Formaldehyde, polymer with benzenamine, hydrogenated

Genetic toxicity: in vitro

Administrative data

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-bis[(4-aminocyclohexyl)methyl]aniline; 2,4-bis[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 2-[(1-aminocyclohexyl)methyl]aniline; 4-[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 4-{[4-({4-[(4-aminocyclohexyl)methyl]cyclohexyl}amino)cyclohexyl]methyl}cyclohexan-1-amine

EC Number:

603-894-6

Cas Number:

135108-88-2

Molecular formula:

Exact identification is not feasible

IUPAC Name:

2,4-bis[(4-aminocyclohexyl)methyl]aniline; 2,4-bis[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 2-[(1-aminocyclohexyl)methyl]aniline; 4-[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 4-{[4-({4-[(4-aminocyclohexyl)methyl]cyclohexyl}amino)cyclohexyl]methyl}cyclohexan-1-amine

Test material form:

liquid: viscous

Details on test material:

Formaldehyde, polymer with benzenamine, hydrogenated of Evonik Degussa Antwerpen N.V., batch no. 180-120417, Molecular weight: Mn approx. 305, Mw approx. 315 g/mol

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The indicator cell used for this study was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay is isolated by ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA.
Stock cultures were obtained from ATCC, and master stocks were maintained in liquid nitrogen. Laboratory cultures were periodically checked for karyotype stability and the absence of mycoplasma contamination by culturing methods. To reduce the background mutant frequency (spontaneous frequency) of TK-/- mutants to a level as low as possible, cell cultures were exposed to conditions that select against the TK-/- phenotype (exposure to aminopterin or methotrexate). Cell cultures were maintained in cleansing medium for one day, placed in recovery medium for one day and then returned to normal growth medium for three to eight days before use.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

In the first experiment without metabolic activation and both experiments with metabolic activation (3-hour exposure):
15.63, 31.25, 62.5, 125 and 250 µg/mL medium
In the second experiment without metabolic activation (24-hour exposure):
7.81, 15.63, 31.25, 62.5 and 125 µg/mL medium.

Vehicle / solvent:

The test item was completely dissolved in dimethylsulfoxide (DMSO).
As recommended by the guidelines at least 10 exp5 cells were suspended in treatment medium and diluted to 5 x 10 exp5 cells/mL.
Fresh preparations of the test and reference item solutions were prepared on each day of biological testing.

Controls

Untreated negative controls:

no

Remarks:

The vehicle dimethylsulfoxide (DMSO) served as the negative control.

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle dimethylsulfoxide (DMSO) served as the negative control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Remarks:

The vehicle dimethylsulfoxide (DMSO) served as the negative control.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the present test conditions, Copolymer of aniline and formaldehyde, hydrogenated, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Copolymer of aniline and formaldehyde, hydrogenated also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that Copolymer of aniline and formaldehyde, hydrogenated, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

Executive summary:

Copolymer of aniline and formaldehyde, hydrogenatedwas assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.

Copolymer of aniline and formaldehyde, hydrogenated was completely dissolved indimethylsulfoxide (DMSO).The vehicle DMSO served as the negative control.

In the preliminary experiment without and with metabolic activation (24-hour or 3-h exposure)cytotoxicity (decreased survival) was noted starting at a concentrations of 100 or 250 µg/mL, respectively.

Hence, in the main studythe concentration-range of 15.63 to 250 µg/mL was usedin theexperiments without and with metabolic activation with a 3-hour exposure time and a concentration-range of 7.81 to 125 µg/mL was used in the second experiment without metabolic activation (24-hour exposure).

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3 -Methylcholanthrene (at 2.5 or 4.0 µg/mL) in the presence of exogenous metabolic activation.

In the main study,cytotoxicity(decreased survival) was noted in the presence and absence of metabolic activation (3-hour exposure) at the top concentration of 250 µg/mL and in the second experiment without metabolic activation (24-hour exposure) at the top concentration of 125 µg/mL.

The values of mutation frequencies of the negative controls ranged from 57.54 to 74.74 per 10⁶ clonable cells in the experiments without metabolic activation and from 55.50 to 69.29 per 10⁶ clonable cells in the experiments with metabolic activationand, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with Copolymer of aniline and formaldehyde, hydrogenated ranged from 67.37to 127.28 per 10⁶ clonable cells (3 hours exposure) and from 49.44 to 60.19 per 10⁶clonable cells (24 hours exposure) in the experiments without metabolic activation and from 63.68 to 95.20 per 10⁶clonable cells (3 hours exposure, first assay) and from 66.66 to 96.51 per 10⁶ clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10⁶ viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.43 to 1.50 for Copolymer of aniline and formaldehyde, hydrogenated treated cells and from 0.67 to 0.95 for the negative controls.

The plating efficiency (PE step 1 and step 2) of the negative control was ≥ 50%, the mean cloning efficiencies (CE) within the range of 65% to 120% two days after treatment, and the mean suspension growth (SG) within the range of 8 to 32 following 3 -hour treatments and between 32 and 180 following 24-hour treatments.

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 1157.15 to 2592.61 per 10⁶ clonable cells in the case of MMS and ranging from 917.29 to 1434.11 per 10⁶ clonable cells in the case of 3 -MC.

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.65 to 2.29 in the case of MMS.