4,4'-methylenebis(cyclohexylamine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the present test conditions the test item tested up to cytotoxic concentrations in the in vitro bacterial mutagenicity assay (Salmonella reverse mutation Assay, OECD 471), the in vitro mammalian mutagenicity assay (HPRT Assay, OECD 476) and the in vitro mammalian chromosome aberration test (CA Test, OECD 473) each assay carried out without and with metabolic activation, revealed no genotoxic activity.

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:

02.04.1982- 31.08.1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study performed according to standard Ames testing guidelines with no major deviations (minor deviations: the test substance was tested up to very slight toxicity, no more than 27% toxicity in strain TA98 (without S9-mix) and 2-aminoanthracene alone is insufficient as a positive control with S9).

Qualifier:

according to guideline

Guideline:

other: Directive 79/831/EEC

Deviations:

yes

Remarks:

: 2-aminoanthracene alone is insufficient as a positive control with S9

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

other: S.typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Clophen A 50 induced rat liver

Test concentrations with justification for top dose:

- Dosing: 4, 12.5, 40, 125, 400 ug/plate
- Additional experiment: 125, 400, 800 ug/plate

Vehicle / solvent:

- Vehicle/solvent used: ethanol (70%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: 70% ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine for TA1537, sodium azide for TA100 and TA1535, 2-nitrofluorene for TA98 and TA1538

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: 70% ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for all strains

Remarks:

with S9

Details on test system and experimental conditions:

PLATE INCORPORATION TEST
The following components were mixed in a sterile tube:
-2 mL top-agar
-0,1 mL of the bacterial overnight culture (0,8- 1,5x 10^8 bacteria/ mL)
-0,01 mL of the test item or solvent
-0,5 mL S9 mix
The mixing was done in triplicate for each bacterial strain and for each concentration of the test material and the mixture was then poured onto the surface of minimal agar plates.
-Incubation: 72 hours, 37 °C and then the number of revertant colonies was counted

Rationale for test conditions:

The toxicity of the test item was tested with and without metabolic activation. Doses from 62,5 µg - 2000 µg/ plate were tested. As the highest test dose 800 µg/ plate were chosen (survival of 10-15 % without and 70 % with metabolic activation)

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:
- Dose related increase in the number of revertant colonies

Statistics:

not applicable

Species / strain:

other: S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 800 ug/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

GENOTOXIC EFFECTS:
- With metabolic activation: negative
- Without metabolic activation: negative,
TA1538 additional experiment; 3-fold increase. However, in one experiment only.

PRECIPITATION CONCENTRATION:
- no precipitate

CYTOTOXIC CONCENTRATION:
- With metabolic activation: very slight toxicity at 800 ug/plate
- Without metabolic activation: very slight toxicity at 800 ug/plate

STATISTICAL RESULTS:
- not applicable

Conclusions:

An Ames-test was performed to assess the mutagenis properties of the test item. The test item was proven to be non mutagenic in the absence and presence of S9 mix.

Executive summary:

The test item was tested for its ability to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were treated with the test compound by the Ames test plate incorporation. Dose levels up to 800 µg/ plate, in triplicate both with and without the addition of metabolising system were employed.

All bacterial strains exhibited mutagenis responses to the appropriate positive control substances. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range. 

A reproducible mutagenic activity of the test compound to any of the tester strains was not observed with and without metabolic activation.

It is therefore concluded, that the test item is not a bacterial mutagen. 

Reference 2

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:

13.08.1997- 25.08.1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Current OECD guideline: 2-Aminoanthracene is no sufficient positive control with S9, at least five strains should be tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the livers of male Wistar rats which received on 3 consecutive days doses of 80 mg/ kg b.w. Phenobarbital plus 80 mg/ kg b.w. ß-Naphtoflavone an were killed 24 hours after the last administration

Test concentrations with justification for top dose:

10-5000 µg/ plate test item

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The tester strains were stored as frzen permanents at -80 °C. Fresh working cultures were prepared for each test day by inoculating bacteria from frozen working cultures into nutrient broth. Cultures were incubated overnight (approx. 18 hrs) at 37 °C with gentle agitation. The bacterial titer was determined by plating 10^-6-dilution of the overnight culture on nutriernt agar plates and counting the resultant colonies after 48 hrs incubation at 37 °C.

