3-aminomethyl-3,5,5-trimethylcyclohexylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance Isophorone diamine was tested in the Ames Salmonella mutagenicity test for any mutagenic activity according to OECD 471 from 1981. Under the conditions of the study the test item isophorone diamine proved to be non-mutagenic, both in the presence and in the absence of Arochlor-induced liver microsomes, for all test strains used in this study (Hüls AG 1990).

Chinese hamster ovary cells (CHO), treated with Isophorone diamine, were evaluated for chromosome aberrations at three dose levels, in duplicate, together with negative and positive controls. Isophorone diamine is considered to be non-clastogenic to CHO cells (SafePharm 1992).

In this HPRT test with Chinese Hamster ovary (CHO) cells according to OECD TG 476 (1984), Isophorone diamine did not significantly increase the mutant frequency of treated cells. Cytotoxicity was not observed. It is concluded that Isophoron diamine, in the presence as well as in the absence of S9 mix, demonstrates no mutagenic potential in this in vitro mammalian cell assay (Hüls AG 1992).

Link to relevant study records

Reference

Reference 1

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:

1992-01-20 - 1992-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO (Chinese hamster ovary) K1 cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced Wistar rat liver S9

Test concentrations with justification for top dose:

0 - 2 mg/ml

Vehicle / solvent:

HO medium, dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

= solvent/ vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

Medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

positive control with metabolic activation: 3-Methylcholanthrene Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
CHO cells that had been subcultured twice were seeded in HlO medium at a concentration of 2x10^5 cells/25 cm^2 flask. Two flasks were seeded for each concentration of the test substance. After incubation at 37 °c for approx. 20 hrs the medium was replaced by HO-medium containing the following concentrations of the test item:
0, 0.02, 0.03, 0.06, 0.12, 0.2, 0.3, 0.6, 1.2 and 2.0 mg/ml for HPRT #1 ± S9;
Exposure was for 4 hrs with and without S-9 mix. At the end of the treatment period the cells were harvested and cells of the same test substance concentration were pooled. Three dishes (60 mm) per culture were seeded with 200 cells/dish in H0 medium. After incubation for 6 days at 37 °c colonies were fixed with methanol, stained with Giemsa and counted. Cell survival is expressed as the cloning efficiency of treated cells relative to untreated cultures. The highest concentration to be used in the main study will be either the solubility limit of the test substance or the concentration resulting in a reduction of the cloning efficiency to approx. 80 %. In the absence of a solubility or toxicity limit, the highest concentration to be used will be 2.0 mg/ml.

MAIN STUDY
To reduce the number of cells harboring spontaneous mutations of the HPRT locus, the cells for the main study were treated in HAT medium for approx. 1 week. Then the procedure described before was carried out using the following concentrations of the test item in the medium:
- HPRT #1 ±S9: 0, 0.02, 0.06, 0.2, 0.6, 2.0 mg/ml
- HPRT #2 ±S9: 0, 0.02, 0.06, 0.2, 0.6, 2.0 mg/ml
Positive controls demonstrating the sensitivity of the test system were ethyl methane sulfonate (EMS, without S9, 300 µg/ml) and 3-methylcholanthren (MCA, with S9, 10 µg/ml). Duplicate cultures were used for each treatment group. After an incubation time of 4 hrs the cells were washed 3 times with phosphate buffered saline (free of calcium and magnesium) and trypsinized. Duplicate cultures were pooled and eel ls were counted. The cells of each treatment groupwere divided into two subgroups. One subgroup was used to determine the cloning efficiency according to the procedure described before. The cells of the other subgroup were cultured in H1O medium for 7 days to allow for expression of the mutated phenotype. At the end of the expression period the cloning efficiency was determined. The selection of mutants was performed using five cultures for each treatment group. 1x10^6 cells were seeded per treatment group (5x10^5 cells/25 cm^2 flask, duplicate
cultures) in H6TG medium. After incubation for 6 days at 37 °C the colonies were fixed with methanol, stained with Giemsa and counted.

