Cyclohexyldimethylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key study addressing reverse mutations in a standard plate test and pre-incubation test using E. coli and S. typhimurium bacterial strains according to the Ames test design showed no mutagenic activity in the presence or absence of metabolic activation. Two additional Ames tests, supporting studies, investigated reverse mutation potential in three test strains-Salmonella, Saccharomyces and Escherichia. The results of the tests were negative in the presence or absence of metabolic activation. No indications of reverse mutation were evident in the available data.

The potential for DCMHA to induce chromosomal aberrations was tested in CHO cells in vitro, in the presence and absence of metabolic activation, at suitable non-cytotoxic levels for metaphase analyses. DMCHA caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations either with or without S-9.N,N-dimethylcylcohexamine showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

DMCHA did not cause any statistically significant increases in the mutant frequency at the thymidine kinase locus of L5178Y mouse lymphoma cells in vitro, in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not stated

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Salmonella typhimurium

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

Escherichia coli

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.

Additional strain / cell type characteristics:

other: The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Escherichia coli WP2 uvrA is a derivative of E . coli WP2 with a deficient excision repair and is used to detect substances which induce base pair
substitutions. The rate of induced back mutations from tryptophan auxotrophy to tryptophan independence is determined.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Arochlor induced rat liver S-9 mix

Test concentrations with justification for top dose:

For the Standard plate test (SPT) the doses were 20, 100, 500, 2500 and 5000 µg/plate, and for the preincubation test (PIT) the doses were 4, 20, 100, 500 and 2500 µg/plate.

Vehicle / solvent:

DMSO for both strains

Untreated negative controls:

yes

Remarks:

with and without S-9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO free

Positive controls:

yes

Positive control substance:

other: with S-9 mix: 2.5 µg 2-aminoanthracene

Remarks:

for the strains TA 100, TA 98, TA 1537 and TA 1535

Untreated negative controls:

yes

Remarks:

with and without S-9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO free

Positive controls:

yes

Positive control substance:

other: with S-9 mix: 60 µg 2-aminoanthracene

Remarks:

for the strain E . coli WP2 uvrA

Untreated negative controls:

yes

Remarks:

with and without S-9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO free

Positive controls:

yes

Positive control substance:

other: without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

for the strains TA 100 and TA 1535

Untreated negative controls:

yes

Remarks:

with and without S-9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO free

Positive controls:

yes

Positive control substance:

other: without S-9 mix: 10 µg 4-nitro-o-phenylendiamine (NOPD)

Remarks:

for the strain TA 98

Untreated negative controls:

yes

Remarks:

with and without S-9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO free

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for the strain TA 1537 Migrated to IUCLID6: without S-9 mix: 100 µg

Untreated negative controls:

yes

Remarks:

with and without S-9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO free

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

for the strain E . coli WP2 uvrA Migrated to IUCLID6: without S-9 mix: 10 µg

Details on test system and experimental conditions:

Three test plates were used per dose or per control. 

Evaluation criteria:

In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements :
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results .

Statistics:

Not relevant

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

An increase in the number of his+ or trp+ revertants was not observed in either the Standard plate test or in the Preincubation test.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at doses >= 2500 µg/plate in the SPT and at doses >= 500 µg/plate in the PIT. 

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

An increase in the number of his+ or trp+ revertants was not observed in either the Standard plate test or in the Preincubation test.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at doses >= 2500 µg/plate in the SPT and at doses >= 500 µg/plate in the PIT. 

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No further details

Remarks on result:

other: all strains/cell types tested

No additional information

Conclusions:

According to the results of the present study, the test substance DMCHA is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions outlined. Based on the results of the in vitro study provided, the test substance, does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

Executive summary:

The substance cyclohexyldimethylamine (DMCHA0 was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test and in the Escherichia coli.- reverse mutation assay.

Strains : TA 1535, TA 100, TA 1537, TA 98 and E . coli WP2 uvrA

Dose range : 20 µg - 5,000 µg/plate (SPT), 4 µg - 2,500 µg/plate (PIT)

Test conditions : Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor induced rat liver S-9 mix).

Solubility : No precipitation of the test substance was found.

Toxicity : A bacteriotoxic effect was observed depending on the strain and test conditions at doses ≥ 2,500 µg/plate (SPT) or at

doses ≥ 500 µg/plate (PIT).

Mutagenicity : No increase ín the number of his+ or trp+ revertants was observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Conclusion :

According to the results of the present study, the test substance DMCHA is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions chosen here.

Based on the results of the in vitro study provided, the test substance, does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September 2015 to 13 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell forward mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Provided by the sponsor (Air Products, USA), batch no. 1920179
- Expiration date of the lot/batch: 02 February 2018
- Purity test date: 99.3% (02 February 2015)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability under test conditions: assumed to be stable during testing; test items dilutions were prepared within 2 hours of application
- Solubility and stability of the test substance in the solvent/vehicle: in-house solubility checks performed prior to testing
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity reported

OTHER SPECIFICS
- Molecular weight: 127.23

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y TK +/- 3.7.2c cells obtained from Dr. J. Cole, MRC Mutation Unit, University of Sussex, Brighton, UK
- Suitability of cells: recommended by guideline
- Cell cycle length, doubling time or proliferation index: 12 hours
- Methods for maintenance in cell culture if applicable: Stock cells stored in liquid nitrogen at approx. -196°C

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B ( 2.5 µg/mL) and 10% donor horse serum (giving R10 media). 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes; before the stock cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. THMG medium contains Thymidine (9 µg/L), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours cells were cultured in THG medium before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

PB/BNF S9 prepared in-house on 01 March 2015 from the livers of male Sprague-Dawley rats. S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCls (8 mM) in R0.

