Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-09-04 to 2013-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study according to OECD/EU guideline. Study was performed with the Racemat (DL-alpha-methylbenzylamine). Read across is done to L-alpha-methylbenzylamine, as the substance is expected to behave similar to the racemat with regard to mutagneic properties. For read-across justification please refer to IUCLID section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD 487 (adopted 22 July 2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Comission Regualtion (EC) No 640/2012; B.49
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
DL-α-methylbenzylamine
EC Number:
210-545-8
EC Name:
DL-α-methylbenzylamine
Cas Number:
618-36-0
IUPAC Name:
1-phenylethanamine
Test material form:
other: liquid

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL), 1% (v/v) amphotericine B (250 μg/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for plating efficiency incl. vital staining
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from S9 fraction of phenobarbital/beta-naphthoflavone induced male rat liver.
Test concentrations with justification for top dose:
1st Experiment:
4 hour exposure, without S9-mix: 6.25 µg/mL, 12.50 µg/mL, 25.00 µg/mL, 50.00 µg/mL, 100.00 µg/mL, 200.00 µg/mL
4 hour exposure, with S9-mix: 40.63 µg/mL, 81.25 µg/mL, 162.50 µg/mL, 325.00 µg/mL, 650.00 µg/mL, 1300.00 µg/mL

2nd Experiment:
4 hour exposure, without S9-mix: 40.63 µg/mL, 81.25 µg/mL, 162.50 µg/mL, 325.00 µg/mL, 650.00 µg/mL, 1300.00 µg/mL
4 hour exposure, with S9-mix: 121.88 µg/mL, 243.75 µg/mL, 487.50 µg/mL, 975.00 µg/mL, 1300.00 µg/mL

3rd Experiment:
4 hour exposure, without S9-mix: 500.00 µg/mL, 600.00 µg/mL, 700.00 µg/mL, 800.00 µg/mL, 1000.00 µg/mL, 1300.00 µg/mL
Vehicle / solvent:
- Vehicle used: Culture medium
- Justification for choice of vehicle:Good solubility of the test item in water
Controls
Untreated negative controls:
yes
Remarks:
Culture medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: 500 and 600 μg/mL ethyl methanesulfonate; With metabolic activation: 2.5 μg/mL cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time: 20 hours
- Fixation time: 24 hours

SPINDLE INHIBITOR: Ethanol:glacial acetic acid, ratio 3:1; +4°C
STAIN: Wrights solution (modified May-Grünwald solution), counterstained with 2.6% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1000 cells per culture, 2000 cells per dose group

DETERMINATION OF CYTOTOXICITY
- Method: Cell count, proliferation index

OTHER EXAMINATIONS:
- pH value: Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the negative control with and without S9 mix.
- Osmolarity: Osmolarity was measured, at least for the top dose and for the negative control with and without S9 mix.
- Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically/ microscopically).
Evaluation criteria:
Micronucleus analysis
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only cells clearly surrounded by a nuclear membrane were scored.
Slides were coded before microscopic analysis. Cultures with few isolated cells were not analysed for micronuclei.

Cell count
The cell count was determined by means of test cultures prepared especially for this purpose in 25 cm2 flasks for all test groups, except the positive controls. The data are given in percentage compared with the current vehicle control.

Relative increase in cell count (RICC)
The cell count was determined by means of test cultures prepared especially for this purpose in 25 cm2 flasks for all test groups, except the positive controls. The relative increase in cell counts was calculated based on the following formula:
RICC = [ (Increase in number of cells in treated cultures (final - starting)) / (Increase in number of cells in control cultures (final - starting)) ] x 100

Cell morphology:
At the end of the treatment period, the test cultures of all test groups were checked microscopically for cell morphology and attachment to the slides as further parameters of toxicity.

Assessment of the slides
The dose selection for the scoring was based on the results of a previous check on
− the quality of the cells
− the number of analyzable cells
− the amount of fragmented cells

Proliferation Index
For the assessment of test substance induced cytotoxicity the proliferation index as measure of the proliferative activity of the cells was determined. The proliferation index was determined in at least 1 000 cells per culture (corresponding to at least 2 000 cells per dose group) in all test groups.
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
If the results of this test were statistically significant compared with the respective negative control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been be printed in the tables.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the stock solutions was adjusted to a physiological value using small amounts of HCl.
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: Yes
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 1 300 μg/mL (approx. 10.7 mM) 1-Phenylethylamine was used as top concentration. The cells were prepared at a harvest time of 24 hours (about 2 cell cycles) after 4 and 24 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix.
The pretest was performed following the method described for the main experiment. As indication of test substance toxicity relative increase in cell count (RICC) and cell attachment (morphology) were determined for dose selection.
In the pretest various additional parameters were checked or determined for all or at least some selected doses. The following parameters are available: pH, osmolarity, solubility.
After 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced relative increase (RICC) of about or below 40 - 50% was observed from 162.5 μg/mL onward. In addition, in the presence of S9 mix, clearly reduced increase in cell count was observed after treatment with 1 300 μg/mL.
Besides, in the pretest with 24 hours continuous treatment in the absence of S9 mix, the relative increase in cell count was clearly reduced after treatment with 81.25 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Historical negative control data range (0.1 – 1.8% micronucleated cells)
- Historical positive control data range (2.3 – 26.6% micronucleated cells)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Proliferation Index:
In this study, in the absence and presence of S9 mix no cytotoxicity indicated by reduced Proliferation Index was observed at the test groups scored for cytogenetic damage.
- Relative increase in cell count:
In addition, no toxic effects were obtained in the 1st Experiment without S9 mix up to the highest applied test substance concentration (200 μg/mL). Besides, in the 2nd and 3rd Experiment in the absence of S9 mix growth inhibition indicated by clearly reduced cell counts (36.5% and 36.6% of control, respectively) was observed at the highest required test substance concentration (1 300 μg/mL). However, due to strong toxic effects the slides of these test groups were not scorable for cytogenetic damage. In the presence of S9 mix no growth inhibition was observed up to the highest required test substance concentration.
- Cell morphology:
In this study, cell morphology/attachment was adversely influenced (grade > 2) in the 1st Experiment at 200 μg/mL and in the 3rd Experiment at 500 μg/mL and above without S9 mix. Besides, in all additional experimental parts no relevant influence on cell morphology/attachment was found.
The slides were not scorable for cytogenetic damage due to strong cytotoxicity and/or poor quality in the 2nd Experiment in the absence of S9 mix at 1 300 μg/mL and in the 3rd Experiment in the absence of S9 mix at 800 μg/mL and above.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental parts without S9 mix