Five dose levels of the test item in the absense and in the presence of metabolic activation were spaced half log intervals.

PLATE INCORPORATION TEST
The following components were mixed in a sterile tube:
-0,1 mL (0,05 mL in #18.1W) of the test material OR 0,1 mL (0,05 mL in #18.1W) of the solvent OR 0,1 mL (0,05 mL for TA 1535) of the strain specific positive control substance
-0,5 mL phosphate buffer OR 0,5 mL S9 mix in the experiment with metabolic activation
-2 mL of molten trace histidine supplemented top agar at approx. 45 °C
-0,1 mL of the bacterial overnight culture

The mixing was done in triplicate for each bacterial strain and for each concentration of the test material and the mixture was then poured onto the surface of minimal agar plates.
-Incubation: 72 hours, 37 °C and then the number of revertant colonies was counted

PREINCUBATION TEST
-In a sterile tube 0,1 mL aliquot of each one of the bacterial overnight was mixed with 0,5 ml volume of S9 mix (for tests with metabolic activation) or phosphate buffer (for tests without metabolic activation)
- either 100 µL (50 µL for TA 1535) of the appropriate positive control, 50 µL of the test substance solution were added
- incubated at 30 °C for 30 min with gentle agitation.
-then 2 mL of molten top agar was added to each tube, mixed briefly and poured onto minimal agar plates -> incubated at 37 °C for 72 hours and then the number of revertant colonies was counted

DATA COLLECTION
Bacterial colonies were counted on an Artek Model 880 Colony Counter which was calibrated for each test to check the counting accuracy. In addition, an examination of the bacterial background lawn on each plate was made. If no background lawn was observed, this was recorded and no plate count was made. If the background lawn was reduced as compared to the solvent control, this was also recorded, but colonies were counted. A reduction in the number of spontaneous revertants is also indicative of toxicity.

Rationale for test conditions:

In the plate incorporation test (#18.1) the test substance at the two highest concentrations (1600 µg/ plate and 5000µg/ plate) induced toxic effects in the bacteria strains TA 98, TA 1535 and TA 1535, the test was repeated with 10/ 30/ 100/ 300/ 1000 µg/ plate (TA 98 without S9-mix, TA1535 and TA 1537 with/ without S9 mix).

Evaluation criteria:

CRITERIA FOR DETERMINATION OF A VALID TEST
The following criteria must be met for the mutagenicity assay to be considered valid:
-in the solvent control, each tester strain culture must exhibit a characteristic mean number of spontaneous revertants
-to ensure that appropriate numbers of bacteria are plated, overnight cultures must be in excess of 10^8 bacteria/ mL
-the mean of rach positive control must exhibit a significant increase in the number of revertants over the mean value of the respective vehicle control
-normally, at least four non-toxic dose levels are required to evaluate the assay data, exceptions from this requirement may be justified

CRITERIA FOR EVALUATION OF TEST RESULTS
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be calles non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-relevant and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Statistics:

For all replicate platings, the mean number of revertants per plate and the standard deviation around the mean were calculated.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

4, 4'-methylenebis (cyclohexylamine) was found to negative in the Ames test for Genetic toxicity in vitro. The test used was Salmonella typhimurium Reverse Mutation Assay with the test item (OECD 471).

Executive summary:

The test item was tested for its ability to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were treated with the test compound by the Ames test plate incorporation (test #18.1, #18.1W) as well as the preincubation method (test #18.2. #18.2 W, #18.2WW, #18.2WWW). Dose levels covering the range of 10 to 5000 µg/ plate, in triplicate both with and without the addition of metabolising system (Phenobarbital/ ß-Naphthoflavane co-induced rat liver S9 mix) were employed.

All four bacterial strains exhibited mutagenis responses to the appropriate positive control substances. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range. 

A reproducible mutagenic activity of the test compound to any of the tester strains TA 98, TA 100, TA 1535 or TA 1537 was not observed with and without metabolic activation.

It is therefore concluded, that the test item is not a bacterial mutagen. 

