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Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 DEC 2021 - 19 SEP 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted 29th July, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU method B.62: In vivo Mammalian Alkaline Comet Assay
Version / remarks:
14 February 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-phenylbutenone
EC Number:
204-555-1
EC Name:
4-phenylbutenone
Cas Number:
122-57-6
Molecular formula:
C10H10O
IUPAC Name:
4-phenylbut-3-en-2-one
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat, HAN:WIST of Wistar origin

Rats are routinely used in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at TOXI-COOP ZRT
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103 Budapest, Cserkesz u. 90.
- Age at study initiation: 8-9 weeks at commencement of the treatment
- Weight at study initiation: 259-283 g. The weight variation in animals involved at the starting point of the test will not exceed ± 20 %.
- Assigned to test groups randomly: yes, under following basis: All animals will be sorted according to body weight by computer and grouped according to weight ranges. There will be an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping will be controlled by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.
- Fasting period before study: no
- Housing: 2 - 3 animals/cage (Type III polypropylene/polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sunflower oil
- Concentration of test material in vehicle: 240, 120 and 60 mg/mL
- Amount of vehicle (if gavage or dermal): 5 mL/kg bw
- Lot/batch no. (if required): 8007816003

The positive control It will be dissolved and applied in ultrapure water (ASTM Type I), at a concentration of 20 mg/mL just before the treatment.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The planned doses are 1200, 600 and 300 mg/kg bw/day, consequently the test item was formulated in the vehicle, sunflower oil in concentrations of 240, 120 and 60 mg/mL. The formulations will be prepared in the laboratory of the Test Facility just before each treatment. For preparation of test item solutions 15 min ultrasonication was applied.
Duration of treatment / exposure:
2 days (test item)
1 day (positive control)
Frequency of treatment:
once daily
Post exposure period:
the sampling will be performed 3-4 hours after the second treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
In the analytical measurements the measured concentration values remained within the ± 10 % of nominal range in the test item solutions at all concentrations; for this reason the nominal concentration values 240, 120 and 60 mg/mL (corresponding dose levels: 1200, 600 and 300 mg/kg body weight/day) were applied and referred throughout the study.
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
In the analytical measurements the measured concentration values remained within the ± 10 % of nominal range in the test item solutions at all concentrations; for this reason the nominal concentration values 240, 120 and 60 mg/mL (corresponding dose levels: 1200, 600 and 300 mg/kg body weight/day) were applied and referred throughout the study.
Dose / conc.:
1 200 mg/kg bw/day (nominal)
Remarks:
In the analytical measurements the measured concentration values remained within the ± 10 % of nominal range in the test item solutions at all concentrations; for this reason the nominal concentration values 240, 120 and 60 mg/mL (corresponding dose levels: 1200, 600 and 300 mg/kg body weight/day) were applied and referred throughout the study.
No. of animals per sex per dose:
6 animals in the dose groups and negative control group, respectively (5 animals analysed)
4 animals in the positive control group (3 animals analysed)
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): In this study a separate positive control group with a known mutagen will be used for the tissues.
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw

Examinations

Tissues and cell types examined:
Glandular Stomach Cells, Duodenum Cells, Liver Cells, Gonadal cells (isolated, not examined)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
For dose selection the corresponding OECD 489 guideline proposal, the information provided by Sponsor; furthermore, the results of non-GLP preliminary dose-range finding tests are taken into consideration. For the correct identification of the maximum tolerated dose (MTD) where no death, evidence of pain, suffering or distress occurs, non-GLP dose range finding tests were performed in the testing laboratory, using the same species, strain, sex, and treatment regimen (administration of doses by oral gavage twice: once on the day 0 and 24 hours thereafter) to be used in the main study. Each test was performed with two animals. The dose range-finding tests were started with 2000 mg/kg bw/day. Because of the observed toxic effects, the dose was lowered gradually to 1000 mg/kg bw/day. At the dose of 2000 mg/kg bw/day definite behavioral changes (reduced activity or hyperactivity, incoordination) appeared after the first treatment and these symptoms escalated to motionless, lying animals without reactions to the end of the observation period after the second treatment. These behavioral changes appeared at the investigated lower dose levels (at 1800 and 1500 mg/kg bw/day) at different timepoints within the observation periods (e.g.: after the first treatment the signs appeared equivocally at 1800 mg/kg bw/day, and no signs were noticed at 1500 mg/kg bw/day), but they reached the same intensity (motionless lying animals) to the end of the observation period after the second treatment. Incoordination, slight piloerection and slightly reduced activity were noticed at the end of the observation period (3-hour) after the second treatment at 1200 mg/kg bw/day, and incoordination and slight piloerection at 1000 mg/kg bw/day. Necropsy was performed on animals of the dose 1500 mg/kg bw/day. At the macroscopic examination of stomach in the forestomach tympanites, hyperaemia, bruising were described; furthermore faint structured glandular stomach was noticed; however, the latter signs were rather subjective observations. The non-GLP preliminary dose-range finding tests were not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of the present study.
Based on the results of the preliminary dose-range finding tests the maximum dose will be 1200 mg/kg bw/day, and in addition to the maximum dose two additional doses, 600 and 300 mg/kg bw/day are selected.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The test item will be administered in doses by oral gavage twice: once on the day 0 and 24 hours thereafter (on day 1). The negative control animals will be treated concurrently with the vehicle only. The positive control animals will be treated by oral gavage once during the experiment (on day 1).
The sampling time is a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hour after the last treatment. In this particular test and based on the experience of the laboratory over previous years the sampling will be performed 3-4 hours after the second treatment (the animals will be euthanized consistent with the effective animal welfare legislation and 3Rs principles and the cells of the target tissues will be isolated) and care will be taken to necropsy all animals at the same time after the last dose.