Test concentrations with justification for top dose:

Preliminary toxicity test: 10 concentrations from 4.96 to 1270 µg/mL (top dose is 10 mM limit dose recommended in OECD 490)
Main test (4-hour exposure): 79.38, 158.75, 317.5, 635, 952.5, 1270 µg/mL (top dose is 10 mM limit dose recommended in OECD 490)
Main test (24-hour exposure): 5, 10, 20, 40, 80, 160, 200, 240, 280, 320 µg/mL (top dose based on evidence of toxicity in preliminary test)

Vehicle / solvent:

R0 medium was chosen as the solvent based on in-house solubility checks

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium in tissue culture flasks
- Cell density at seeding: Preliminary toxicity test, 5 x 10E5 cells/mL (4-hour exposure) and 1.5 x 10E5 cells/mL (24-hour exposure). Main test, 1 x 10E6 in 10 mL medium for the 4-hour exposure and 0.3 x 10E6 cells/mL in 10 mL medium for the 24-hour exposures.

DURATION
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): 4 µg/mL 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Relative Suspension Growth (%RSG), Viability (%V) and Relative Total Growth (RTG)

Rationale for test conditions:

As prescribed by guideline OECD 490

Evaluation criteria:

A test item was considered to demonstrate a mutagenic response if it produced a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The IMF must also exceed the Global Evaluation Factor (GEF) of 1.26 x 10E-6. Therefore any test item dose level that has a mutation frequency greater than the corresponding vehicle control by the GEF of 126 x 10E-6 and demonstrates a positive linear trend will be considered positive. If a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF and is not reproducible or part of a dose-related response then it may be considered to have no toxicological significance. When a test item induces modest reproducible increases in mutation frequencies that do not exceed the GEF then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Standard acceptability criteria (recommendations of the IWGT (International Workshops on Genotoxicity Testing)) were considered).

Statistics:

Data were analysed using computer program Mutant 240C by York Electronic Research, which follow the statistical guidelines recommended by the UKEMS.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Modest toxicity observed in the 4-hour exposure groups (tested up to limit concentrations) and marked toxicity in the 24-hour group

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
There was evidence of modest reductions in %RSG in treated cells compared to vehicle controls in the 4-hour exposure groups, and marked reductions in the 24-hour exposure group. Precipitation of the test material was not observed. Based on the %RSG values observed, the maximum dose selected for the main test was the 10 mM limit dose for the 4-hour exposure groups, and limited by test-item induced toxicity in the 24-hour exposure group.

MUTAGENICITY (MAIN) TEST
There was evidence of modest toxicity in the 4-hour exposure groups, and marked toxicity in the 24-hour exposure group. There was also evidence of modest reductions in viability and the 24-hour exposure group, indicated by the %RSG and RTG values. There was also evidence of modest reductions in viability in the 24-hour exposure group indicating that residual toxicity had occurred. Based on the RTG and %RSG values observed, it was considered that optimum levels of toxicity were achieved in the 24-hour exposure group. The excessive toxicity at 320 µg/mL in the 24-hour exposure group resulted in this dose level not being plated for viability and 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substances.
The test item did not induce any statistically significant or dose related (linear trend) increases in the mutant frequency x 10E-6 per viable cell at any of the dose levels, including the 10 mM limit dose in the 4-hour exposure groups, and a dose level that achieved optimum levels of toxicity in the 24-hour exposure group. Precipitate of the test item was not observed.

Summary of mutagenicity results

Concentration (µg/mL)	4-hour exposure without S9			4-hour exposure with S9
	%RSG	RTG	MF^2	%RSG	RTG	MF^2
0	100	1.00	115.31	100	1.00	127.43
79.38	100	1.20	126.49	95	0.92	127.60
158.75	105	1.14	117.24	99	1.09	110.62
317.5	93	0.95	135.24	89	0.85	170.44
635	65	0.72	125.24	81	0.79	156.63
952.5	51	0.54	138.40	66	0.75	112.66
1270	35	0.38	120.76	41	0.55	104.53
Linear trend	Non-significant			Non-significant
Positive control^1	72	0.42	1014.27	62	0.37	1245.96

 

Concentration (µg/mL)	24-hour exposure without S9
	%RSG	RTG	MF^2
0	100	1.00	127.33
5 Ø	67	-	-
10 Ø	72	-	-
2 0Ø	82	-	-
40	94	0.92	132.29
80	107	0.98	91.37
160	58	0.77	106.07
200	52	0.61	115.47
240	29	0.38	167.56
280	15	0.26	123.89
320 Ø	8	-	-
Linear trend	Non-significant
Positive control^1	29	0.17	1736.47

¹Ethylmethanesulphonate without S9; Cylophosphamide with S9

²Positive wells per tray, 96 wells plated

Ø Not plated for viability or 5-TFT resistance

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at any dose level, therefore the test item is not considered to be mutagenic under the conditions of this test.

