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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Short description of key information:
N,N-dimethylbenzylamine does not induce mutagenic activity in the Ames test and in the HPRT test in vitro with Chinese Hamster V79 cells but induces chromosome aberrations in Chinese Hamster Ovary cells in the presence of a metabolic activation system but not in the absence of a metabolic activation system. However, the In-vivo Micronucleus Test in mouse bone marrow cells yielded a negative result. Therefore N.N-dimethylbenzylamine is considered to be a Non-mutagen.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline test and GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
- preincubation method
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimuriun TA98. TA100. TA1535, TA1537, TA1538
Additional strain / cell type characteristics:
other: Salmonella typhimuriun TA98. TA10,. TA1535, TA1537, TA1538
Metabolic activation:
with and without
Metabolic activation system:
S9-mix of male Wistar rat liver after induction with Aroclor 1254
Test concentrations with justification for top dose:
0 - 1.58 - 5 - 15.8 - 50 - 158 -500 -1580 - 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Methyl methansulfonatem 9-Aminoacridine, 2-Nitrofluorene, N-ethyl-Nnitro-N.nitrosoguanidine, 2-Aminoanthracene, Benzo(a)pyrene
Details on test system and experimental conditions:
IUCLID4 Type: Ames test, according to the respective guieline: preincubation procedere
Evaluation criteria:
A material is identified as a mutagen in this test system if ther was a reproducible demonstration of a dose-effect relation with a 2-fold increase in the number of revertants compared with the concurrent negative control in at least 1 strain. With the strain TA100 a 1.5-fold increase was the criterion for a positive result.
Statistics:
no data
Species / strain:
other: Salmonella typhimuriun TA98. TA100. TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+/- S9-mix: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Remarks:
Migrated from field 'Test system'.
Conclusions:
negative
Executive summary:

N,N-Dimethylbenzylamine diluted in DMSO was tested for point mutations in the Ames-test according to OECD TG 471 and GLP using Salmonella typhimuriun TA98, TA100, TA1535, TA1537, TA1538 in the presence and in the absence of a metabolic activation system and concentrations ranging between 1.58 and 5000 µg/plate. Cytotoxicity was observed at the highest test concentration. Under the conditions of this test N,N-dimethylbenzylamine did not induce point mutations (BG Chemie 1986).

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
HPRT
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
other: Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
liver S9-mix fron 6-12 week old male Wistar rats induced by phenobarbital/ß-naphthoflavone
Test concentrations with justification for top dose:
85.0, 170.0, 340.0, 680.0, 1360.0 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: EMS (ethylmethane sulfonate), DMBA (7,12-dimethylbenz(a)anthracene
Details on test system and experimental conditions:
according to the respective OECD test guideline: 2 seperate experiments: incubation time with and without S9-mix: 4 hours
Evaluation criteria:
A test item producing neither a biologically relevant concentration-related increase of the mutant frequecy nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
Statistics:
linear regression
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Chinese Hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

N,N-dimethylbenzylamine was tested for genotoxicity in Chinese hamster V79 cells according to OECD TG 376 in the presense and in the absence of a metabolic activation system (S9 -mix from rat liver). Based on the negative result N,N-dimethylbenzylamine was considered to be non-mutagenic in this test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: main text in Japanese
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
- S9 mix (continuous exposure): 0, 75.0, 150, 300 µg/ml;
-S9 mix (short-term exposure): 0, 375, 750, 1500 µg/ml;
+S9 mix (short-term exposure): 0, 188, 375, 750 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9 mix. Mitomycin C; +S9 mix, Cyclophosphamide
Details on test system and experimental conditions:
Chromosome Aberration test according to Guideline in the presence and in the absence of a metabolic activation system: up to cytotoxicity:
count of 200 metaphase per concentration,
Evaluation criteria:
the test substance is considered to be positive when assay cultures with the test substance show a significantly higher incidence of cells with chromosomal aberrations as compared with the negative control and when the effect is reasonably reproducible or dose-dependent.
Statistics:
yes, main text in Japanese
Species / strain:
other: Chinese Hamster Lung (CHO) cells
Metabolic activation:
with and without
Genotoxicity:
other: positive with metabolic activation; negative without metabolic activation
Cytotoxicity / choice of top concentrations:
other: Lowest concentration producing cytogenetic effcts in vitro: With methabolic activation: 0.213 mg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese Hamster lung cells
Remarks:
Migrated from field 'Test system'.

