1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-01-22 to 2001-03-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

A limit study was performed and not a full study with two sexes and three dose levels as is recommended by the authorities, if the test substance is genotoxic

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

yes

Remarks:

A limit study was performed and not a full study with two sexes and three dose levels as is recommended by the authorities, if the test substance is genotoxic

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1-(3,5-dihydroxyphenyl)-2-{[1-(4-methoxyphenyl)propan-2-yl]amino}ethan-1-one hydrobromide

EC Number:

928-779-0

Cas Number:

1178555-23-1

Molecular formula:

C18-H21-N-O4 x HBr

IUPAC Name:

1-(3,5-dihydroxyphenyl)-2-{[1-(4-methoxyphenyl)propan-2-yl]amino}ethan-1-one hydrobromide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: No details on the source of the test material were provided. Batch number: 375.
- Expiration date of the lot/batch: 01 March 2001 (allocated by testing facility, 1 year after reciept of the test substance).
- Purity test date:No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Stability under test conditions:Stable
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in the vehicle was not indicated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No details reported.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

BR (SPF)

Details on species / strain selection:

Recommended test system in international guidelines.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 - 8 weeks old
- Weight at study initiation: 30.6 +- 1.5 to 31.8 +- 1.9 g
- Assigned to test groups randomly: yes
- Housing: Group housing of 5 animals per sex per cage in labeled polycarbonate cages containing purified sawdust
- Diet (e.g. ad libitum): free access, (Altromin (code VRF 1), Lage, Germany).
- Water (e.g. ad libitum): free access, tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light, 12 hours dark

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil (OPG, Utrecht, The Netherlands) for the test substance and physiological saline (Fresenius B.V., ´s-Hertogenbosch, The Netherlands)
- Concentration of test material in vehicle: 150 mg/ml suspension

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in corn oil (OPG, Utrecht, The Netherlands). The test item concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. The test item concentrations were dosed within 4 hours after preparation

Duration of treatment / exposure:

The mice received an intraperitoneal injection of a maximum tolerated dose. The route of administration was chosen to maximize the chance of the test article reaching the target tissue. The dosing volume was 10 ml/kg body weight. The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.

Frequency of treatment:

single (dosing volume 10 ml/kg bw)

Doses / concentrations

Dose / conc.:

150 mg/kg bw (total dose)

Remarks:

nominal conc.

No. of animals per sex per dose:

2 male mice in each treatment group were used in the dose range finding study
5 male mice per sampling time in each treatment group were used in the micronucleus test.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CP; CAS 50-18-0; Endoxan, Asta-Werke, F.R.G.)

Examinations

Tissues and cell types examined:

micronucleated polychromatic erythrocytes (bone marrow)

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing of the test substance, 24 h after dosing of the vehicle and 48 h after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approx. 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the study identification number and the animal ID number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approx. 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears:
The slides were automatically stained using the 'Wright-stain-procedure' in an 'Ames' HEMA-tek slide stainer (Miles, Bayer Nederland B. V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

Analysis of the bone marrow smears for micronuclei:
All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100* for regions of suitable technical quality, i. e. where the cells were well spread, undamaged and well stained.. Slides were scored at a magnification of 1000*. The number of micronucleated polychromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in poly chromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.

Statistics:

The Wilcoxon Rank Sum Test, two-sided test at P< 0.05 and historical control data range were used to verify the results, taking into account the characteristics of the micronucleus assay. A probability of P < 5% (cells with micronuclei in any dose group) was considered statistically significant.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

substance caused clinical signs of toxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY 1
- Dose range: 2000, 1000, 250 mg/kg bw
- Number of animals: 6 (2 males per group)
- Clinical signs of toxicity in test animals: refer to table 1

RESULTS OF Micronucleus Test 1
- Dose: 250 mg/kg bw
- Number of animals: 5 males per group, 3 additional animals to correct for possible deaths
- Clinical signs of toxicity in test animals: during the first hour after dosing all animals were lethargic. Within 20 hours after dosing ten animals died, two animals were only lethargic and one animal was lethargic, had a rough coat and a hunched posture
- Rationale for exposure: based on the results of the range-finding study 1

RESULTS OF RANGE-FINDING STUDY 2
- Dose range: 150 mg/kg
- Number of animals: 2 males
- Clinical signs of toxicity in test animals: within 2 hours after dosing both animals showed ataxia and one animal showed also tremors and salivation. Within 20 hours after dosing both animals showed no abnormalities.
- Rationale for exposure: since the dose level of 250 mg/kg bw was too toxic for the mice, this additional dose range finding study was performed.