Reference 3

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:

22.08.1994 to 18.01.1195

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

April 1984

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cells, obtained from Flow Laboratories, Mackenheim, Germany

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver of male Wistar rats which received a single injection of Aroclor 1254)

Test concentrations with justification for top dose:

0, 20, 60, 200, 600 and 1000 µg /ml of test material with and without S9

Vehicle / solvent:

culture medium:
H10: Ham´s F12 medium with 10 % FCS, 2 mM L-Glutamine, 100 IU/ mL Penicillin, 10 µg/ L Streptomycin
H0: Ham´s F12 medium with 2 mM L-Glutamine, 100 IU/ mL Penicillin, 10 µg/ L Streptomycin
H6TG: Ham´s F12 medium with 10 % FCS, 2 mM L-Glutamine, 100 IU/ mL Penicillin, 10 µg/ L Streptomycin, 10 µg/ mL 6-thioguanine (does not contain hypoxanthine)
HAT: Ham´s F12 medium with 10 % FCS, 2 mM L-Glutamine, 100 IU/ mL Penicillin, 10 µg/ L Streptomycin, 200 µM glycin, 5 µM thymidine, 10 µM hypoxanthine, 3,2 µM aminopterin

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

H0 medium (H0 medium + S9 mis in the assay with metabolic activation)/ DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
-CHO cells that had been subcultured twice were seeded in H10 medium (2x 10^5 cells/ cm^2 flask)
-two flaskes were seeded for each cencentration
-incubation at 37 °C for approx. 20 hrs, then medium was replaced by H0 medium containing 0, 1, 2, 4, 6, 100, 200, 400, 600, 1000 µg/ mL +/- S9 mix
-4 hrs exposure time, pooling of harvested cells of the same test substance concentration
-three dishes (60 mm) per culture were seeded wirh 200 cells/ dish in H10 medium
-after 7 days at 37 °C: fixing with mehanol, staining with Gisema and counting
-Cell survival = cloning efficiency of treated cells relative to untreated cells

MAIN STUDY
-cells for the main study were treated in HAT medium for approx. 1 weeks to reduce the number of cells harboring spontaneous mutations of the HPRT locus
- procedure described above was carried out at concentrations of 0, 20, 60, 200, 600, 1000 µg/ mL with and without S9 mix
-positive controls: Ethylmethanesulfonate (without S9) and 3-Methylcholanthrene (with S9)
-duplicate cultures with a total of 1x 10^6 cells were seeded for each treatment group
-4 hrs incubation time, then 3 times washed with phosphate buffered saline and trypsinized
-duplicate cultures were pooled and cells were counted, cells of each treatment group were divided into two subgroups (one to determine the cloning efficiency and one to allow expression of the mutated phenotype (H10 medium for 7 days/ 9 days with S9), cells were subcultured after 3 days at approx. 2x 10 ^6 cells/ flask cell density)
-at the end of expression period: determination of cloning efficiency
-selection of mutants was performed using 5 cultures for each treatment group, 1x 10^6 cells/ 75 cm^2 flask were seeded in H6TG medium and after 6 days at 37 °C fixed with methanol and stained with Giemsa and counted

Rationale for test conditions:

Solubility limit of 1000 µg test item per ml cell culture, therefore 9 different concentrations in the range of 1 to 1000 µg/ mL test item were employed in the main study.

Evaluation criteria:

For a test to be valid, all five criteria must be met:
1. the cloning efficiency of the negative control must be 20% or higher.
2. the mutation frequency in the negative control should not exceed 20 cells/10^6 cells.
3. the mutation frequency of the positive control must be elevated.
4. Four doses must be examined with the highest dose displaying toxicity of cells (about 20% cloning efficiency).
5. Results must be reproducible in a second experiment.

Criteria for evaluation of results: A substance iwill be reported as being mutagenic in the HPRT test with CHO cells if it causes a statistically significant, dose related increase in mutant frequency at concentrations of the test substance resulting in greater than 20 % cell survival. In addition, a positive response is claimed only, of the mean mutant frequency in treated cultures reaches a value significantly above the maximum spontaneous mutant frequency (approx. 20/10^6 viable cells)

Calculation of cloning efficiency and mutation frequency: absolute cloning efficiency (%)= (mean number of colonies/ dish x 100)/ (number of plated cells/ dish (200)), relative cloning efficiency (%) = (absolute C.E. of treated cells x 100)/ (absolute C.E. of solvent control), mutation frequency= (counted colonies (mean of 5 dishes))/ (absolute cloning efficiency (in %))

Statistics:

t-test.