Executive summary:

The mutagenic potential of cyclohexyldimethylamine was evaluated in vitro on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, according to OECD 490. The test item dose range for the mutagenicity experiment was selected based on a preliminary cytotoxicity study. In the main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells were treated with the test item at up to ten dose levels in duplicate, together with vehicle (R0 medium) and positive controls. The exposure periods were 4-hours both in the absence and presence of metabolic activation (S9-mix) and 24-hours in the absence of metabolic activation. Following exposure and an expression period of 2 days the number of small and large colonies was scored.

The maximum dose level used in the 4-hour exposure period was the limit dose (10 mM), but was limited by toxicity in the 24-hour exposure group. Precipitation of the test item was not observed. The vehicle controls demonstrated acceptable mutant frequencies for this cell line. The positive controls induced marked increases in the mutant frequency indicated the satisfactory performance of the test. The test item did not induce any statistically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Therefore, cyclohexyldimethylamine is not considered to be mutagenic to mammalian cells in vitro, under the conditions of this study.

Reference 3

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

Study period:

January 12th to February 19th, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to guideline study; GLP. However, only 100 cells were scored per concentration (guideline: 200 metaphases per concentration). Doses were tested as single exposures (no duplicates)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

1. Cytotoxicity was not determined, only assessed from confluency. 2. Only 100 cells are scored per concentration (guideline: 200 metaphases per concentration). 3. Doses are tested as single exposures (no duplicates)

Principles of method if other than guideline:

Not indicated

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not documented

Species / strain / cell type:

other: Chinese hamster ovary (CHO) cell line WBI

Details on mammalian cell type (if applicable):

CHO cells for this assay were grown in McCoy's 5a medium supplemented with 10% foetal calf serum, (FCS), L-glutamine, and antibiotics. Cultures were set up approximately 24 hours prior to treatment by seeding 1.5 x 10E6 cells per glass culture, flask in 10 ml of fresh medium .

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 mix (Aroclor 1254 induced)

Test concentrations with justification for top dose:

Without metabolic activation: Dose ranging from 125 nl/ml to 3 ul/ml, which is approx. equivalent to 106 - 2547 ug/ml.
With metabolic activation: Dose ranging from 400 nl/ml to 4 ul/ml, which is approx. equivalent to 340 - 3396 ug/ml.

Doses used for evaluation:
First test:
Without S9: 250, 500, 750, 1000 and 2000 nl/ml (= 212, 424, 636, 849 and 1698 ug/ml) (8 h exposure + 2.5 hours in fresh medium in the presence of colcemid)
With S9:  600, 800, 1000, 2000 and 3000 nl/ml (509, 679, 849, 1698 and 2547 ug/ml) (2 h exposure + 10 h in fresh medium without test substance, during the last 2.5 h in presence of colcemid)
Second test:
Without S9: 1500, 1750 and 2000 (1274, 1486 and 1698 ug/ml (6 h exposure + 2.5 h in presence of colcemid).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Remarks:

nothing added to the cells (only culture medium)

Negative solvent / vehicle controls:

yes

Remarks:

Cells exposed to the solvent (ethanol).

True negative controls:

not specified

Positive controls:

yes

Remarks:

Without S9: mitomycin C With S9: cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Without metabolic activation: approximately 8 hours With metabolic activation: 2 hours
- Expression time (cells in growth medium): 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells):2.5 hours after the addition of colcemid


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATES: one culture per treatment group

NUMBER OF CELLS EVALUATED: 100 cells were scored for dosing

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

The following factors were taken into account in evaluation:
- The estimated number of breaks involved in production of the different types of aberrations observed
- The frequency of cells with more than one aberration.
- Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response

Statistics:

Statistical method: Newman-Kuels test

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Without S9: 2000 nl/ml (1698 µg/ml); With S9: 3000 nl/ml (2547 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No additional information

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Chinese hamster ovary (CHO) cell line WBI

PRECIPITATION CONCENTRATION: 
not indicated

% of cells with aberrations: 

without S9-mix: 1-4%
with S9-mix: 1-5%

Conclusions:

It was concluded that DMCHA showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

A study (Galloway, 1982) was performed to assess the ability of cyclohexyldimethylamine (DMCHA) to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells cultured in vitro.

Cultured CHO cells were exposed to the test substance both in the presence and absence of metabolic activation (S-9 mix derived from rat livers). Negative, solvent and positive control cultures were also prepared. After the appropriate treatment time cell division was arrested using colcemid, the cells harvested and slides prepared, so that metaphase figures could be examined.

In order to assess the cytoxicity of DMCHA, cultures were examined for degree of confluence and presence of large rounded (mitotic) cells. On the basis of these data the dose levels selected for the metaphase analysis were 250 nl/ml to 2 µl/ml in the absence of S-9 mix and 600 nl/ml to 3 µl/ml in the presence of S9-mix.

In both the presence and absence of S-9 mix DMCHA caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the untreated controls. The aberration frequencies in the negative and solvent controls were within the normal background range for this laboratory.

Both positive control compounds, mytomycin and cyclophosphamide, caused large statistically significant increases in chromosomal aberrations. This demonstrates the sensitivity of the test system and the efficacy of the S-9 mix.

It was concluded that DMCHA showed no evidence of clastogenic activity in this in vitro cytogenetic test system. Based on these results, the test substance does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

 

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

January 8, 1982 - January 25, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 1. No repeat assay 2. One plate per dose was used

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

not indicated

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

trp gene

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

The Escherichia coli strain WP2uvrA- carries a defect in one of the genes for tryptophan biosynthesis and lacks excision repair.