Exp.

Exposure period

Test groups

S9 mix

Precipitation in culture medium

Genotoxicity Micronucleated cells [%] *

Cytotoxicity

Proliferation Index (PI)

Relative increase in cell count [%]

1

4 hrs

Negative control

 -

n.d.

n.d.

n.d.

100.00

 

 

6.25 µg/mL

 -

 -

n.d.

n.d.

80.10

 

 

12.50 µg/mL

 -

 -

n.d.

n.d.

79.40

 

 

25.00 µg/mL

 -

 -

n.d.

n.d.

87.50

 

 

50.00 µg/mL

 -

 -

n.d.

n.d.

91.50

 

 

100.00 µg/mL

 -

 -

n.d.

n.d.

83.80

 

 

200.00 µg/mL

 -

 -

n.d.

n.d.

85.80

 

 

Positive control1

 -

n.d.

n.d.

n.d.

n.t.

 

 

 

 

 

 

 

 

2

4 hrs

Negative control

 -

n.d.

0.7

2.39

100.00

 

 

40.63 µg/mL

 -

 -

n.d.

n.d

76.10

 

 

81.25 µg/mL

 -

 -

n.d.

n.d

57.80

 

 

162.50 µg/mL

 -

 -

1.1

1.98

82.60

 

 

325.00µg/mL

 -

 -

0.6

2.07

60.40

 

 

650.00 µg/mL +

 -

 -

2.9s

1.95

83.50

 

 

1300.00 µg/mL

 -

 -

n.s.

n.s.

36.50

 

 

Positive control1

 -

n.d.

3.7s

2.16

n.t.

 

 

 

 

 

 

 

 

3

4 hrs

Negative control

 -

n.d.

0.8

2.18

100.00

 

 

500.00 µg/mL

 -

 -

2.0s

2.14

103.90

 

 

600.00 µg/mL

 -

 -

4.3s

2.07

102.50

 

 

700.00 µg/mL

 -

 -

4.9s

1.89

89.50

 

 

800.00 µg/mL

 -

 -

n.s.

n.s.

71.30

 

 

1000.00 µg/mL

 -

 -

n.s.

n.s.

58.10

 

 

1300.00 µg/mL

 -

 -

n.s.

n.s.

36.60

 

 

Positive control1

 -

n.d.

3.4s

2.14

n.t.

 

 

*          Relative number of micronucleated cells per 2000 cells scored per test group, in general

+         Due to inhomogeneous data an increased sample of 4 000 cells was scored

s         Frequency statistically significant higher than corresponding control values

n.d.     Not determined

n.s.     Not scorable due to strong cytotoxicity

n.t.      Not tested

1          EMS 500μg/mL

 

Experimental parts with S9 mix

Exp.

Exposure period

Test groups

S9 mix

Precipitation in culture medium

Genotoxicity Micronucleated cells [%] *

Cytotoxicity

Proliferation Index (PI)

Relative increase in cell count

1

4 hrs

Negative control

 +

n.d.

1.2

1.85

100.00

 

 

40.63 µg/mL

 +

 -

n.d.

n.d

96.10

 

 

81.25 µg/mL

 +

 -

n.d.

n.d

114.30

 

 

162.50 µg/mL

 +

 -

n.d.

n.d

108.80

 

 

325.00µg/mL

 +

 -

1.4

1.73

102.50

 

 

650.00 µg/mL

 +

 -

1.7

1.83

99.50

 

 

1300.00 µg/mL

 +

 -

1.5

1.74

100.30

 

 

Positive control1

 +

n.d.

4.9s

1.51

n.t.

 

 

 

 

 

 

 

 

2

4 hrs

Negative control

 +

n.d.

1.2

2.15

100.00

 

 

121.88 µg/mL

 +

 -

n.d

n.d.

109.60

 

 

243.75 µg/mL

 +

 -

n.d

n.d.

84.50

 

 

487.50 µg/mL

 +

 -

1.5

2.27

100.60

 

 

975.00 µg/mL

 +

 -

1.0

1.94

91.00

 

 

1300.00 µg/mL

 +

 -

1.4

2.05

78.40

 

 

Positive control1

 +

n.d.

4.3s

1.75

n.t.

 

*         Relative number of micronucleated cells per 2000 cells scored per test group

s        Frequency statistically significant higher than corresponding control values

n.d.    Not determined

n.s.    Not scorable due to strong cytotoxicity

n.t.     Not tested

1        CPP 2.5μg/mL

Applicant's summary and conclusion