Species / strain:

Chinese hamster Ovary (CHO)

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

HPRT Test without Metabolic Activation 

-negative controls within the range

-positive controls, EMS, significantly higher mutant frequencies, thus demonstrations the sensitivity of the test system

-mutant frequncies of the test item were within the range of historical negative controls

-statistical significance was obtained in 4 of 5 test item concentrations, but has no biological meaning and is not indicative of a mutagenic potential

 

HPRT Test with Metabolic Activation

-negative controls within the range

-positive controls, MCA, significantly higher mutant frequencies, thus demonstrations the sensitivity of the test system

-mutant frequencys of test item treated cells were within the range of historical negative controls, the statistical significance obtained with several dose groups in test #1 must therefore be considered to have no biological meaning

Conclusions:

4, 4'-methylenebis (cyclohexylamine) was found to negative in the in vitro mammalian cell gene mutation assay test for Genetic toxicity in vitro. The test used was "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test" (OECD 476).

Executive summary:

The test item was tested for its ability to induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro both in the presence and in the absence of exogenous activation by Arclor 1254-induced rat liver S9.

In a dose finding study, the test item was tested at concentrations from 1 µg/ mL up to the solubility limit of 1000 µg/ mL. In the absence and presence of S9 mix no significant influence on the cloning efficiency of CHO cells was observed.

In the two experiments of the main study, the mutant frequencies of the negative controls (culture medium) were consistent with historical control data for this test system. Treatment of the cells with the positive controls resulted in highly significant increases of the mutant frequencies of the HPRT locus, thus demonstrating the sensitivity of the test item and the activity of the S9 mix. 

The test item was tested at concentrations of 20, 60, 200, 600 an 100 µg/mL. Treatment of the cells with the test item did not result in reproducible biologically significant increase of the mutant frequncy of the HPRT locus. There was no indication of a mutagenic effect of the test item, neither in the absence nor in the presence of a metabolic activation system. 

Reference 4

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:

August- October 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to guideline and under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

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):

Cloned V79 cells with a modal chromosome number of 22 and a cell cycle length (on glass microscopic slides) of approx. 18 hrs were used. Theses were originally derived from lungs of a male Chinese hamster. Cells were obtaines from Dr. Engelhardt, BASF, Ludwigshafen; Germany and the stock culture was maintained under liquid oxygen.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver of male Wistar rats which received a single injection of Aroclor 1254)

Test concentrations with justification for top dose:

In a seperate experiment solubility testing revealed a limit of solubility of 1000 µg test item per mL of cell culture. Therefore, 10 different concentrations between 10 to 1000 µg/ mL were emploxed. In the presence as well as in the absence of S9 mix, treatment wit the test item dose dependently reduced the number of cells undergoing mitosis.
Without S9 mix: reduction of the mitotic indes of approx. 50 % at 60 µg/mL
With S9 mix: 40 % reduction at 600 µg/ mL and 50 % reduction at 800 µg/ mL
The following concentrations were therefore scored for chromosomal aberration induction: without S9 mix: 0, 10, 40, 60 µg/ mL with S9 mix: 0, 60, 400, 600 µg/ mL

Vehicle / solvent:

cell culture medium/ 1% DMSO:
MEM5: MEM (Eagle) medium with 5% FCS, 2 mM/L-Glutamine, 100 IU/ml Penicillin, 100 µg/ml Streptomycin, 20 mM HEPES.
MEM0: MEM (Eagle) medium with 2 mM L-Glutamine, 100 IU/ml penicillin, 100 µg/ml Streptomycin, 20 mM HEPES.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

MEM5 medium/ MEM0 medium plus S9 mix in the assay with metabolic activiation

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

without metabolic activation (Mitomycin C): 0.02 and 0,04 µg/ mL (test # 1) 0,02 and 0,03 µg/ mL (test # 2), with metabolic activation (Cyclophosphamide): 2 and 4 µg/ mL (test # 1) and 2 and 3 µg/ mL (test # 2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
-cells were removed from the liquid nitrogen container, thawed at 37 °C and transferred into MEM5 medium
- 37 °C, 5% CO2 and approx. 95 % rel. humidity with a first culture medium exchange after approx. 24 hrs
-subculutured 4 and 7 days after deeding
-during the second subculture, cells were seeded onto microscopic slides
-2,5 x10^4 sells were seeded onto each slide and incubates for approx. 24 hrs at 37 °C
-treatment with 10 different test item concentrations, Cyclophosphamide and Mitomycin C
- Exposure duration: 3 h exposure WITH S9, after which medium was exchanged for fresh MEM5. For treatments WITHOUT S9, duration was 20 (experiment 1)and 43 hours (experiment 2)
-treatment was terminated by addition of 50 mM KCl
- Fixation time (start of exposure up to fixation or harvest of cells): Colcemid (0,2 µg/ mL final conc.) was added 2 h prior to cell collection.
-Hypotonic treatment was terminated after 20 mins by fixation in methanol/ glacial acetic acid (3 parts methanol, 1 part acetic acid; precooled to 4 °C)
-3 consecutive fixation steps of 15, 10 and 5 min were performed
-Staining with Giemsa after air drying for 15-20 min and drying at 60 °C for approx. 1 hr