RESULTS OF Micronucleus Test 2 (main test)
- Dose 150 mg/kg bw
- Number of animals: 5 males per group
- Clinical signs of toxicity in test animals: within two hours after dosing three animals showed ataxia. Within 20 hours after dosing one animal showed a hunched posture and one animal had a rough coat and a hunched posture. Within 44 hours after dosing one animal still showed a hunched posture; the other animals showed no abnormalities.
- Ratio of PCE/NCE (for Micronucleus assay): no decrease in the ratio of polychromatic erythrocytes (refer to Table 3)
- Statistical evaluation: Wilcoxon Rank Sum Test, P <= 0.01

Any other information on results incl. tables

Dose range finding study 1

In the dose range finding study 6 animals (2 males per group) were dosed intraperitoneally with 2000, 1000 and 250 mg/kg body weight (groups A, B and C respectively). The results of this dose range finding study are presented in Table 1.

Table 1 Mortality and systemic toxic signs after treatment of the test item in the dose range finding study

Group	Sex	Dose mg/kg	Systemic toxic signs^* day				day 2
			15 min	30 min	1 hr	1.5 -2 hr
A	Male	2000		CE	A
A	Male	2000		CE	A
B	Male	1000			B	A
B	Male	1000			B	CE; A
C	Male	250	CF			B	B
C	Male	250	CF			B	A

^*) A=died; B=showed no abnormalities; C=ataxia; E=tremor; F=lethargy

Micronucleus Test 2 (main test):

Micronucleated polychromatic erythrocytes

The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of polychromatic to normochromatic erythrocytes are presented in Table 2. The indivudual data are described in Table 3. The mean number of micronucleated polychromatic erythrocytes scored in the test item treated groups were compared with the corresponding solvent control group.

No biologically significant increase in the frequency of micronucleated polychromatic erythroytes was observed in the polychromatic erythrocytes of the bone marrow of the test item treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

Table 2: Mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes and ratio of polychromatic/norchromatic erythrocytes

Group	Treatment	Dose (mg/kg bw)	Sampling time (hours)	Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes (mean +- S.D.)*	Ratio polychromatic erythrocytes (mean +- S.D.)*
A	vehicle	0	24	0.2 +- 0.4	1.17 +- 0.17
B	Test item	150	24	1.4 +- 1.3	0.89 +- 0.11
C	Test item	150	48	1.0 +- 1.4	1.05 +- 0.03
D	CP	50	48	23.8 +- 16.9**	0.42 +- 0.09

Vehicle = corn oil

CP = Cyclophoshamide

* Five animals per treatment group

** Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P <= 0.01)

Table 3: Individual data

Group	Animal number	Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes	Ratio polychromatic erythrocytes
A	1	0	1.29
A	2	0	1.26
A	3	1	1.34
A	4	0	0.97
A	5	0	1.00
B	6	2	1.00
B	7	0	0.86
B	8	3	0.94
B	9	2	0.71
B	10	0	0.94
C	11	0	1.05
C	12	3	1.02
C	13	2	1.04
C	14	0	1.04
C	15	0	1.11
D	16	16	0.56
D	17	27	0.38
D	18	12	0.43
D	19	12	0.31
D	20	52	0.40

A: intraperitoneal injection of corn oil

B-C: intraperitoneal injection of the test item

D: intraperitoneal injection of Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

The males that were exposed to the test substance showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of the compound on the erythropoesis. It is concluded that the test substance is not mutagenic in the micronucleus test under the experimental conditions.