The indicator strain is kept at 4°C on standard methods agar plates or minimal medium plates supplemented with an excess of tryptophan. New stock culture plates are made as often as needed from the frozen master culture or from single colony reisolates that were rechecked for appropriate genetic markers and characteristics. For each experiment an inoculum from the stock culture plate was grown overnight at 37 °C in nutrient broth (Oxoid CM67) and used.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

0, 0.02, 0.04, 0.07, 0.15, 0.29, 0.59, 1.17, 2.34, 4.69, 9.38, 18.75, 37.50, 75.0 and 150.0 µl(µg)/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Untreated negative controls:

yes

Remarks:

solvent used for preparing the stock solution and subsequent dilutions of the test material

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG)

Remarks:

Nonactivation in DMSO

Untreated negative controls:

not specified

Remarks:

solvent used for preparing the stock solution and subsequent dilutions of the test material

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other:  Activation: 2-anthracine

Remarks:

in DMSO

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Test method: Direct plate assay

ADMINISTRATION: 
- Number of replicates: one plate/dose

DESCRIPTION OF FOLLOW UP REPEAT STUDY: no repeat assay

Evaluation criteria:

Positive if the dose response is observed over a minimum of three test concentrations and the increase in revertants achieves a doubling of the spontaneous background. If a positive result in one test cannot be reproduced in additional runs, the positive result loses significance.

Statistics:

Statistical methods are not currently used, and evaluation is based on the criteria included in the protocol.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: - With metabolic activation: No toxicity up to the dose level 18.75 µl/plate - Without metabolic activation: 18.75 µl/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The test compound was examined for mutagenic activity in a series of in-vitro microbial assays employing E. coli w P2uvrA indicator organism. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats.

A negative control consisting of the solvent used for preparing the stock solution and subsequent dilutions of the test material and specific positive compounds were also assayed concurrently with the test material. The negative control data was used as the base for evaluating the results obtained w ith the test material.

-DOSE RANGE
A preliminary toxicity study conducted on the test material at 14 doses of 0.02 µl to 150.0 µl per plate using the E. coli w P2uvrA strain exhibited 100% toxicity at 18.75 µl dose ( Table 1 ) . As such , the mutagenicity assays were conducted at 7 doses of 0.29 µl to 18.75 µl per plate.

The results of the tests conducted on the test material in the absence of a metabolic activation system were negative. The results of the tests conducted on the test material in the presence of a rat liver activation system were negative.

Remarks on result:

other: all strains/cell types tested

Table 1: Toxicity results

Test Compound µl/Plate	Number of colonies/plate	% survival relative to control
0 (control)*	687.0**	100.00
0.02	713.0	103.78
0.04	691.0	100.58
0.07	701.0	102.04
0.15	720.0	104.80
0.29	664.0	6.65
0.59	683.0	99.42
1.17	646.0	94.03
2.34	661.0	96.22
4.69	442.0	64.64
3.38	96.0	13.97
18.75	0.0	0.00
37.50	0.0	0.00
75.0	0.0	0.00
150.0	0.0	0.00

* Solvent control (100 µl/Plate)

** Average of two plates

Conclusions:

The test material, DMCHA Lot No. 20-133 CJ did not exhibit genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions according to the evaluation criteria . Based on the results of the in vitro study provided, the test substance, does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

Executive summary:

The objective of this study was to evaluate a test article for mutagenic activity in a bacterial assay with and without a mammalian S9 activation system.

The Escherichia coli strain WP2uvrA- used in this assay carries a defect in one of the genes for tryptophan biosynthesis and lacks excision repair.

The following concentrations were used in the experiment: 0, 0.02, 0.04, 0.07, 0.15, 0.29, 0.59, 1.17, 2.34, 4.69, 9.38, 18.75, 37.50, 75.0 and 150.0  µl(µg)/plate.

The results of the tests conducted on the test material in the absence of a metabolic activation system were negative. The results of the tests conducted on the test material in the presence of a rat liver activation system were negative.

Therefore, the test material, cyclohexyldiemthylamine (DMCHA) Lot No. 20-133 CJ did not exhibit genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions according to the evaluation criteria .

Based on the results of the in vitro study provided, the test substance, does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 12th to March 1st, 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Similar to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)

Principles of method if other than guideline:

Not indicated

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

Not documented

Species / strain / cell type:

hepatocytes: hepatocyte primary cultures of adult male Fischer 344 rats

Details on mammalian cell type (if applicable):

- Type and identity of media:
The cell cultures are established in Williams' Medium E supplemented with 5% foetal bovine serum, 2mM L-glutamine, 2.4 µM dexamethasone
100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 150 µg/ml gentamicin. After the establishment period, the dexamethasone and serum components are removed.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not applicable

Metabolic activation system:

Not relevant

Test concentrations with justification for top dose:

5, 10, 25, 50, 100, 250, 500 and 1000 nl/ml, which is approx. equivalent to 4.2, 8.4, 21, 42, 84, 210, 420 and 849 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: N,N-dimethylcyclohexylamine dissolves easily at a concentration of 100 µl/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

No additional information Migrated to IUCLID6: 0.05µg/ml

Details on test system and experimental conditions:

- No. of cells analyzed: 150 cells/dose level

ADMINISTRATION: 
- Number of replicates:  5 cultures per treatment, of which 2 are used for cytotoxicity measurements. Each treatment contains the positive and negative controls.
- Application:  Freshly prepared rat hepatocytes were incubated for 18-19 hours to the test substance. The UDS assay was terminated by washing the cell monolayers twice with WME containing 1mM thymidine and were further processed. Two cultures were used to monitor the toxicity of each treatment and were re-fed with WME and returned to the incubator. At 20-24 hours after the initiation of the treatments, viable cell counts (trypan blue exclusion) were determined to estimate cell survival relative to the negative control. Cover slips were immersed in 1% sodium citrate for 10 minutes to allow the nuclei to swell. The cells were fixed in acetic acid:ethanol (1:3) and were dried for at least 24 hours. After fixation, cover slips were dipped in photo emulsion and incubated for 7-10 days at 4°C.  The emulsion was developed, fixed and stained.  The number of nuclear grains was counted in 50 cells/cover slip.

Evaluation criteria:

A test substance is considered active in the UDS assay if: 
- mean net nuclear grain count exceeded at least 6 grains/nucleus in excess of the concurrent negative control value and /or
- the percent of nuclei with 6 or more grains to increase above 10% of the examined population, in excess of the concurrent negative control, and/or 
- the present of nuclei with 20 or more grains to reach or exceed 2% of the examined population
A dose-related increase in UDS for at least two consecutive applied concentrations is also desirable to evaluate a test material as active in this assay. I n some cases, UDS can increase with dose and then decrease to near-zero with successively higher doses. If this behavior is associated with increased toxicity, the test material can be evaluated as active. If an isolated increase occurs for a treatment far removed from the toxic doses, the UDS will be considered spurious.
The test material is considered inactive in this assay if none of the above conditions are met and if the assay includes the maximum applied dose or other doses that are shown to be toxic by the survival measurements. If little or no toxicity is demonstrated for any of the applied doses and the test material remains soluble in the culture medium, the assay may be considered inconclusive and may be repeated with higher doses.

Statistics:

Not applicable

Key result

Species / strain:

hepatocytes:

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

849 µg/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS:  negative.; 0.59 - 0.93 grains/nucleus at non-toxic concentrations (solvent control: 0.87 grains/nucleus); positive controls were within expected ranges

CYTOTOXIC CONCENTRATION:  1000 nl/ml (34% survival after 22 h)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Rat hepatocytes

The cell survival ranged between 34.4% and 104.1%. No dose response was reported during the test.

Conclusions:

The test material did not induce any significant changes in the nuclear labeling of primary rat hepatocytes at a concentration range between 1000 nl/ml to 5 nl/ml.

Executive summary:

In an unscheduled DNA synthesis test, cyclohexyldimethylamine (DMCHA) was tested for potential nuclear changes at concentration between 1000 nl/ml to 5 nl/ml in ethanol. Freshly prepared hepatocytes obtained by perfusion of rat livers were treated with 2.5 mL WME containing 1% foetal bovine serum, 1uCi/ml 3H-thymidine and the test material at the relevant concentration. Each treatment included positive and negative controls and were performed on five cultures. After 18 -19 hours, the UDS assy is terminated. Three of the cultures from each treatment are washed with WME containing 1mM thymidine and are further prepared for analysis. The other two cultures used to monitor the toxicity of each treatment. At 20 -24 hours after the initiation of the treatments, viable cell counts were determined to estimate cell survival relative to the negative control.The nuclei were exposed to 1% sodium citrate for 10 minutes and then the cells were fixed in acetic acid:ethanol and dried for at least 24 hours. The coated slides were stored for 7 -10 days at 4C. The cells were then examined microscopically. UDS is measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count. The net nuclear grain count is determined for 50 randomly selected cells. DMCHA was moderately-to-highly toxic at 1000 nl/ml. The number of viable cells was 34.4% of the number in the solvent control. The test material was slightly to non cytotoxic between 500 nl/ml to 0.025 nl/ml. The test was therefore carried out between 1000 to 5 nl/ml. The positive control 2 -AAF induced a clear positive response confirming the validity of the test. The test material did not induce any significant changes in the nuclear labelling of primary rat hepatocytes. Based on these results, the test substance does not require classification according to Directive 67/548/EEC or Regulation EC No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An vivo test was conducted and gave negative results, confirming the absence of genotoxic potential observed in the in vitro studies. The ability of DMCHA to induce micronuclei in polychromatic erythrocytes from rat bone marrow was assessed in vivo. DMCHA did not induce micronuclei in rat polychromatic erythrocytes and is therefore not clastogenic in vivo. However, due to the lack of response observed in the positive control, it validity is questionable.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 21st, 1981 to January 20th, 1982

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Similar to guideline, GLP. The positive control in females did not induce a significant increase in micronuclei.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

1. The proportion of micronucleated PCEs was determined for 500 PCEs / animal. OECD 474 (1983) or OECD 474 (1997) indicate 1000 or 2000 PCEs /animal, respectively. 2. The positive control in females did not induce a significant increase in micronuclei.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: Adult
- Weight at study initiation: First study: 248.7 g (males) and 209.2 (females). Second study: 191.2 g (males) and 186.2 g (females)
- Assigned to test groups randomly: yes
- Housing: Animals were housed up to 4 rats per cage.
- Diet (e.g. ad libitum): Purina Laboratory Chow ad libitum
- Water (e.g. ad libitum): ad libitum

IN-LIFE DATES: From: December 21st, 1981 To: January 20th, 1982

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Initial doses were based upon LD50 information and were 80% of the LD50 for high dose and one-half of the high dose for the low dose. High mortality at the high dose prompted a second study, using a third dose of one-fourth the high dose. The three doses employed were thus: 80% of the LDS0
or 450 mg/kg/day, 225 mg/kg/day and 112 mg/kg/day.