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Geimsa

NUMBER OF REPLICATES: 2

DETERMINATION OF MITOTIC INDEX AND SCORING OF ABBERATIONS
-Examination at a magnification of x200
-the proportion of metaphases was determined, always starting with the highest test substance conc.
-scoring of at least 3000 cells (1500 per slide)
-dose level reducing the metaphase indes to approx. 50 % of the solvent control was used as the highest dose level for the metaphase analysis
-three doses of the test compound over approx. a one-log dose range were employed (without S9 mix: 0, 10, 40, 60 µg/ mL with S9 mix: 0, 60, 400, 600 µg/ mL )
- where possible 100 metaphases from each culture (200 per experimental point) were analysed for chromosomal aberrations
-scoring of cells with good chromosome morphology and having no overlap with other nuclei or debris
-only cells with 20-24 centrometers acceptable for analysis
-polypoid or endoreduplicated cells were also noted and recorded separately, but aberrations in these cells were not scored
-aberrations were classified according to the scheme described by Scott et al. (1990)

TREATMENT OF DATA
Aberrant cells from each culture were categorized as follows:
A: Cells with structural aberrations incl. gaps
B: Cells with structural aberrations excl. gaps
C: Sells with exchange figures
D: Polypoid cells or cells with endoreduplications
On the basis of the categors totals for the negative controls the acceptability of the assay was determinded. The number of aberrant cells in each replica was used to establish acceptable homogeneity between replicates by means of binominal dispersion test (Richardson et al., 1989). The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negatove controls in the Chi square test. Probability values of p <0,05 were accepted as statistically significant.

ACCEPTANCE CRITERIA
The assay with V79 cells was considered valid if:
1. The number of cells with chromosomal aberrations (excluding gaps) in the negative control group falls within the normal range (< 5%). The percentage of polyploid or endoreduplicated cells should be < 10%.
2. Approximately 200 cells per treatment group (100 per each positive control) are analyzable for chromosomal aberrations
3. Treatment with positive controls (MMC and CPP) in at least one concentration tested leads to clear increases in the frequency of cells with structurally altered chromosomes (>>5% excluding gaps)
4. There is acceptable homogeneity between replicate cultures by the binomial dispersion test.

Rationale for test conditions:

In a seperate experiment solubility testing revealed a limit of solubility of 1000 µg test item per mL of cell culture. Therefore, 10 different concentrations between 10 to 1000 µg/ mL were emploxed. In the presence as well as in the absence of S9 mix, treatment wit the test item dose dependently reduced the number of cells undergoing mitosis.
Without S9 mix: reduction of the mitotic indes of approx. 50 % at 60 µg/mL
With S9 mix: 40 % reduction at 600 µg/ mL and 50 % reduction at 800 µg/ mL
The following concentrations were therefore scored for chromosomal aberration induction: without S9 mix: 0, 10, 40, 60 µg/ mL with S9 mix: 0, 60, 400, 600 µg/ mL

Evaluation criteria:

The test chemical is to be considered clastogenic in this assay if
1. it induces chromosomal aberrations (excl. gaps) in a statistically significant manner in one or more concentrations
2. the induced prportion of aberrant cekks at such test substance concentrations exceeds the normal range of the test system (i.e. >> 5%)
3. positive results can be varified in an independent experiment
-increases in the proportion of cells with gaps or increases in the numbers of cells wirh structural aberrations not exceeding the normal range are considered on a case by case basis. The possible influence of pH, S9 mix or osmolality on the occurance of chromosomal aberrations will also be considered. As this assay is not designed to detect numerical aberrations, polyploidy and endoreduplications are reportes when seen, but these data are not used for any kind of interpretation.

