2,3-epoxypropyl isopropyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity:

In vitro: Gene mutation (Bacterial reverse mutation test / Ames test): S. typhimurium TA 100, TA 1535, TA 98, TA 1537, ±S9 (hamster, rat): S. typhimurium TA 100, TA 1535, positive ±S9, S. typhimurium TA 98: negative ±S9 (rat), S. typhimurium TA 98: ambiguous +S9 (hamster), S. typhimurium TA 1537: negative ±S9 (equivalent to OECD 471)

In vitro: SOS Chromotest: E. coli PQ37: positive

In vitro: SCE assay: V79 cells –S9: positive (equivalent to EU method B.19)

In vitro: SLRL assay:postmeiotic and meiotic germ cells of male Drosophila melanogaster: positive (sex-linked recessive lethal assay)

In vitro: Chromosome aberration (micronucleus test): human lymphocytes ±S9: positive (OECD 487, GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: publication

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted equivalent to OECD guideline 471 on the registered substance itself with minor deficiencies. There were only four strains tested, however, this deficiency is considered to be minor as the fifth strain was added to the guideline because the former common four strains may not detect certain oxidising mutagens, cross-linking agents and hydrazines. Based on the chemical structure, this mode of action is not to be expected from the test item. There are also only minor deficiencies in documentation, but the given data indicate that the study was well-performed.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

HAMSTER, LIVER, S-9, AROCLOR 1254 (10% / 30%)

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333, 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): ≙ exposure duration

SELECTION AGENT (mutation assays): minimal Histidine

NUMBER OF REPLICATIONS: three plates per dose, experiment was repeated no less than 1 week after initial test

DETERMINATION OF CYTOTOXICITY
- Method: One or more parameters were used as an indication of toxicity: reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn.

OTHER: Testing was performed as set out in Haworth et al., Environmental Mutagenesis Supplement 1:3 -142 (1983), "Salmonella Mutagenicity Test Results for 250 Chemicals", at Case Western Reserve University

Evaluation criteria:

Testing was performed at 5 doses, using triplicate plates. Tests were repeated at least once; a chemical was not designated positive or negative unless the results were reproducible. A positive response was defined as a reproducible, dose-related increase in his+ revertants over the solvent control level; it was not necessary for the increase to equal 2-fold over background. A response was considered equivocal ("?") if a test was not reproducible; when a low-level, non-dose-related response was obtained; or when an increased response was seen at only one dose.
A chemical was considered mutagenic if at least one strain/activation combination yielded a reproducible positive response.

Statistics:

Mean of three replicates and SEM was calculated

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10 mg/plate, there was complete clearing of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Since the mutagens were all positive in TA100, and most were mutagenic in TA1535 as well, only the data from those strains are presented in detail, whereas data on all four strains is given in short summary.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The available literature was assessed as Klimisch 2. Data was gathered within an interlaboratory comparison aiming to assess the mutagenicity of aliphatic epoxides in Salmonella. A good intra- and interlaboratory reproducibility was seen in the results. Hence, the results are considered sufficiently reliable to assess the mutagenicity of Isopropyl glycidyl ether in Salmonella typhimurium. The substance was tested positive in two out of four tester strains, both with and without metabolic activation. Hence, Isopropyl glycidyl ether must be considered mutagenic in bacteria.

Executive summary:

In a reverse gene mutation assay in bacteria equivalent to OECD 471, strains TA98, TA100, TA1535, and TA 1537 of S. typhimurium were exposed to Isopropyl glycidyl ether in water at concentrations of 0, 100, 333, 1000, 3333, 10000 µg/plate in the presence and absence of mammalian metabolic activation (hamster and rat liver S9 mix) in the preincubation method.

 

Isopropyl glycidyl ether was tested up to limit concentration 10000 µg/plate. There was a dose-related increase of induced mutant colonies over background in strains TA100 and TA1535 with and without metabolic activation, whereas the test item was negative in strains TA98 and TA1537. The positive controls induced the appropriate responses in the corresponding strains.

 

This study is classified as acceptable and satisfies in principle the requirement for Test Guideline OECD 471 (version of 1983) for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting 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.

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

Target gene:

not applicable

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.

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

no information available, additional information (OECD 474) required

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Mode of Action Analysis / Human Relevance Framework

Objective

The present MoAA aims to assess human relevance of effects related to genetic damage. Currently, there is only in vitro data available, showing that IPGE has a genotoxic potential. Without having in vivo data available, the definitive relevance for humans cannot be predicted, just roughly estimated.

 

Hypothesised Mode of action Statement

Most likely the epoxy group of IPGE is responsible for the observed effects. Epoxides are known to undergo nucleophilic additions on the DNA, potentially leading to bulky adducts. Those are repaired e.g. by the nucleotide excision repair, which induces DNA strand breaks during the repair process.

IPGE was tested positive in the micronucleus assay in human lymphocytes. Although being acceptable, positive responses over historical control data were predominantly present in already cytotoxic ranges, indicating that IPGE is not a too potent genotoxic agent. Micronuclei are either induced by clastogens or aneugens. As the substance also induces gene mutations in bacteria, an interference with the spindle apparatus is less likely compared to a direct interaction with chromosomes. As, due to repair mechanisms, DNA strand breaks are formed, this is the more like reason for induction of micronuclei and the substance should be considered as clastogen.

IPGE was also tested positive in the Ames test in strains S. typhimurium TA 100 and TA 1535, both with and without metabolic activation. Those strains detect transitions and transversions (base substitutions at the G:C basepair, TA 100 also some frameshifts. The latter mechanism is less likely, as it was tested negative in strains TA 98 and TA 1537, which detect frameshifts at (or near) GCGCGCGC (TA 98) or GGGGG (TA 1537). In consequence, it can be assumed that both the observed mutations and DNA-strand breaks inducing micronuclei originate from small excisions during repair.

From the available information regarding the SOS chromotest no information on the mode of action can be derived, and the positive result in the available SCE-assay further indicates that repair mechanisms took place

 

Relevance, (in)consistency, data gaps

The effects noted in two different in vitro genotoxicity test systems, Ames test and micronucleus assay being the most relevant ones, allow the conclusion that for both the underlying mechanism was the covalent binding of IPGE on the DNA followed by small incisions during repair, and consequently leading to both DNA strand breaks leading to the formation of micronuclei, and error-prone repair: However, DNA adducts may also lead to mispairing during DNA synthesis, and other effects leading to the formation of micronuclei cannot be excluded, are nevertheless considered to be less likely.

To which extent those positive results however are relevant for in vivo systems including humans, cannot definitively be deducted from the present in vitro data. Provided that IPGE reaches non-modified, i.e. with intact epoxy group, the DNA, similar adducts may occur. However, as in vivo systems have a large amount of possibly modifying systems, including Cytochrome P450 metabolism, various redox-systems or possibly elevated hydrolysis after oral application, no conclusion may be drawn on a possible in vivo mutagenicity. Hence, an additional in vivo micronucleus test should be conducted ahead.

 

Conclusion

In conclusion, it may be assumed that IPGE acts as direct mutagen and clastogen by direct DNA-addition via the epoxy group. However, the relevance for humans cannot be absolutely determined, and in vivo genotoxicity testing is required ahead.

Justification for classification or non-classification

There are indications that IPGE acts as direct mutagen and clastogen by direct DNA-addition via the epoxy group, but taking into account its potential to also bind directly to Proteins or being modified in vivo via additional systems not being present in in vitro genotoxicity tests, a definitive conclusion on its mutagenicity in vivo cannot be drawn. Before determining the classification, an in vivo genotoxicity test must be performed ahead.