2-Propenoic acid, 4-[[(4-benzoylphenoxy)carbonyl]oxy]butyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Bacterial Reverse Mutation Assay: The test substance did not induce gene mutations in the S. typhimurium and E. coli strains. In vitro Mouse Lymphoma Test: The test substance is not mutagenic in the mouse lymphoma thymidine kinase locus assay after pulse treatment up to the maximal evaluated concentrations and after continuous treatment except at highly toxic concentrations. This effect may well represent an artefact caused by selection of cells during continuous treatment and the first 24 hours after treatment rather than a true mutagenic effect. The substance was thus judged to be not mutagenic in this assay. In vivo Micronucleus Test: In an in vivo micronucleus test in rat bone marrow no genotoxic effect of the test substance was observed.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Weight at study initiation: mean weight of 28.2 g
- Assigned to test groups randomly: no
- Housing: individual housing in Makrolon cages, type Ml
- Diet: Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

DMSO
- Vehicle/solvent used: DMSO
- Choice of solvent/vehicle:
Due to the insolubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.

Details on exposure:

For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 hour) and then deep-frozen until they were evaluated analytically. This analytical investigation was carried out by means of photometry.

Duration of treatment / exposure:

2 treatments at a 24 h interval, samples taken 24 h after the second treatment

Frequency of treatment:

two administrations

Post exposure period:

none

Remarks:

Doses / Concentrations:
200, 400, 800 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed down to a dose of 600 mg/kg body weight (at 600 mg/kg and 800 mg/kg body weight only a few animals died on the 7th day after the 2nd administration). Evident clinical signs were observed both using male and female animais. However, species specific differences were not observed and thus, only male animais were used for the cytogenetic investigations. Therefore, a dose of 800 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 400 mg/kg and 200 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID, W..
The two femora were prepared by dissection and removing all soft tissues. After cutting oft the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 3700 (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5 % Giemsa solution for 15 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
Microscopic analysis and determination of the number of polychromatic erythrocytes containing micronuclei and normochromatic erythrocytes containing micronuclei and determination of the the number of small micronuclei (d < DM4) and of large micronuclei (d > DM4) (d = diameter of micronucleus, D = cell diameter).

Evaluation criteria:

The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test systemn if:
- There was no significant incmease in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
- The frequencies of cells containing micronuclei were within the historical control range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft). The number of micronuclei in polychromatic erythrocytes was analyzed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. This test was performed one-sided.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Signs of toxicity were observed in all dose groups.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: Deaths were observed down to a dose of 600 mg/kg body weight. Evident clinical signs were observed both using male and female animals.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The test substance did not lead to any increase in the rate of micronuclei.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro studies:

Bacterial test:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay following OECD guideline 471 and EU method B13 and B14. The study was conducted under GLP compliance. Standard plate test (SPT) and preincubation test (PIT) both with and witheut metabolic activation (Aroclor-induced rat liver S-9 mix) were conducted. A weak bacteriotoxic effect was observed under all test conditions. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. The test substance was not determined as mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen.

Mouse lymphoma test:

The test was performed to assess the ability of the test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5 178Y in the 96- microwell cloning version (microtiter assay). The assay was performed in two independent experiments, using two parallel cultures. The first main experiment was accomplished with and without liver microsomal activation. The second experiment was solely performed without metabolic activation and a continuous treatment of 24 h. The test article was tested at the following concentrations: Experiment 1 (with and without S9 mix): 9.4; 18.8; 37.5; 75; 150 and 300 µg/mL, experiment 2 (without S9 mix): 4.7, 9.4, 18.8, 37.5; 75; and 150 µg/mL.

Precipitation of the test article occurred at 150 µg/ml and above. According to the preexperiment on toxicity the cancentration range was selected. In this pre-experiment strong toxic effects were observed at 156.25 µg/mL and above with and without S9 mix. In experiment 1 strong toxic effects occurred at concentrations of 150.0 µg/mL and above in´the absence and presence of metabolic activation. In the second experiment strong toxic effects were observed at 150 µg/ml. The cell growth observed at the lowest concentrations was approximately in the range of the negative control.

No substantial and reproducible dose dependent increase in mutant colony numbers and no relevant shift of the ratio of small versus large colonies was observed in the first experiment up to the maximal concentration tested, neither in the absence nor in the presence of metabolic activation. In the second experiment a substantial and reproducible increase of the number of mutant colonies was solely observed at the maximal concentration above the limit of solubility, showing strong toxic effects. The ratio of small versus large colonies is shifted towards the small colonies at the maximal concentration indicating chromosome aberrations. Although this effect was reproduced, it occurred only at highly toxic concentrations under precipitation of the test article. Precipitation is a known source of artefacts in the mouse lymphoma assay since particles may be phagocytised leading to high local concentrations within the cells with the possible result of lysosomal damage and a subsequent release of nucleases. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies.

In vivo studies:

Micronucleus test:

The test substance was tested for clastogenicity and for the ability to induce spindle poison effects in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in DMSO, was administered twice intraperitoneally to male animals at dose levels of 200 mg/kg, 400 mg/kg and 800 mg/kg body weight in a volume of 4 ml/kg body weight in each case. As a negative control, DMSO was administered to male mice, by the same route, and gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance up to800mg/kg led to evident signs of toxicity.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2,000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2,000 polychromatic erythrocytes were also registered.

According to the results of the present study, the intraperitoneal administration of the test substance did not Iead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups.

An inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected at a dose of 800mg/kg body weight.

Thus, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.

Justification for selection of genetic toxicity endpoint
only one in vivo study available, guideline and GLP conform

Justification for classification or non-classification

The test substance was not mutagenic either in the in vitro studies or in the in vivo micronucleus test.

Dangerous Substance Directive (67/548/EEC)
The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation No 605/2014.