A subchronic dosing regimen was used. It consisted of two administrations approximately 24 hours apart. Harvest was at 48 and 72 hours after the first administration of the test article, and at 48 hours after the first administration of the control articles. Equal numbers of males and females were used at each dose/kill -time combination. The route of administration was oral gavage.

Duration of treatment / exposure:

Two administrations at an interval of approximately 24 hours

Frequency of treatment:

Two treatments at a 24-hour interval

Post exposure period:

Not documented

Dose / conc.:

225 mg/kg bw/day (nominal)

Dose / conc.:

450 mg/kg bw/day (nominal)

Dose / conc.:

112 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 per sex (2 per sex per sampling time).

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine at 0.5 mg/kg dissolved in saline
Triethylenemelamine
- Route of administration: Intraperitoneal injection
- Doses / concentrations: at 0.5 mg/kg dissolved in saline

Tissues and cell types examined:

% of polychromatic erythrocytes (PCE) in 500 erythrocytes. % micronucleated PCEs in 500 PCEs.
Number of cells scored: 500 PCEs were scored per animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The test compound was initially tested at 433 mg/ml in corn oil for the low dose. Due to the high morality of the high dose animals (450 mg/kg) wherein 15 out of 16 animals died, the study was repeated with a lower dose and concurrent 48-hour negative control.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): three doses were administered: high dose 450 mg/kg bw, 225 mg/kg bw and 112 mg/kg bw. Sampling time were carried out at 48 and 72 hours after the first treatment and at 48 hours after the first administration of the control articles.

DETAILS OF SLIDE PREPARATION: Following centrifugation to pellet the tissue, the supernatant was drawn off, cells resuspended in a drop of serum, and suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa,
and rinsed in deionized water (Schmid, 1975).

METHOD OF ANALYSIS: Based on total numbers of PCE in the optic fields. The frequency of PCEs versus mature erythrocytes (RBCs) was
determined by scoring the number of RBCs observed in the optic fields while scoring the f i r s t 200 PCEs for micronuclei.

Evaluation criteria:

A test article is positive if two treatment groups are positive (p < 0.05). If only one treatment group is positive, the result is designated as unconfirmed positive.

Statistics:

Student t test and ANOVA using mean square for error (MSE)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death:  6 at medium dose, 15 at high dose and one positive control animal were found dead during the study. Time to death not recorded.
- Clinical signs:  all medium and high dose animals showed convulsions after dosing on the first day. After seizure, animals were lethargic. 

EFFECT ON PCE/NCE RATIO: 
%PCE at 112 and 225 mg/kg was 82 and 117, respectively (males, 48 h); 61 and 113% (males 72h).
%PCE at 112, 225 and 450 mg/kg was 62, 126 and 88, respectively (females, 48h), and 110 and 55% respectively (females, 72 h, all females of 450 mg/kg died)

GENOTOXIC EFFECTS: 
Mean number of micronucleated PCE: at 48 h: at 112, 225 and 450 mg/kg: 0.65, 0.4 and 0.4 respectively (male and female)
at 72h:  at 112 and 225 mg/kg: 0.28 and 0.52 respectively (male and female).

MUTANT/ABERRATION/mPCE/ POLYPLOIDY FREQUENCY:  MPCE: no treatment related effects

All medium and high dose animals convulsed after dosing on first day; after seizure, animals were lethargic.

Conclusions:

Although there was no increase in micronuclei in the females treated with the positive controls, DMCHA does not induce micronuclei in rat polychromatic erythrocytes and is therefore not clastogenic.

Executive summary:

A study (Cimino, 1982) was performed to assess the ability of DMCHA to induce micronuclei in polychromatic erythrocyte from rat bone marrow.

DMCHA was administered via intraperitoneal injection to groups of 4 animals/sex/dose level. Negative and positive control cultures were also prepared. Triethylenemelanine was used as the positive control at a dose level of 0.5 mg/kg bw. A preliminary study was carried out and due to the high mortality at 450 mg/kg bw, the study was repeated with a lower dose and concurrent 48 hour negative control. A total of three doses were administered 450 mg/kg bw/day, 225 mg/kg bw/day and 112 mg/kg bw/day. Two administrations were performed at a 24 hour intervals. Harvest of the cells was performed at 48 and 72 hours after the first administration and at 48 hours after the first administration of the control articles.

The animals were sacrificed and the bone marrow was extracted for preparation. The tissue was centrifuged and the supernatant was collected, resuspended in serum and spread on slides. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa and rinse in deionised water prior to analysis for scoring.