Statistics:

The number of aberrant cells in each replica was used to establish acceptable homogeneity between replicates by means of a binomial dispersion test (Richardson, et .al., 1989). The proportion of cells that was treated with the test substance and harbored structural aberrations (excluding gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 800 microgram/ml with S9 and ≥ 80 microgram/ml without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in the frequency of chromosomal aberrations at concentrations which were nontoxic (50% reduction in mitotic index).

Conclusions:

4, 4'-methylenebis (cyclohexylamine) was found to negative chromosomal aberrations test in vitro, because it did not induce biologically significant increases in the chromosome aberration frequency of V79 Chinee hamster cells. The test used was Chinese Hamster Lung V79 Assay for chromosomal aberrations (OECD 473).

Executive summary:

Cytotoxicity

In a seperate experiment solubility testing revealed a limit of solubility of 1000 µg test item per mL of cell culture. Therefore, 10 different concentrations between 10 to 1000 µg/ mL were emploxed. In the presence as well as in the absence of S9 mix, treatment wit the test item dose dependently reduced the number of cells undergoing mitosis.
Without S9 mix: reduction of the mitotic indes of approx. 50 % at 60 µg/mL
With S9 mix: 40 % reduction at 600 µg/ mL and 50 % reduction at 800 µg/ mL
The following concentrations were therefore scored for chromosomal aberration induction: without S9 mix: 0, 10, 40, 60 µg/ mL with S9 mix: 0, 60, 400, 600 µg/ mL

 

Chromosome Aberration Study

Negative controls: chromosomal aberration frequencies of 0- 2,5 % which is consistent with spontaneous aberration frequencies for the V79 cell line in this laboratory (max. acceptable spontaneous aberration frequencie <5 %)

Positive controls: Mitomycin C and Cyclophosphamide led to biologically significant increases in the frequency of aberrations, so the metabolic activation system was satisfactory and the test method itself was operating as expected

Treatment with the test item, without as well as with S9, at both sampling times did not result in biologically significant increases, although statistically significance was reached with two test compound concentrations in the frequency of chromosome aberrations. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Under the experimental conditions of the in vivo mammalian erythrocyte micronucleus test (BG Chemie, 1984), the test substance has no chromosome damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

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:

key study

Study period:

2.11.1983- 27.01.1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was performed according to accepted but older guidelines and under GLP.

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Cited as Directive 84/449/EEC, B.12

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Lippische Versuchstierzucht, Extertal, Germany
- Strain: NMRI-SPF (HAN/Bö)
- Sex: male and female
- Age: 7 to 8 weeks
- Weight at study initiation: 33-40 g (males) & 30-35 g (females)
- Housing: Makrolon cages
- Acclimation period: 10 days
- Diet and water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C
- Relative humidity: 45-55 %
- Light: 12 hour light/ dark cycle

Route of administration:

intraperitoneal

Vehicle:

DMSO

Details on exposure:

TEST GROUP
- The animals received a single intraperitoneal injection of the test substance dissolved in DMSO (stock solution: 50 mg of the test item + 1 mL DMSO + 9 mL NaCl-solution (0,9%))
- The dose level of 50 mg/ kg used as the test dose was selcted to produce mortality of about 10% (based on preliminary LD50 study)
- Volume injected: 0.1 mL/10 g bw

NEGATIVE CONTROL GROUP
- The animals received a single intraperitoneal injection of DMSO (stock solution: 1 mL DMSO + 9 mL NaCl-solution (0,9%))
- Volume injected: 0.1 mL/10 g bw

POSITIVE CONTROL GROUP
- The animals received a single intraperitoneal injection of Cyclophosphamide in DMSO (stock solution: 20 mg Cyclophosphamide + 1 mL DMSO + 9 mL NaCL-solution (0,9%))
- Volume injected: 0.1 mL/10 g bw

Duration of treatment / exposure:

single application

Frequency of treatment:

once

Post exposure period:

24, 48 and 72 h after administration, 6 animals of each group were sacrificed, after 24 h animals from the positive control group were sacrificed

Dose / conc.:

50 other: mg/ kg

No. of animals per sex per dose:

negative control group: 20
positive control group: 8
test group: 22

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (20 mg/kg)

