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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:
08 January 2016 - 15 February 2016 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP OECD 487 guideline study without deviations on the registered substance itself.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals (2014) No. 487 "In Vitro Mammalian Cell Micronucleus Test"
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-epoxypropyl isopropyl ether
EC Number:
223-672-9
EC Name:
2,3-epoxypropyl isopropyl ether
Cas Number:
4016-14-2
Molecular formula:
C6H12O2
IUPAC Name:
2-[(propan-2-yloxy)methyl]oxirane
Test material form:
other: liquid
Details on test material:
- Substance type: pure substance
- Storage condition of test material: Room temperature, under nitrogen, in the dark

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 32 years
Main Experiment: male, aged 25 years
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/β-Naphthoflavone induced male rat liver S9
Test concentrations with justification for top dose:
0, 4.54, 9.08, 18.15, 36.3, 72.6, 145.2, 290.4, 580.8, 1161.6 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
- Justification for choice of solvent/vehicle: The test item was soluble in Minimal Essential Medium (MEM) at 11.62 mg/mL in solubility checks performed in-house.
Controls
Untreated negative controls:
yes
Remarks:
Minimal Essential Medium
Negative solvent / vehicle controls:
other: not required
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine (DC)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): exposure duration plus 24h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: minimum of approximately 500 cells per culture (for CBPI), scoring for micronuclei: 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI) for cytostasis
Evaluation criteria:
Acceptability Criteria
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed

Data Evaluation
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response. Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two.
Statistics:
Statistical Analysis
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Water solubility: not exceeded
- Precipitation: none
- Other confounding effects: none stated

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 4.54, 9.08, 18.15, 36.3, 72.6, 145.2, 290.4, 580.8 and 1161.6 μg/mL. The maximum dose was the maximum recommended dose level the 10 mM concentration.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 580.8 μg/mL in the 4-hour exposure groups, both in the presence and absence of metabolic activation (S9). The maximum dose with binucleate cells present in the 24-hour continuous exposure was 145.2 μg/mL. The test item induced evidence of toxicity in all three exposure group and demonstrated a very sharp toxicity curve, particularly in the 4-hour exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was the maximum recommended dose level (1161.6 μg/mL) for the 4-hour exposure group in the presence of S9, 726 μg/mL for the 4-hour exposure group in the absence of S9 and 290.4 μg/mL for the 24-hour exposure group.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 2,3-epoxypropyl isopropyl ether to induce micronuclei in human lymphocytes. The test item induced a statistically significant increase in the frequency of binucleate cells with micronuclei in both the absence and presence of a metabolizing system. The test item was hence considered to be able to induce chromosome breaks and/or gain or loss to human lymphocytes in vitro.
Executive summary:

An in vitro study for the detection of the clastogenic and aneugenic potential of 2,3-epoxypropyl isopropyl ether on the nuclei of normal human lymphocytes was conducted according to OECD TG 487 under GLP.

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows:

4-hour without S9:0, 72.6, 145.2, 290.4, 363, 435.6, 508.2, 580.8, 726 µg/ml

4-hour with S9 (2%): 0, 72.6, 145.2, 290.4, 580.8, 726, 871.2, 1016.4, 1161.6 µg/ml

24-hour without S9: 0, 18.15, 36.3, 72.6, 108.9, 145.2, 181.5, 217.8, 290.4 µg/ml

 

All vehicle (Minimal Essential Medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item demonstrated toxicity in the preliminary toxicity test and the main experiment where near optimum toxicity was achieved in each of the exposure groups. The test item induced statistically significant increases in the frequency of binucleate cells with micronuclei, in the three exposure groups of the Main Experiment.

 

The test item was considered to be able to induce chromosome breaks and/or gain or loss to human lymphocytesin vitro.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenetic mutagenicity data.