DETAILS OF SLIDE PREPARATION:
Isolation of Glandular Stomach Cells
The stomach was cut open and washed free shortly from food using cold phosphate buffered saline. The forestomach was removed and discarded. The glandular stomach was then placed into cold mincing buffer and incubated on ice in a short period of time. After the incubation, the surface epithelia were gently scraped using a scalpel blade. This layer was discarded and the gastric mucosa was rinsed with cold mincing buffer. The stomach epithelia were then carefully scraped at least 4-5 times with scalpel blade. The cell suspension was stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant was used to prepare the comet slides.

Isolation of Duodenum Cells
A representative duodenum sample was removed, cut lengthwise open and washed thoroughly with cold phosphate buffered saline. The duodenum sample will then be placed into cold mincing buffer and incubated on ice in a short period of time (< 1 minute), thereafter the surface epithelia will be gently scraped using tweezers and/or scalpel blade. The obtained cell suspension will be stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant will be used to prepare the comet slides.

Isolation of Liver Cells
A portion of the left lateral lobe of the liver was removed and washed in the cold mincing buffer until as much blood as possible was removed; thereafter placed in mincing buffer (ice cold HBSS containing 20 mM EDTA and 10 % DMSO), minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant was used to prepare the comet slides.

Isolation of Gonadal Cells
The left testis was removed, rinsed with cold phosphate buffered saline; the tunica layers were opened; removed, thereafter the seminiferous tubules were placed into ice cold phosphate buffered saline and the tightly coiled seminiferous tubules were gently agitated, shaken with tweezers for getting a loose structure. The loosen structure of seminiferous tubules were cut with a pair of fine scissors; thereafter the obtained parts (a volume of ~ 0.5 mL) transferred with tweezers, pipettes into an Eppendorf tube. The seminiferous tubules were suspended shortly with pipette (using an adequate tip). The cell suspension was stored on ice for about 30 seconds to allow the large clumps to settle. The obtained supernatant was used to prepare the comet slides.
The prepared slides are stored adequately, at room temperature, until potential analysis. In the case of additional analysis, its details will be provided in an amendment to the study plan. If additional analysis will not be required, the samples will be discarded after delivery of the final report.


METHOD OF ANALYSIS:
Coded slides were stained and blind scored. The slides were examined with an appropriate magnification (200x) using fluorescent microscope (Nikon Upright Microscope Eclipse Ci-S) equipped with Nikon-INTENSILIGHT C-HGFI Precentered Fiber Illuminator an appropriate excitation filter (TRITC) and with an Andor-Zyla sCMOS camera.
For image analysis, the Andor Komet GLP 7.1.0 (Andor Technology) was used.
For each tissue sample, fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, 750 per vehicle control and 450 per positive control).
DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length.
The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity.
Additionally, the OTM and tail length values were collected. The OTM is expressed in arbitrary units and is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail.
In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum.
Ghost cells, also known as clouds or hedgehogs, are morphological indicative of highly damaged cells and their presence is often associated with severe genotoxicity, necrosis and apoptosis. Ghost cells were excluded from the image analysis data collection, however determining of their frequency is useful for the data interpretation. The ghost cells were recorded for each slide per animal, per treatment and per tissue

