4-phenylbutenone

Genetic toxicity: in vivo

Administrative data

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

Study period:

experimental starting date: 2012-03-28, experimental completion date: 2012-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report Date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The relative humidity in the animal rooms ranged between 35 – 65% instead of 45 – 65%. This deviation, however, does not affect the validity of the study.

Test guideline 2

Qualifier:

according to

Guideline:

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

Deviations:

yes

Remarks:

The relative humidity in the animal rooms ranged between 35 – 65% instead of 45 – 65%. This deviation, however, does not affect the validity of the study.

GLP compliance:

yes (incl. certificate)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): 4-phenylbutenone
- Substance type: organic
- Physical state: solid
- Analytical purity: > 98.0 % (dose calculation was not adjusted to purity)
- Storage condition of test material: at room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Pre-experiment: Harlan Laboratories B.V., Horst / The Netherlands
Main experiment: Charles River Laboratories, Research Models and Services Germany GmbH, Sulzfeld, Germany
- Number of Animals used: 42
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: mean value 35.0 g (SD +/- 1.7 g)
- Assigned to test groups randomly: [yes]
- Housing: single
- Diet (e.g. ad libitum): ad libitum (pelleted standard diet)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 35 - 65 %
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

According to the suppliers assurance the animals were in healthy condition. The animals were under acclimation in the animal house of Harlan CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO; polyethylene glycol
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: On the day of the experiment, the test item was dissolved in 30% DMSO and 70% PEG 400.
- Amount of vehicle (if gavage or dermal): All animals received a single standard volume of 10 mL/kg body weight orally.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was dissolved in 30% DMSO and 70% PEG 400. All animals received a single standard volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

once orally (Amount given: 10 mL/ kg bw)

Frequency of treatment:

once

Post exposure period:

24 or 48 hours, respectively

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

7 males/ dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally once
- Doses / concentrations: 40 mg/kg b.w. (Amount given: 10 mL/kg b.w.)
- Other: The stability of CPA at room temperature was sufficient. At 25 °C only 3.5 % of its potency is lost after 24 hours.

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The administered volume was 10 mL/kg b.w..
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively. The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe.

DETAILS OF SLIDE PREPARATION:
The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

The study is considered valid if the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the negative control.

Evaluation of Results
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group in comparison to the laboratory’s historical data range. Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 mg/kg bw
- Solubility: test item was dissolved in 30 % DMSO / 70 % PEG 400
- Clinical signs of toxicity in test animals: none (no ruffled fur, no reduction of spontaneous activity

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): test item PCE with micronuclei: 0.042 versus 0.05 % in the vehicle control

Any other information on results incl. tables

Pre-Experiment on Toxicity

A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.

The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Since no gender specific differences on acute toxic symptoms were observed in agreement with the sponsor the main study was performed using males only.

Dose Selection

It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.

The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.

The administered volume was 10 mL/kg b.w..

Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Pre-Experiments for Toxicity

The animals treated in the pre-experiments received the test item 4-phenylbutenone dissolved in 30% DMSO / 70% PEG 400 once orally. The volume administered was 10 mL/kg b.w.. The following dose levels were tested and expressed toxic reactions were shown in the table:

Table 1: results of the pre-experiments for toxicity

	hours post-treatment
	1	2-4	6	24	30	48
1^stPre-experiment: 1000 mg/kg b.w. ; males / females
reduction of spontaneous activity	0/0	0/2	0/2	0/2	0/2	0/0
ruffled fur	0/0	0/2	0/2	0/2	0/2	0/0

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable as highest dose.

No substantial sex specific differences were observed with regard to clinical signs. In agreement with the sponsor the main study was performed using males only.

Toxic Symptoms in the Main Experiment

In the main experiment for each test item dose groups 7 males received the test item 4-phenylbutenone dissolved in 30% DMSO / 70% PEG 400 once orally. The volume administered was 10 mL/kg b.w.. The symptoms of toxicity observed following treatment are shown in the following table for each dose group, which indicates the number of males with findings. The animals treated with the negative control (30% DMSO / 70% PEG 400) did not express any toxic reaction.

Table 2: Toxic symptoms in the main experiment

	hours post-treatment (males)
	1	2-4	6	24	48*  h
High dose: 2000 mg/kg b.w. (24 h and 48 h)
reduction of spontaneous activity	3	4	4	2	0
abdominal position	3	1	1	0	0
eyelid closure	3	2	2	2	0
ruffled fur	3	3	3	4	0
death	0	1**	0	0	0
Medium dose: 1000 mg/kg b.w. (24 h)
reduction of spontaneous activity	1	1	1	0	-
abdominal position	1	0	0	0	-
eyelid closure	1	1	1	0	-
ruffled fur	1	1	1	1	-
Low dose: 500 mg/kg b.w. (24 h)
abdominal position	3	0	0	0	-
eyelid closure	4	0	0	0	-
excitement	2	0	0	0
ruffled fur	4	0	0	0	-

*   data only from 7 male animals

**  animal no. 40

-   no observation made

Table 3: Summary of Micronucleus Test Results

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
vehicle	0	24	0.050	0 -2	1288
test item	500	24	0.114	0 -3	1333
test item	1000	24	0.100	0 -5	1245
test item	2000	24	0.093	1 -3	1234
positive control	40	24	1.964	32 -49	1240
test item	2000	48	0.042	0 -2	1099

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The in vivo micronucleus test was conducted in NMRI mice, according to OECD 474 and EU Method B12 (with only non-significant deviations) and therefore considered to be of the highest quality. No increases in the incidence of micronucleated PCEs were observed in males exposed to 4-phenylbutenone. It is therefore considered that 4-phenylbutenone not clastogenic in the mouse micronucleus test.

Executive summary:

The test item 4-phenylbutenone was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (Roth, 2012). The test item was dissolved in 30% DMSO / 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w. and 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

As estimated by pre-experiments 2000 mg 4-phenylbutenone per kg bw was suitable as top dose. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw and 48 h preparation interval: 2000 mg/kg bw.

As a result , the mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that 4-phenylbutenone did not have any cytotoxic properties in the bone marrow. However, one male (animal no. 40) of the high dose group (48 h treatment interval) died approximately 3 hours after treatment with the test item.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 4-phenylbutenone were near to the value of the vehicle control group. However, the statistically significant increase above the vehicle control group observed in the low dose group was considered to have no biological relevance, as the value was well within the laboratory’s historical vehicle control data. Additionally, no dose dependent increase in the frequency of detected micronuclei was observed with increasing dosages.

Cyclophosphamide administered once orally (40 mg/kg b.w.) was used as positive control which showed a substantial and biologically relevant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, 4-phenylbutenone is considered to be non-mutagenic in this micronucleus assay.