Tissues and cell types examined:

femoral bone marrow smears

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION
- The bone marrow preparations were prepared, stained and evaluated based on the method of Schmid
- The two femora were excised from the test animals, the attached tissue was removed and the femora were dissected at the condyles
- The bone marrow was rinsed with fetal calf serum by means of a syringe and cannula and transferred to a centrifuge tube with 4 mL calf serum
- The cell suspension was centrifuged at 100 g for 5 minutes, and the serum was sucked off except for a small amount
- The cells were resuspended by careful pipetting, dropped onto clean microscopic slides and smeared

METHOD OF ANALYSIS
- After one day, the microscopic slide preparations were stained with May-Gruenwald and Giemsa solution, rinsed in distilled water, dried and mounted in DePeX via xylene
- The coded preparations were evaluated under the microscope at a 500 fold enlargement
- 1000 erythrocytes were counted and differentiated according to polychromatic (young) and normochromatic (old) erythrocytes
- The fraction of polychromatic cells indicates the function of erythropoiesis and allows detecting possible cytostatic or cytotoxic effects of the test substance
- The proportion of cells carrying miconuclei was determined in 2000 polychromatic erythrocytes
- The mean and standard deviation were calculated from the individual values of the 6 animals of each group

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:
- Significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male and female mice

Statistics:

- The test group and control group were investigated for differences by means of the U-test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality : Male: 2 out of 22, Female: 3 out of 22
- Clinical signs: slight to moderate convulsion and piloerection

STATISTICAL RESULTS:
% PCE and %PCE with micronucleus were not increased in males or females at 24, 48, and 72 hours after treatment.

EFFECT ON PCE/NCE RATIO (%PCE):
Males
- Control group: 52.9, 53.3, 51.8 at 24, 48 and 72 hours, respectively
- Treatment group: 50.4, 48.4, 53.6 at 24, 48 and 72 hours, respectively
- Positive control: 52.2 at 24 hours

Females
- Control group: 53.4, 51.9, 52.2 at 24, 48 and 72 hours, respectively
- Treatment group: 53.0, 53.2, 54.1 at 24, 48 and 72 hours, respectively
- Positive control: 51.7 at 24 hours

GENOTOXIC EFFECTS:
Mean number of micronucleated PCE:
Males
- Control group: 0.16, 0.27, 0.21 at 24, 48 and 72 hours, respectively
- Treatment group: 0.26, 0.24, 0.20 at 24, 48 and 72 hours, respectively
- Positive control: 1.40 at 24 hours

Females
- Control group: 0.13, 0.25, 0.19 at 24, 48 and 72 hours, respectively
- Treatment group: 0.17, 0.22, 0.21 at 24, 48 and 72 hours, respectively
- Positive control: 1.39 at 24 hours

Conclusions:

Under the experimental conditions chosen here, the test substance has no chromosome damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

Executive summary:

According to the test results available, there are thus no significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the dose group (50 mg/ kg body weight) or between the different sacrifice intervals (24, 48 and 72 hours). Thus, under the experimental conditions chosen here, the test substance has no chromosome damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis. The positive control is valid. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity:

in vitro studies

 

PACM (chemical name: 4,4'-Methylenebis(cyclohexylamine), was tested for mutagenicity in the Ames test (GLP study) according to OECD 471 (Huels AG, 1997). The tested strains were TA 1535, TA 100, TA 1537, TA 98. The test conditions were without and with metabolic activation. The test substance was dissolved in DMSO. An increase in the number of his+ revertants could not be observed either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance 4,4'-Methylenebis(cyclohexylamine) is not mutagenic in the Ames test under the experimental conditions chosen.

There are four additional genotoxicity tests available according or similar to OECD 471. Each assay, carried out with 5 strains and without and with metabolic activation, revealed no genotoxic activity.

 

PACM was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (Huels AG, 1995). The experiments were carried out both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). The test concentrations were 0, 20, 60, 200, 600 and 1000 µg/ml medium. Under the experimental conditions of this study, the substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

PACM was investigated for its potential to induce chromosome-damaging effects in an in vitro cytogenetic study according to OECD 473 (Huels AG, 1994). The test substance did not lead to a biologically relevant increase in the number of structural chromosomal aberrations (including and excluding gaps) either without S9 mix or after the addition of a metabolizing system. The types and frequencies of structural chromosome aberrations were close to the range of the concurrent vehicle control values and within the range of the historical negative control data. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances Mitomycin and Cyclophosphamide clearly demonstrated the sensitivity of the test system and the metabolic activity of the used S9 mix. Thus, PACM is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.

 

in vivo studies

 

The substance PACM was tested for its mutagenic potential in NMRI mice using the micronucleus test method (key study; GLP; BG Chemie, 1984). The acute LD50 of PACM was determined in a pretest in male mice after a single intraperitoneal injection. A dose of 50 mg/kg bw was selected to be used in the micronucleus test (mortality was expected to be 10%). During the main study, clinical signs observed were slight to moderate convulsions and piloerection; 2 of 22 males and 3 of 22 females died in test substance treated groups. The proportion of polychromatic erythrocytes in the bone marrow was not affected after administration of Dicykan. The administration of the positive control Cyclophosphamide (20 mg/kg bw) has also not affected the proportion of polychromatic erythrocytes but led to the expected increase in the number of polychromatic erythrocytes containing micronuclei. After single intraperitoneal administration of 50 mg/kg bw Dicykan, the rate of micronuclei was always in the same range as that of the concurrent control at all 3 sacrifice intervals (24, 48 and 72 h). Thus, under the experimental conditions chosen, the test substance does not have any chromosome damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.

 

In a supporting study, PACM was tested for its mutagenic potential in NMRI mice using the micronucleus test method (non GLP; BASF, 1983). For this purpose, PACM, dissolved in DMSO, was administered once intraperitoneally to male and female animals at a dose level of 50 mg/kg bw. As a control, male and female mice were administered merely the solvent by the same route. As a positive control, male and female animals were administered 40 mg Cyclophosphamide/kg bw dissolved in distilled water once by intraperitoneal administration. Animals which were administered the solvent DMSO or the positive control substance Cyclophosphamide did not show any clinical signs of toxicity. However, 50 mg PACM/kg bw led to piloerection, apathy, irregular respiration and spastic gait in some cases. After 30 - 60 minutes, a narcotic-like state was observed. On the day after test substance administration, the animals still showed clinical signs such as apathy and piloerection; in some cases, these symptoms were observed throughout the observation period. A total of 5 male and 4 female mice died during the study period. The positive control substance Cyclophosphamide, as expected, led to the clear increase in the number of polychromatic erythrocytes containing micronuclei. The single intraperitoneal administration of PACM at a dose of 50 mg/kg bw did not lead to any increase in the number of polychromatic erythrocytes containing micronuclei. The rate of micronuclei was always in the same range as that of the concurrent control at all 3 sacrifice intervals (24, 48 and 72 h). A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected in individual animals of the 48 h sacrifice interval and in most animals of the 72 h sacrifice interval. Thus, under the experimental conditions chosen, the test substance is not considered mutagenic.

 

There are further in vivo micronucleus tests available. In a non-GLP study (BASF, 1981), 5 male and 5 female NMRI-mice were treated with a single intraperitoneal dose of 4,4'-methylenebis(cyclohexylamine) at10 mg /kg bw. No significant increase in the number of polychromatic erythrocytes containing micronuclei was observed. Only 1000 PCEs were evaluated (the current OECD guideline requires an evaluation of 2000 PCEs) and no positive control group was investigated.

In another non-GLP study (BG Chemie, 1982), NMRI mice were treated with a single intraperitoneal dose of 50 mg/kg bw. The treated animals showed slight to moderate paresis a few hours after the administration; 6 of 25 animals died in the group of the males within 3 days, whereas none of the 25 females died. The number of micronuclei showed a slight increase up to twice the control values after administration of PACM. This increase was however statistically significant only in the females on days 1 and 2 after treatment. Only 1000 PCEs were evaluated in this study, the current OECD guideline requires an evaluation of 2000 PCEs. Due to the lack of historical control data, the relevance of the effect cannot be assessed.

The combination of a negative in vitro chromosomal aberrations test, with 3 of 4 in vivo mutagenicity tests (including a modern guideline assay according to GLP), offer a weight of evidence that the substance is not clastogenic or otherwise genotoxic.

Justification for classification or non-classification

Because all in vitro and in vivo genotoxicity studies revealed clearly negative results, it can be concluded that the test item is not genotoxic in vitro and in vivo and therefore must not be classified according to the criteria of EC Regulation 1272/2008.