Evaluation criteria:
Providing that all validity criteria are fulfilled, the test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

Providing that all validity criteria are fulfilled, the test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;

There is no requirement for verification of the clearly negative or positive response. In the case of equivocal results, the further process will be discussed with the Sponsor’s monitor.
Statistics:
The statistical significance of % tail DNA values; furthermore, the number of ghost cells was carried out using the appropriate statistical method, using SPSS software.
The homogeneity of variance between groups was checked by Levene's Test for Equality of Variances, the normal distribution of data was examined by Kolmogorov-Smirnov test. Following these analyses, a one-way analysis of variance following by Dunnett’s test was used to compare the vehicle control value and the corresponding values of test item doses or the inter-group comparisons were performed using non-parametric Mann-Whitney U test.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1800, 1500, 1200, 1000 mg/kg bw/d
- Clinical signs of toxicity in test animals: At the dose of 2000 mg/kg bw/day definite behavioral changes (reduced activity or hyperactivity, incoordination) appeared after the first treatment and these symptoms escalated to motionless, lying animals without reactions to the end of the observation period after the second treatment. These behavioral changes appeared at the investigated lower dose levels (at 1800 and 1500 mg/kg bw/day) at different timepoints within the observation periods (e.g.: after the first treatment the signs appeared equivocally at 1800 mg/kg bw/day, and no signs were noticed at 1500 mg/kg bw/day), but they reached the same intensity (motionless lying animals) to the end of the observation period after the second treatment. Incoordination, slight piloerection and slightly reduced activity were noticed at the end of the observation period (3-hour) after the second treatment at 1200 mg/kg bw/day, and incoordination and slight piloerection at 1000 mg/kg bw/day.
- Evidence of cytotoxicity in tissue analysed: Necropsy was performed on animals of the dose 1500 mg/kg bw/day. At the macroscopic examination of stomach in the forestomach tympanites, hyperaemia, bruising were described; furthermore faint structured glandular stomach was noticed; however, the latter signs were rather subjective observations.
- Rationale for exposure: For the correct identification of the maximum tolerated dose (MTD) where no death, evidence of pain, suffering or distress occurs non-GLP dose range finding tests were performed in the testing laboratory, using the same species, strain, sex, and treatment regimen (administration of doses by oral gavage twice: once on the day 0 and 24 hours thereafter) to be used in the main study. Each test was performed with two animals.

RESULTS OF DEFINITIVE STUDY
All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met.
No mortality was observed during the treatments and expression period in any dose group up to the limit dose of 1200 mg/kg body weight/day and in the controls. The observed clinical signs: strongly decreased activity (lying in cage floor, no responses), incoordination, increased respiration rate at the highest examined dose of 1200 mg/kg body weight/day after the second treatment, confirmed the adequate dose selection.
All of the group mean median % tail DNA values of each dose remained well within the vehicle control range at the examined tissues, and the slightly higher or lower values did not differ statistically significantly from that of the vehicle control up to the highest dose of 1200 mg/kg body weight/day. Furthermore, the mean median % tail DNA values (groups mean values) of the test item doses in the stomach, duodenum and liver samples were in line with the corresponding historical control data ranges (within the 95 % confidence intervals, C-charts) and in accordance with referred literature. The obtained tail length and OTM values (group means of dose groups), similarly to the main parameter % tail DNA values were in line with the corresponding historical control data ranges (within the 95 % confidence intervals, C-charts). The outlier values of the above parameters were further analyzed, compared with laboratory’s existing robust databases and were considered acceptable and to represent biological variability of the applied test system.

Any other information on results incl. tables

Summary Results (Stomach)






























































 



% Tail DNA



Tail length (µm)



OTM



Ghost cells (%)



Negative (vehicle) control



6.18 ns



14.84



0.70



13.92



300 mg/kg body weight/day



6.65 ns



17.58



0.71



15.87



600 mg/kg body weight/day



5.55 ns



13.15



0.57



17.97



1200 mg/kg body weight/day



6.16 ns



15.86



0.68



20.02



Positive (EMS) control



30.06 *



41.87



6.60



21.45



Negative control 95 % confidence intervals, C-charts



2.70 – 16.88



4.50 – 69.56



0.00 – 6.22



Negative control
Min- max range: 9 – 17



Positive control 95 % confidence intervals, C-charts



18.00 – 43.14



37.50 −145.25



0.19 – 25.53



Positive control
Min – max range: 12 − 30



Remark:        the table contains the group mean values of each parameter


Statistical analysis of %tail DNA values:


