:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
3-hydroxypropyl octanoate
EC Number:
844-232-8
Cas Number:
102731-54-4
Molecular formula:
C11H22O3
IUPAC Name:
3-hydroxypropyl octanoate

Method

Species / strain
Species / strain / cell type:
lymphocytes: human lymphocytes, primary culture
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: 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.
- Sex, age and number of blood donors: Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (29 years old in Experiment I and 30 years old in Experiment III) and from a male donor (21 years old) for Experiment II.
- Mitogen used for lymphocytes: PHA

MEDIA USED
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.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the ICCR-Roßdorf SOP for rat liver S9 preparation.
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 30.2 mg/mL.
- quality controls of S9: 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.
Test concentrations with justification for top dose:
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.
This preliminary test was designated Experiment I, since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.
Vehicle / solvent:
- Solvent used: Ethanol, the final concentration of Ethanol in the culture medium was 0.5 %.
- Justification for choice of solvent: 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: demecolcine (without metabolic activation)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (pulse exposure), 20 hours (continuous exposure)
- Harvest time after the end of treatment: 36 hours (pulse exposure), 20 hours (continuous exposure)
- Identity of cytokinesis blocking substance: Cytochalasin B (4 μg/mL), duration and period of cell exposure: 20 hours
- Methods of slide preparation and staining technique: The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- Criteria for scoring micronucleated cells: The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index


Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− 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% control limit realized as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− 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% control limit realized as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:
Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of “R”, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline was not evaluable for cytogenetic damage, due to strong cytotoxic effects. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration which showed phase separation. In Experiment III in the absence of S9 mix, clear cytotoxicity (47.2% cytostasis) was observed at the highest evaluable concentration. In Experiment I in presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In Experiment I the absence of S9 mix, statistically significant increases in micronucleated cells were observed after treatment with all evaluated concentrations. The value of 1.65% micronucleated cells after treatment with 653 μg/mL clearly exceeded the range of the historical control data (0.00 – 1.04% micronucleated cells) and dose dependency, tested by trend test, was observed.
In the confirmatory Experiment II in the absence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. The value 1.15% micronucleated cells after treatment with 299 μg/mL, however, exceeded the range of the 95% control limit of the historical control data (0.00 – 1.04% micronucleated cells), but was clearly within the min-max range (0.05 – 1.20% micronucleated cells). Neither a statistically significant increase nor dose dependency, tested by trend test, was observed. In Experiment III in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells and no dose dependency, tested by trend test, were observed after treatment with the test item.

Any other information on results incl. tables

Table 2 Summary of results







































































































































































 

Exp.



Preparation interval



Test item  concentration in µg/mL



Proliferation index CBPI



Cytostasis  in %*



Micronucleated cells in %**



95% Ctrl limit



Exposure period 4 h without S9 mix



I



40 h



Solvent control 1/#



1.46



 



0.55



0.00-1.04



Positive control 2



1.56



n.c.



10.25S



213#



1.50



n.c.



1.03S



373#



1.44



4.3



0.98S



653#



1.45



3.2



1.65S



Trend test: p-value 0.491 T



II



40 h



Solvent control 1



1.56



 



0.95



0.00-1.04



Positive control 3



1.51



10



9.50S



171



1.66



n.c.



0.60



299



1.47



15.7



1.15



522PS



1.46



17.8



0.40



Trend test: p-value 0.491



Exposure period 20 h without S9 mix



III



40 h



Solvent control 1#



1.74



 



0.43



0.00-0.86



Positive control 4



1.70



6.6



3.60S



135#



1.71



5.2



0.65



237#



1.66



11.6



0.40



356#



1.39



47.2



0.68



Trend test: p-value 0.522



Exposure period 4 h with S9 mix



I



40 h



Solvent control 1



1.49



 



0.65



0.00-1.03



Positive control 5



1.37



26



3.40S



213



1.51



n.c.



0.40



373



1.50



n.c.



0.80



653PS



1.46



5.9



0.65



Trend test: p-value 0.754



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


** 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


PS Phase separation occurred at the end of treatment


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


T Trend analysis via linear regression is significant (p ˂ 0.05)


n.c. Not calculated as the CBPI is equal or higher than the solvent control value


solvent control 1 Ethanol 0.5 % (v/v)


positive control 2: MMC 1.0 μg/mL


positive control 3 MMC 0.8 μg/mL


positive control 4 Demecolcine 50 ng/mL


positive control 5 CPA 15.0 μg/mL

Applicant's summary and conclusion

Conclusions:
The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Executive summary:

The test item dissolved in ethanol was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. In each experimental group, two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.


The highest applied concentration in this study (2000 μg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline was not evaluable for cytogenetic damage, due to strong cytotoxic effects. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration which showed phase separation. In Experiment III in the absence of S9 mix, clear cytotoxicity (47.2% cytostasis) was observed at the highest evaluable concentration.


In Experiment I in presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In Experiment I in the absence of S9 mix, statistically significant increases in micronucleated cells were observed after treatment in all evaluated concentrations. The value of 1.65% micronucleated cells after treatment with 653 μg/mL clearly exceeded the range of the historical control data (0.00 – 1.04% micronucleated cells) and dose dependency, tested by trend test, was observed. In the confirmatory Experiment II in the absence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. The value of 1.15% micronucleated cells after treatment with 299 μg/mL, however, exceeded the range of the 95% control limit of the historical control data (0.00 – 1.04% micronucleated cells), but was clearly within the min-max range (0.05 – 1.20% micronucleated cells). Neither a statistically significant increase nor dose dependency, tested by trend test, was observed.


Taken together, none of the findings in Experiment I were confirmed in the confirmatory experiment and these can therefore be regarded as biologically irrelevant.


In Experiment III in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells and no dose dependency, tested by trend test, were observed after treatment with the test item.


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

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