Five hundred PCEs per animal were scored.The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The frequency of PCEs versus mature erythrocytes (RBCs) was determined by scoring the number of RBCs observed in the optic fields while scoring the first 200 PCEs for micronuclei. The percentage micronuclei in marrow from the negative controls were 0.45% and 1.00% for males, and 0.85% and 0.65% for females, in the first and second studies respectively. The positive control compound, triethylenemelamine (TEM),induced an increase in frequency of micronuclei in males but not in females. The mean number of micronucleated PCE at 48 h were 0.65 at 112, 0.4 at 225 and 0.4 at 450 mg/kg (male and female). At 72 hours, the mean numbers of micronucleated PCE were  0.28 at 112 and and 0.52 at 225 mg/kg:  (male and female). 

 

Although there was no increase in micronuclei in the females treated with the positive controls, DMCHA does not induce micronuclei in rat polychromatic erythrocyte and is therefore not clastogenic. The negative response in the in vivo assay confirmed the negative in vitro responses in a battery of mutagenicity assays.

Based on these results, the test substance does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Cyclohexyldimethylamine (DMCHA) was tested for its mutagenic potential in a battery of in vitro tests.

The key study addressing reverse mutations in a standard plate test and pre-incubation test using E. coli and S. typhimurium bacterial strains according to the Ames test design showed no mutagenic activity in the presence or absence of metabolic activation. Two additional Ames tests, supporting studies, investigated reverse mutation potential in three test strains- Salmonella, Saccharomyces and Escherichia. The results of the tests were negative in the presence or absence of metabolic activation. No indications of reverse mutation were evident in the available data.

The potential for DCMHA to induce chromosomal aberrations was tested in CHO cells in vitro, in the presence and absence of metabolic activation, at suitable non-cytotoxic levels for metaphase analyses. DMCHA caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations either with or without S-9. N,N-dimethylcylcohexamine showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

DMCHA did not cause any statistically significant increases in the mutant frequency at the thymidine kinase locus of L5178Y mouse lymphoma cells in vitro, in the presence and absence of metabolic activation.

DMCHA was also tested for potential to induce nuclear changes in an unscheduled DNA synthesis test in rat hepatocytes.

The net nuclear grain count was determined for 50 randomly selected cells. DMCHA was moderately-to-highly toxic at 1000 nl/ml. The test material was slightly to non-cytotoxic between 500 nl/ml to 0.025 nl/ml. The test was therefore carried out between 1000 to 5 nl/ml. The test material did not induce any significant changes in the nuclear labelling of primary rat hepatocytes.

None of the in vitro assessments of mutagenicity showed any indications of a genotoxic effect of exposure to DMCHA.

An vivo test was conducted and also gave negative results, confirming the absence of genotoxic potential observed in the in vitro studies. The ability of DMCHA to induce micronuclei in polychromatic erythrocytes from rat bone marrow was assessed in vivo. DMCHA did not induce micronuclei in rat polychromatic erythrocytes and is therefore not clastogenic in vivo. However, due to the lack of response observed in the positive control, it validity is questionable.

In vitro

Engelhardt, G., 1998 (Key Study):

Cyclohexyldimethylamine (DMCHA) was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test and in the Escherichia coli.- reverse mutation assay.

Strains : TA 1535, TA 100, TA 1537, TA 98 and E . coli WP2 uvrA

Dose range : 20 ug - 5,000 ug/plate (SPT), 4 ug - 2,500 µg/plate (PIT)

Test conditions : Standard plate test (SPT) and pre-incubation test (PIT) both with and without metabolic activation (Aroclor induced rat liver S-9 mix).

Solubility : No precipitation of the test substance was found.

Toxicity : A bacteriotoxic effect was observed depending on the strain and test conditions at doses ≥ 2,500 µg/plate (SPT) or at

doses ≥ 500 µg/plate (PIT).

Mutagenicity : An increase ín the number of his+ or trp+ revertants was not observed both in the standard plate test and in the pre-incubation test either without S-9 mix or after the addition of a metabolizing system.

Conclusion :

DMCHA was not mutagenic in the Ames test or in the Escherichia coli - reverse mutation assay under these experimental conditions.

Jagannath, D.R. 1982:

Cyclohexyldimethyl amine (DMCHA) was evaluated for mutagenic activity in a bacterial assay with and without a mammalian S9 activation system in the Escherichia coli strain WP2uvrA-. The concentrations were used in the experiment: 0, 0.02, 0.04, 0.07, 0.15, 0.29, 0.59, 1.17, 2.34, 4.69, 9.38, 18.75, 37.50, 75.0 and 150.0 ul(ug)/plate.

The results of the tests conducted in the absence of a metabolic activation system were negative. The results in the presence of metabolic activation were also negative.

DMCHA Lot No. 20-133 CJ did not exhibit genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.

Galloway, 1982

Cyclohexyldimethylamine (DMCHA) was evaluated for potential to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells culturedin vitro.

Cultured CHO cells were exposed to the test substance both in the presence and absence of metabolic activation (S-9 mix derived from rat livers). Negative, solvent and positive control cultures were also prepared. After the appropriate treatment time cell division was arrested using colcemid, the cells harvested and slides prepared, so that metaphase figures could be examined.

In order to assess the cytotoxicity of DMCHA, cultures were examined for degree of confluence and presence of large rounded (mitotic) cells. On the basis of these data the dose levels selected for the metaphase analysis were 250 nl/ml to 2 µl/ml in the absence of S-9 mix and 600 nl/ml to 3 µl/ml in the presence of S9-mix.

