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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Remarks:
Communication number: CCH-D-2114504234-63-01/D_Additional information included.
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No OECD Guideline defined.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987
Reference Type:
secondary source
Title:
Dimethylbenzylamine - Micronucleus test in bone marrow cells of the mouse
Author:
Völkner W.
Year:
1987
Bibliographic source:
LMP 262 (1987)||cited in:|Heiman H-G, Huber W|Mutagenitätsprüfungen von Arbeitsstoffen mit wesentlicher Bedeutung und Verbreitung|GUM, 4/89: 2-6 (1989)
Reference Type:
publication
Title:
Mutagenitätsprüfungen von Arbeitsstoffen mit wesentlicher Bedeutung und Verbreitung
Author:
Heimann K.-G- et al.
Year:
1998
Bibliographic source:
GUM 4/89

Materials and methods

Principles of method if other than guideline:
The test substance was assessed for mutagenic properties in the micronucleus test with bone marrow cells of the mouse. The dosing was 15, 50, 150 mg/kg bw. (24 h); 150 mg/kg bw (48h); 150 mg/kg bw (72 h). The administration was per os (stomach tube); single. Preparation of the bone marrow cells was done 24, 48 and 72 h after the administration of the test substance.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TS-Freetext:
Purity: 99.34 %

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 2.5-4 months
- Housing: individually
- Diet ad libitum
- Water ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 40-60
- Air changes (per hr):10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Details on exposure:
Single oral administration of the test substance diluted in DMSO by gavage.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
24, 48, 72 hours, respectively
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 50, 150 mg/kg bw
Basis:

No. of animals per sex per dose:
10
Control animals:
yes
Positive control(s):
cyclophosphamide

Examinations

Tissues and cell types examined:
mouse bone marrow cells
Details of tissue and slide preparation:
The animals were killed by cervical dislocation. The femora were removed and freed from muscles and tissue. The epiphyses were cut off with scissors and the marrow was fluxhed out with fetal calf serum. The cell suspension was centrifuged and the pellet spread on a slide . The smear was air-dryed and then stained with May-Grünwald/Giemsa. Cover slips were mounted with Eukitt. 3 Slides were made from each animal.
Evaluation criteria:
no data

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
150 mg/kg bw: reduction of spontaneous activity and reduced food and water consumption in a pre-test.
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity:
150 mg/kg produced reduction of spontaneous activity and reduced food and water consumption in a pre-test.

Any other information on results incl. tables

Result of the dose range finding “pre-experiment” as indicated in the study report:

Dosing was chosen with regard to the results of a pre-experiment. 150 mg/kg b.w. of the test substance was the highest non-lethal dose. With this dose the animals expressed the following toxic reactions but no mortality: reduction of spontaneous activity, no food and water uptake for 1 hour after administration.

With higher doses of the test substance animals died in the dose range finding “pre-experiment”. The following results were reported:

200 mg/kg b.w. dosing: 1 out of 6 treated animals died.

250 mg/kg b.w. dosing: 1 out of 6 treated animals died.

300 mg/kg b.w. dosing: 3 out of 6 treated animals died.

Main study:

Therefore the following doses in the main study were used:

 Group Substance

Dose

[mg/kg b.w.

Preparation hours

p. admin.

 Number of animals analyses

Males : females = 5 : 5

per group

 1

 solvent

 0

 24

 10

 2

 solvent

 0

 48

 10

 3

 solvent

 0

 72

 10

 4

 CPA

 30

 24

 10

 5

 test substance

 15

 24

 10

 6

 test substance

 50

 24

 10

 7

 test substance

 150

 24

 10

 8

 test substance

 150

 48

 10

 9

 test substance

 150

 72

 10

CPA = Cyclophosphamide

The requirements specified in the OECD guideline no. 474 are as follows:

If a preliminary range-finding study is performed because there are no suitable data already available to aid in dose selection, it should be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study. The study should aim to identify the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia)” are fulfilled.

Based on the data of the dose range finding “pre-experiment” the selected dose of 150 mg/kg is considered appropriate according to the OECD TG  474.

In addition, reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow might be considered to conclude if a compound reached the target tissue. Within the main study the relationship PE/NE (polychromatic erythrocytes (PE) / normochromatic (NE) was examined.

It is considered that a decrease of the ratio of polychromatic erythrocytes  to normochromatic erythrocytes  (PE/NE) in the micronucleus test is an indicator to conclude if a compound reached the target tissue.

 Group  Substance

 Dose

[mg/kg b.w.]

 Preparation hours

p. admin.

 Polychromatic

Erytrocytes

with

micronulei

Range 

 PE/NE

 1

 solvent

 0

 24

 0.14%

 0 - 5

 1000/455

 2

 solvent

 0

 48

 0.06%

 0 - 3

 1000/885

 3

 solvent

 0

 72

 0.09%

 0 - 2

 1000/435

 4

 CPA

30 

 24

 1.22%

 4 - 24

 1000/644

 5

 test substance

15

 24

 0.15%

 0 - 4

 1000/498

 6

 test substance

 50

 24

 0.18%

 0 - 4

 1000/516

 7

 test substance

 150

24 

 0.13%

0 - 2 

 1000/441

 8

test substance 

 150

 48

 0.10%

 0 - 2

 1000/702

 9

 test substance

 150

 72

 0.10%

 0 - 3

 1000/442

The effects of a decrease of the ratio of PE/NE (polychromatic erythrocytes / normochromatic erythrocytes in the highest dose applied (150 mg/kg b.w.) is not evident compared to the solvent at 24 and 72 hours after administration of the test substance, however a clear effect exists at 48 hours.

The mean value of the solvent   is 592 (at 24, 48 and 72 h NE is (455 + 885 + 435) : 3) whereas the mean value of the test substance at 150 mg/kg bw  is 528 (at 24, 48 and 72 h NE is (441 + 702 + 442) : 3).

This means, the ratio of polychromatic erythrocytes to normochromatic erythrocytes = (PE/NE) decreased from 592 to 528.

Conclusion:

In a range finding “pre-experiment” the maximum non-lethal tolerated dose (MTD) was 150 mg/kg b.w. At higher doses (200 mg/kg b.w. and above) mortality was observed. Consequently the selected high dose of 150 mg/kg b.w. in the main experiment is considered appropriate according to the OECD TG  474 and the negative result in this guideline study is considered valid.

Applicant's summary and conclusion

Conclusions:
The test substance did not show any mutagenic activity as determined by the micronucleus test with mouse bone marrow cells .
Executive summary:

The test substance was assessed for mutagenic activity in the micronucleus test in vivo with bone marrow cells of the mouse.

Male and female mice were given single oral doses by gavage 0, 15, 50, 150 mg/kg bw. (24 h); 150 mg/kg bw (48h); 150 mg/kg bw (72 h).diluted in DMSO. Toxicity was examined in a pre-experiment. 150 mg/kg bw was the highest non-lethal dose but animals showed reduction of spontaneous activity and no food and water uptake was observed for up to 1 hour post application in that test..

Preparation of mice bone marrow cells was done 24, 48 and 72 h after the administration of the test substance.

With no dose at no preparation time any enhanced micronucleus rates were found. The sensitivity of the test system was shown by administration of cyclophosphamide (positive control) which induced a significantly higher micronucleus rate as compared to the negative controls.

The test substance did not show any mutagenic activity as determined by the micronucleus test with bone marrow cells.