Genetic effects:

         clastogenicity        polyploidy
 +  -
  Without metabolic activation  X
  With methabolic activation  X  /  /  /

 X

Executive summary:

N,N-Dimethylbenzylamine was tested in a chromosome aberration test with and without metabolic activation in Chinese Hamster (CHO) cells according to Japanese Guidelines for Screening Mutagenicity Testing of chemicals. This chemical induced structural chromosomal aberrations in the presence of a metabolic activation system. The lowest concentration producing cytogenetic effects in vitro was with metabolic activation: 0.213 mg/ml (MHLW 1997).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The In-vivo Micronucleus Test in mouse bone marrow cells yielded a negative result.

Link to relevant study records
Reference
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.
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
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

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
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
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
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.

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

N,N-Dimethylbenzylamine diluted in DMSO was tested for point mutations in the Ames-test according to OECD TG 471 and GLP using Salmonella typhimuriun TA98, TA100, TA1535, TA1537, TA1538 in the presence and in the absence of a metabolic activation system and concentrations ranging between 1.58 and 5000 µg/plate. Cytotoxicity was observed at the highest test concentration. Under the conditions of this test N,N-dimethylbenzylamine did not induce point mutations (BG Chemie 1986).

Furthermore, N,N-dimethylbenzylamine diluted in DMSO was tested for genotoxicity in Chinese hamster V79 cells according to OECD TG 376 (HPRT-test) in the presense and in the absence of a metabolic activation system (S9 -mix from rat liver). The concentrations used ranged between 85.0 and 1360.0 µg/ml. Based on the negative result N,N-dimethylbenzylamine was considered to be non-mutagenic in this test system (Harlan CCR, 2011, at the request of Lanxess Deuschland GmbH).

N,N-Dimethylbenzylamine was tested in a chromosome aberration test with and without metabolic activation in Chinese Hamster ovary (CHO) cells according to Japanese Guidelines for Screening Mutagenicity Testing of chemicals in concentrations up to 3000 µg/ml.. This chemical induced structural chromosomal aberrations in the presence of a metabolic activation system. The lowest concentration producing cytogenetic effects in vitro was with metabolic activation: 0.213 mg/ml.(MHLW 1997).

However, N,N-dimethylbenzylamine was also assessed for its mutagenic potenial in vivo using the Micronucleus Test in vivo with bone marrow cells of the mouse. The doses of the test substance administered orally by gavage were 15, 50 , 150 mg/kg bw . 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.

The preparation of cells in the main test was done 24, 48 and 72 hours after the treatment with the high dose and 24 hours after the treatment with the medium and low dose. In each experimental group 1000 cells from each of 10 animals were scored.

There was no enhancement of the number of cells with micronuclei in the groups treated with the test substance as coompared with the negative controls treated with the solvent. On the other hand the sensitivity of the test system was demonstrated by significantly enhanced micronuclei rates after treatment with 30 mg/kg bw cyclophposphamide (positive control).

The results allow to draw the conclusion that the test substance did not induce micronuclei in mouse bone marrow cells under the experimental conditions described (BG Chemie 1987).

Overall, based on the available results, N,N-dimethylbenzylamine is considered to be non-mutagenic


Short description of key information:
N,N-dimethylbenzylamine does not induce mutagenic activity in the Ames test and in the HPRT test in vitro with Chinese Hamster V79 cells but induces chromosome aberrations in Chinese Hamster Ovary cells in the presence of a metabolic activation system but not in the absence of a metabolic activation system. However, the In-vivo Micronucleus Test in mouse bone marrow cells yielded a negative result. Therefore N.N-dimethylbenzylamine is considered to be a Non-mutagen.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

N,N-dimethylbenzylamine does not induce mutagenic activity in the Ames test and in the HPRT test in vitro with Chinese Hamster V79 cells but induces chromosome aberrations in Chinese Hamster Ovary cells in the presence of a metabolic activation system but not in the absence of a metabolic activation system. However, the In-vivo Micronucleus Test in mouse bone marrow cells yielded a negative result. Therefore. N.N-dimethylbenzylamine is considered to be a Non-mutagen.

Up to now N,N-dimethylbenzylamine is not classified in one of the categories of mutagenic substances.

Based on the available results no classification or labelling is required.