In both the presence and absence of S-9 mix DMCHA caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the untreated controls. The aberration frequencies in the negative and solvent controls were within the normal background range for this laboratory.

Both positive control compounds, mytomycin and cyclophosphamide, caused large statistically significant increases in chromosomal aberrations. This demonstrates the sensitivity of the test system and the efficacy of the S-9 mix.

It was concluded that DMCHA showed no evidence of clastogenic activity in thisin vitro cytogenetic test system. Based on these results, the test substance does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

 

Myhr, 1982:

In an unscheduled DNA synthesis test, cyclohexyldimethylamine (DMCHA) was tested for potential nuclear changes at concentration between 1000 nl/ml to 5 nl/ml in ethanol. Freshly prepared hepatocytes obtained by perfusion of rat livers were treated with 2.5 mL WME containing 1% foetal bovine serum, 1 µCi/ml 3H-thymidine and the test material at the relevant concentration. Each treatment included positive and negative controls and were was on five cultures. After 18 -19 hours, the UDS assay is terminated. Three of the cultures from each treatment are washed with WME containing 1mM thymidine and are further prepared for analysis. The other two cultures used to monitor the toxicity of each treatment. At 20 -24 hours after the initiation of the treatments, viable cell counts were determined to estimate cell survival relative to the negative control.The nuclei were exposed to 1% sodium citrate for 10 minutes and then the cells were fixed in acetic acid:ethanol and dried for at least 24 hours. The coated slides were stored for 7 -10 days at 4°C. The cells were then examined microscopically. UDS is measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count. The net nuclear grain count is determined for 50 randomly selected cells. DMCHA was moderately-to-highly toxic at 1000 nl/ml. The number of viable cells was 34.4% of the number in the solvent control. The test material was slightly to non cytotoxic between 500 nl/ml to 0.025 nl/ml. The test was therefore carried out between 1000 to 5 nl/ml. The positive control 2 -AAF induced a clear positive response confirming the validity of the test. The test material did not induce any significant changes in the nuclear labelling of primary rat hepatocytes.

Flanders, 2015:

The mutagenic potential of cyclohexyldimethylamine was evaluatedin vitroon the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, according to OECD 490. The test item dose range for the mutagenicity experiment was selected based on a preliminary cytotoxicity study. In the main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells were treated with the test item at up to ten dose levels in duplicate, together with vehicle (R0 medium) and positive controls. The exposure periods were 4-hours both in the absence and presence of metabolic activation (S9-mix) and 24-hours in the absence of metabolic activation. Following exposure and an expression period of 2 days the number of small and large colonies was scored.

The maximum dose level used in the 4-hour exposure period was the limit dose (10 mM), but was limited by toxicity in the 24-hour exposure group. Precipitation of the test item was not observed. The vehicle controls demonstrated acceptable mutant frequencies for this cell line. The positive controls induced marked increases in the mutant frequency indicated the satisfactory performance of the test. The test item did not induce any statistically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Therefore,cyclohexyldimethylamine is not considered to be mutagenic to mammalian cellsin vitro, under the conditions of this study.

In vivo

Cimino, 1982:

The ability of cyclohexyldimethylamine (DMCHA) to induce micronuclei in polychromatic erythrocyte from rat bone marrow was assessed.

DMCHA was administered via intraperitoneal injection to groups of 4 animals/sex/dose level. Negative and positive control cultures were also prepared. Triethylenemelanine was used as the positive control at a dose level of 0.5 mg/kg. A preliminary study was carried out and due to the high mortality at 450 mg/kg bw, the study was repeated with a lower dose and concurrent 48 hour negative control. A total of three doses were administered 450 mg/kg bw/day, 225 mg/kg bw/day and 112 mg/kg bw/day. Two administrations were performed at 24 hour interval. Harvest of the cell was performed at 48 and 72 hours after the first administration and at 48 hours after the first administration of the control articles.

The animals were sacrificed and the bone marrow was extracted for preparation. The tissue was centrifuged and the supernatant was collected, re-suspended in serum and spread on slides. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa and rinse in deionised water prior to analysis for scoring.

Five hundred PCEs per animal were scored.The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The frequency of PCEs versus mature erythrocytes (RBCs) was determined by scoring the number of RBCs observed in the optic fields while scoring the first 200 PCEs for micronuclei.

 

The percentage micronuclei in marrow from the negative controls were 0.45% and 1.00% for males, and 0.85% and 0.65% for females, in the first and second studies respectively. The positive control compound, triethylenemelamine (TEM),induced an increase in frequency of micronuclei in males but not in females. The mean number of micronucleated PCE at 48 h were 0.65 at 112, 0.4 at 225 and 0.4 at 450 mg/kg (male and female). At 72 hours, the mean numbers of micronucleated PCE were  0.28 at 112 and and 0.52 at 225 mg/kg:  (male and female). 

 

Although there was no increase in micronuclei in the females treated with the positive controls, DMCHA does not induce micronuclei in rat polychromatic erythrocytes and is therefore not clastogenic. However, due to the lack of resposne observed in the positive control, the validity of htis study is questionable.

Short description of key information:
Several in vitro and in vivo study reports are available, including Ames test using Salmonella typhimurium and E.coli strains, an unscheduled DNA synthesis test in rat hepatocytes and a chromosomal aberration test in Chinese Hamster Ovary cells. The in vivo study was conducted in a micronucleus assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the in vitro studies provided, the test substance does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.