DL-α-methylbenzylamine

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:

03 June 2013 - 02 July 2013 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study conducted in compliance with GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

DL-α-methylbenzylamine

EC Number:

210-545-8

EC Name:

DL-α-methylbenzylamine

Cas Number:

618-36-0

Molecular formula:

C8H11N

IUPAC Name:

1-phenylethan-1-amine

Details on test material:

- Name of test material (as cited in study report): 1-Phenylethylamine
- Analytical purity: 99.8 corr. area%
- Lot/batch No.: O 2932
- Physical state/ appearance: liquid / colorless, clear
- Stability under test conditions: confirmed indirectly by dose formulation analytics
- Storage condition of test material: at room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Details on strain: Crl: NMRI
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 35.6 g
- Assigned to test groups randomly: yes
- Housing: single housing in Makrolon cages, type M II/III with wire mesh top
- Diet (ad libitum): pelleted standard diet, ad libitum Harlan Laboratories B.V.
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 45 - 75
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: due to its non-toxicity for the animals
- Amount of vehicle: 10 mL/kg bw
- Concentration of test material in vehicle: 6.25, 12.5, and 25 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h
after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Homogeneity of the test item in vehicle
was maintained during treatment using a magnetic stirrer.The vehicle was chosen due to its relative non-toxicity for the animals. All animals receive
a single standard volume orally.


ANALYSIS OF TEST SUBSTANCE PREPARATION:
The dose formulation analytics was performed as a separate study under the responsibility of the sponsor and the results will be reported in a separate report (BASF study code 04Y0728/11Y130). As a clear solution was achieved in the vehicle, samples of all dose formulations and of the vehicle
control (1 x 1 mL, each) were taken on the first treatment day immediately after the last application of the test item. Also, reserve samples of all dose formulations and the vehicle control were taken (1 x 1 mL, each). Corresponding reserve samples were also taken. The aliquots were stored
at ≤ -18 °C at Harlan CCR until shipment. Furthermore a small amount of the test item was also taken as a sample.

The determined concentration of 1-Phenylethylamine in Corn oil was found to be in the range between 90 % and 110 % of the nominal concentration

Duration of treatment / exposure:

once administered by gavage

Frequency of treatment:

once

Post exposure period:

24 h: vehicle control; 62.5, 125, 250 mg test substance/kg bw;
positive controls: 40 mg cyclophosphamide (CPA)/kg bw
48 h: vehicle control; 250 mg test substance/kg bw

Doses / concentrations

Remarks:

Doses / Concentrations:
62.5, 125, 250 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

7 males per test group (except the vehicle and positive control groups with 5 males only)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA);

- Route of administration: orally (CPA)
- Doses / concentrations: CPP: 40 mg/kg bw (10 mL/kg bw in deionized water)

Examinations

Tissues and cell types examined:

The animals of all dose groups, except the positive control were examined for clinical signs at intervals of around 1 h, 2 - 4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
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. 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- /Giemsa. Cover slips were
mounted with EUKITT. At least one slide was made from each bone marrow sample.
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate 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.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
based on range-finding study 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.

DETAILS OF SLIDE PREPARATION:
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. 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- /Giemsa. Cover slips were
mounted with EUKITT. At least one slide was made from each bone marrow sample.
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate 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.


METHOD OF ANALYSIS:
To investigate 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.

Evaluation criteria:

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. Statistical methods (nonparametric Mann-Whitney test (8)) are used as an aid in evaluating the results, if necessary. 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:

yes

Remarks:

Reduction of spontaneous acivity, ruffled fur 125 and 250 mg/kg; addominal posture 250 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
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 orally with the test item at in sequential experiments at dose levels of 1000, 125, 500, and 250 mg/kg b.w. and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 5-6 h, 24 h, 30 h and 48 h after administration of the test item.
1000 mg/kg:Two animals died 10 minutes after treatment. Two animals were found moribund and were euthanized.
500 mg/kg all animals were euthanized due to severity of systemic toxicity.
250 mg/kg: ruffled fur, reduction of spontaneous activity, abdominal position and tippy toe walk
125 mg/kg: no clinical signs of toxicity were observed.

On the basis of these data 250 mg/kg b.w. were estimated to be suitable as highest dose level and considered as maximum tolerated dose leve

Any other information on results incl. tables

DISCUSSION:

The test item 1-Phenylethylamine was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 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 (except the vehicle and positive control groups with 5 males

only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate 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.

The following dose levels of the test item were investigated, based on results of a preexperiment:

24 h preparation interval: 62.5, 125, and 250 mg/kg b.w.

48 h preparation interval: 250 mg/kg b.w.

After treatment with the test item at 48h preparation interval the mean number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that 1-Phenylethylamine did not induce cytotoxic effects in the bone

marrow. Clinical signs comprised ruffled fur and reduction of spontaneous activity at 125 and 250 mg/kg b.w. and abdominal posture at 250 mg/kg b.w. in the main experiment, observed 1 h (125 mg/kg b.w.) or up to 2-4 h (250 mg/kg b.w.) after treatment.

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 1-Phenylethylamine were below or near to the value of the vehicle control group and all values were very well within the historical vehicle control data range.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, 1-Phenylethylamine did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, 1-Phenylethylamine did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.