Evaluation criteria:

The validity of an experiment is determined by the following criteria:
- The cloning efficiency of the negative control at the end of the expression period must be at least 50 %.
- The mutation frequency of the negative control should not exceed the maximum spontaneous mutation frequency of approx. 20/10^6 cells
- The mutation frequencies of the positive controls must be significantly elevated.
- At least 4 dose levels must be examined, the highest dose being eather significantly toxic (cloning efficiency approx. 80 %) or being the solubility limit of the test substance. Concentrations higher than 2 mg/ml will not be tested.
- The results must be reproduced in a second independent experiment.

Criteria for evaluation of results:
- A test compound will 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 50 % cell survival. In addition, a positive response is claimed only, if the mean mutant frequency in treated cultures reaches a value significantly above the maximum spontaneous mutant frequency (of approx. 20/10^6 viable cells)

Statistics:

t-test

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

none over test concentration range

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: CHO (Chinese hamster ovary) K1 cells

no further remarks

Conclusions:

In this HPRT test with Chinese Hamster ovary (CHO) cells according to OECD TG 476 (1984), Isophorone diamine concentrations of
20 - 2,000 mg/l (+/- S9 mix from Aroclor 1254 induced rat livers) did not significantly increase the mutant frequency of treated cells. Cytotoxicity was not observed. It is concluded that Isophoron diamine, in the presence as well as in the absence of S9 mix, demonstrates no mutagenic potential in this in vitro mammalian cell assay at any of the concentrations tested.

Executive summary:

The test item Isophorone diamine 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 the absence of exogenous metabolic activation(by Arochlor 1254 -induced rat liver S9). The substance was tested in concentrations of 20 mg/l to the solubility limit of 2000 mg/l with and without S9 mix. In this concentration range, Isophorone diamine did not induce any detectable cytotoxicity. Compared to the negative control treatment with Isophorone diamine in both assays (with and without metabolic activation) did not result in any reproducible statistically or biologically significant increase of the mutation frequency of the HPRT locus. It is concluded that Isophoron diamine, in the presence as well as in the absence of S9 mix, demonstrates no mutagenic potential in this in vitro mammalian cell assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-02-22 - 1990-03-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study, comparable to OECD guideline 471 with acceptable restrictions (TA 102 or E.coli WP2 were not tested, not required by applied 1983 guideline version)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Cited as Directive 84/449/EEC, B.14

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

TA 102 or E.coli WP2 were not tested, not required by applied 1983 guideline version

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, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat S9 liver, male Bor: WISW (SPF/Cpb)

Test concentrations with justification for top dose:

8 - 5000 µg/plate

Vehicle / solvent:

water / dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

=Solvent/ vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO; water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: Nitrofluorene (TA 98; TA 1538), Sodium azide (TA 100; TA 1535), Aminoacridine (TA 1537); with metabolic activation: Aminoanthracene (TA 100)

Details on test system and experimental conditions:

METHODS:
Two tests were carried out :
1. Main test with and without the use of an Aroclor-induced metabolic system.
2 . Preincubation test with and without the use of an Aroclor- induced metabolic system (preincubation for 30 minutes at 30°C).

The incubation conditions were:
- Incubation time 96 hours
- Incubation temperature 37°C

Constituents of this testing were:
- determination of the spontaneous reverse mutation rate (negative controls) as triplicate determination
- solvent controls (negative controls), as triplicate determination
- determination of bacterial counts of overnight culture
- triplicate determination of each concentration stage
- the positive controls, in triplicate

CHARACTERISATION OF THE TEST SYSTEM
To carry out the mutagenicity test, we used five Salmonella typhimurium mutants obtained directly from Ames. The strains are the following : TA 98 , TA 100 , TA 1535, TA 1537 , TA 1538.

DISSOLUTION OF THE TEST SUBSTANCE
In the main test, the product was dissolved in water at 50 g/l , and in the preincubation test, at 40 g/l. The highest tested concentration was 5000 μg/plate .

DOSAGE
- main test: 8 / 40 / 200 / 1000 / 5000 µg/plate (+/- metabolic activation)
- preincubation test: 125/250/500/1000/2000 µg/plate (+/- metabolic activation)

COMPOSITION OF THE MEDIA USED
- