Dimethyl(octyl)amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An OECD 490 in vitro mammalian cell gene mutation tests using the thymidine kinase gene study was performed, AD1 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

An OECD 487 in vitro mammalian micronucleus assay was carried out. AD 1 did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.

Therefore, AD 1 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

An OECD 471 bacterial reverse mutation assay was performed. AD-1 was examined for mutagenic activity in two histidinedependent auxotrophs of Salmonella typhimurium, strains TA 98 and TA 100, using pour-plate assays. The test material, did not exhibit any mutagenic activity under the conditions of test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The study was performed according to the AMES test methodologies but predated the current OECD 471 guideline

Principles of method if other than guideline:

This report describes experiments performed to assess the mutagenic activity of Farmin DM08P in two histidine-dependent strains of Salmonella typhimurium using the procedures developed by Ames et al (1975) and later refined by Maron and Ames (1983).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

300 g sample of Fannin DM08P, of lot No. 1318, a clear courless liquid, was received from Kao Corporation on 21 November 1995. It was stored at ambient temperature until required.

Target gene:

rfa wall mutation, uvrB and pKM101 in Salmonella typhimurium. Tester strains TA98, TA100,

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

Young male CD rats, ca. 200 g BW, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent. Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity.

Test concentrations with justification for top dose:

The maximum concentration of test material employed was selected with the aid of a preliminary toxicity test with strain TA 98.
Visible thinning of the background lawn ofnon-revertant cells was obtained following exposure to the test item at 5000 μg per plate only. This wastherefore selected as the top exposure level for use in the main tests.
Maximum dose to be plated in the presence of metabolic activation =5,000 ug per plate for both TA98 and TA100.

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Remarks:

negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains

Negative solvent / vehicle controls:

yes

Remarks:

absence of effects of spontaneous reversionon rates when ethanol included

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

benzo(a)pyrene

Details on test system and experimental conditions:

A solution of Farmin DM08P was prepared in ethanol at 50 mg/ml, and four half-log dilutions were prepared from this solution. Aliquots (0.1 ml) of each concentration of Farmin DM08P were placed in sterile tubes. Molten, histidine-deficient top-agar (2 ml), maintained at 45°C, and bacterial culture (0.1 ml) were then added. The tubes were mixed by inversion and 0.5 ml rat liver microsomal preparation (S-9 mix) or O.IM phosphate buffer was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 ml).
Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of ethanol (0.1 ml) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates. Aliquots (0.1 ml) of a 10^·6 dilution of the overnight cultures were spread on
the surface of plates of complete medium to measure the viability and celldensity of each culture. All plates were prepared in duplicate, allowed to solidify and incubated at 37°C for 2 days. After incubation, numbers ofrevertant colonies were counted, either manually or with an automated colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified.
All plates and tubes were identified by the use of numbers indelibly marked on the plates and test tube racks.

Evaluation criteria:

The plates were incubated at 3 7°C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material is shown by absence or thinning of the background lawn. The level oftest material chosen as the top level for pour-plate tests is normally the lowest level causing visible thinning of the lawn. (In the absence of such thinning, a top level of 5 mg/plate would be selected).

Statistics:

For all replicate platings, the mean number of revertants per plate was calculated

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Conclusions:

The test material, Farmin DM08P, did not exhibit any mutagenic activity under the conditions of test.

Executive summary:

The material Farmin DM08P was examined for mutagenic activity in two histidinedependent auxotrophs of Salmonella typhimurium, strains TA 98 and TA 100, using pour-plate assays.

The studies, which were conducted in the presence and absence of an activating system derived from rat liver (S-9 mix), employed a range of levels of Farmin DM08P from 50 to 5000 μg per plate, selected following a preliminary toxicity test in strain TA 98. The tests included solvent (ethanol) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with either of the bacterial strains at the Farmin DM08P levels tested, either in the presence or absence of S-9 mix.

Inhibition of bacterial growth, observed as slight thinning of the background lawn of non-revertant cells and reduction in revertant colony numbers, occurred in both strains following exposure to Farmin DM08P at 5000 μg per plate.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene and sodium azide when examined under similar conditions.

It was concluded that Farmin DM08P did not exhibit any mutagenic activity under the conditions of test.

Reference 2

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

Study period:

2015-11-30 - 2015-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

other: OECD 490 (In vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IWGT Recommendations

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment without and with metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10 mM

Main Experiment
without metabolic activation: 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00 and 1.25 mM
with metabolic activation: 0.2, 0.4, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mM

Vehicle / solvent:

RPMI cell culture medium was used as solvent (RPMI + 5% HS).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

other: benz(a)pyrene 3.5 µg/mL

Positive control substance:

other: ethylmethanesulfonate 300 µg/mL

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: one experiment, cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

Evaluation criteria:

The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative controls.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item AD-1 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item AD1 was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the main experiment without metabolic activation 1.25 mM and with metabolic activation 1.8 mM were selected as the highest concentrations. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation:

0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00 and 1.25 mM

and with metabolic activation:

0.2, 0.4, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mM

No precipitation of the test item was noted in the experiment.

Growth inhibition was observed in the experiment without and with metabolic activation.

In the main experiment without metabolic activation the relative total growth (RTG) was 17.3% for the highest concentration (1.25 mM) evaluated. The highest concentration evaluated with metabolic activation was 1.8 mM with a RTG of 12.3%.

In the main experiment no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed.

Additionally, in the main experiment colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisifes the requirements for Test Guidelines OPPTS 870.5300, OECD 490 forin vitromutagenicity (mammalian forward mutation) data.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: clastogenicity/aneugenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-27 to 2016-04-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Pre-Experiment:
with and without metabolic activation: 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM
Main Experiments:
Experiment I:
without metabolic activation: 0.5, 1.0 and 1.5 mM
with metabolic activation: 1.0, 1.5, 2.0 and 2.5 mM
Experiment II (long term):
without metabolic activation:0.1, 0.3, 0.5 and 0.7 mM

Vehicle / solvent:

- Justification for choice of solvent/vehicle:
A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 10 mM. The test item was dissolved in cell culture medium within 1 hour prior to treatment. After an ultrasonication treatment for 5 min at 37 °C a clear solution was obtained. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

clastogenic control without metabolic activation [900 and 1200 µg/mL]

Positive controls:

yes

Positive control substance:

other: colchicine

Remarks:

aneugenic control without metabolic activation [0.08 and 2.0 µg/mL]

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

clastogenic control with metabolic activation [5.0 µg/mL ]

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation)
24 hours (Experiment II without metabolic activation)

FIXATION INTERVAL:
24 hours (Main Experiment)

STAIN (for cytogenetic assays): acridin orange

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 2000 cells per concentration (1000 cells per culture)

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI)

Evaluation criteria:

There are several criteria for determining a positive result:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the study described and under the experimental conditions reported, AD 1 did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, AD 1 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In order to investigate AD 1 for a possible potential to induce micronuclei in Chinese hamster V79 cells an in vitromicronucleus assay was carried out.

The selection of the concentrations was based on data from the pre-experiment. In the main experiment I without and with metabolic activation a concentration of the test item of 2.5 mM and in main experiment IIwithout metabolic activation a concentration of the test item of 1.5 mM was selected as the highest concentration.

The following concentrations were evaluated for micronuclei frequencies:

Experiment Iwith short-term exposure (4 h):

withoutmetabolic activation: 0.5, 1.0 and 1.5mM

withmetabolic activation: 1.0, 1.5,2.0 and 2.5 mM

Experiment II with long-term exposure (24 h):

without metabolic activation: 0.1, 0.3, 0.5 and 0.7mM

No precipitate of the test item was noted in all concentration groups evaluated in experiment I and II.     

According to the OECD Guideline 487 the maximum of cytotoxicity should not exceed the limit of 55% ± 5%. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. According to laboratory experience the limit for discrimination between not cytotoxic and a cytotoxic effect is a CBPI value of 70% compared to the negative/solvent control which corresponds to 30% of rel. cytostasis.

In experiment Iwithout metabolic activation no increase of the relative cytostasis above
30 % was noted up to a concentration of 0.5 mM. At a concentration of 1.0 mM a relative cytostasis of 33% and at a concentration of 1.5 mM a relative cytostasis of 54% was noted.

In experiment I with metabolic activation no increase of the relative cytostasis above
30 % was noted up to a concentration of 1.0 mM. At a concentration of 1.5 mM a relative cytostasis of 32%, at a concentration of 2.0 mM a relative cytostasis of 49% and at a concentration of 2.5 mM a relative cytostasis of 60%was noted.

In experiment II without metabolic activation no increase of the relative cytostasis above
30 % was noted up to a concentration of 0.3 mM. At a concentration of 0.5 mM a relative cytostasis of 42% and at a concentration of 0.7 mM a relative cytostasis of 58% was noted.

In the main experiment Iwith and without metabolic activation and in the main experiment IIwithout metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametric chi² Test was performed to verify the results in both experiments. No statistically significant increase (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated except for the test item concentration of 2.5 mM in experiment I with metabolic activation. This value was statistically significantly increased compared to the concurrent negative control. However, the corresponding micronucleus frequency was within the historical control limits of the negative control and no dose-response relationship was observed.

 

The chi² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.

Ethylmethanesulfonate (EMS, 900 and 1200 µg/mL) and cyclophosphamide (CPA, 5.0 µg/mL) were used as clastogenic controls. Colchicine (0.08 and 2.0 µg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 487 for In Vitro Mammalian Cell Micronucleus Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)