* :     Statistically significant (Mann-Whitney U Test)


ns:    Statistically not significant (Mann-Whitney U Test)


 


Summary Results (Duodenum)






























































 



% Tail DNA



Tail length (µm)



OTM



Ghost cells (%)



Negative (vehicle) control



7.44 ns



16.23



0.97



11.42



300 mg/kg body weight/day



5.31 ns



14.96



0.67



10.87



600 mg/kg body weight/day



7.26 ns



18.35



0.86



12.23



1200 mg/kg body weight/day



6.59 ns



13.98



0.80



11.39



Positive (EMS) control



27.89 *



42.57



7.11



14.86



Negative control 95 % confidence intervals, C-charts



0.36 – 21.05



2.44 – 78.53



0.00 – 6.73



Negative control
Min- max range: 8 – 11



Positive control 95 % confidence intervals, C-charts



10.10 – 49.67



25.62 −143.13



1.80 – 21.40



Positive control
Min – max range: 7 − 28



Remark:        the table contains the group mean values of each parameter


Statistical analysis of %tail DNA values:


* :     Statistically significant (Mann-Whitney U Test)


ns:    Statistically not significant (Mann-Whitney U Test)


 


Summary Results (Liver)






























































 



% Tail DNA



Tail length (µm)



OTM



Ghost cells (%)



Negative (vehicle) control



3.51 ns



7.07



0.35



4.26



300 mg/kg body weight/day



2.77 ns



7.29



0.31



3.95



600 mg/kg body weight/day



3.28 ns



7.05



0.34



4.89



1200 mg/kg body weight/day



3.09 ns



6.80



0.32



3.89



Positive (EMS) control



19.81 *



33.62



3.91



5.22



Negative control 95 % confidence intervals, C-charts



2.01 – 8.90



2.42 – 43.62



0.26 – 2.91



Negative control
Min- max range: 2 – 20



Positive control 95 % confidence intervals, C-charts



10.98 – 34.46



36.84 −126.44



1.22 – 15.80



Positive control
Min – max range: 5 − 31



Remark:        the table contains the group mean values of each parameter


Statistical analysis of %tail DNA values:


* :     Statistically significant (Mann-Whitney U Test)


ns:    Statistically not significant (Mann-Whitney U Test)


 

Applicant's summary and conclusion

Conclusions:
The test item 4-Phenyl-3-buten-2-one was investigated by the means of the in vivo comet assay on isolated stomach, duodenum and liver cells under alkaline conditions in the male HAN:WIST rats. The test item was administered twice via oral gavage at the dose levels 1200, 600 and 300 mg/kg body weight/day. Sampling was performed about 3 to 4 hours after the second treatment. Under the experimental conditions presented in this report, the test item 4-Phenyl-3-buten-2-one did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum and liver cells. Concurrent controls confirmed the sensitivity and validity of the test. The investigated test item 4-Phenyl-3-buten-2-one did not show genotoxic activity in the examined tissues in this Comet Assay.
Executive summary:

A study according to OECD Guideline 489 and under GLP conditions was performed with the registered substance. Male Wistar rats were treated with the test item dissolved in sunflower oil at doses of 1200, 600 and 300 mg/kg bw twice (at 0 h and at 24 h). A concurrent vehicle control and positive control (Ethyl methanesulfonate) was included.


The observed symptoms were in correspondence to already observed signs in the dose-range finding tests. After the second treatment: incoordination, motionless lying animals at the highest examined dose level, and no or slight symptoms at the lower doses. At the necropsy no signs of test item effect were noticed at the controls and lower doses, but signs of local test item effects (tympanites, significant amount of bedding material, liquid in the stomach, but no signs of affected mucous membranes) were noticed in animals at 1200 mg/kg bw dose.
The viability values of liver, stomach and duodenum cell suspensions (Trypan blue technique, screening) remained in the same vehicle control range at all test item treatment doses and positive control. The screened average viability values varied between 94-98 % at the liver cell preparations, between 72-80 % at the stomach cell preparations, it was 73-76 % at the duodenum preparations (slight, dose dependent average viability decrease (the average viability value was 10% lower at the highest test item dose, than in the vehicle control).


Under the experimental conditions presented in this report, the test item 4-Phenyl-3-buten-2-one did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum and liver cells. Concurrent controls confirmed the sensitivity and validity of the test. The investigated test item 4-Phenyl-3-buten-2-one did not show genotoxic activity in the examined tissues in this Comet Assay.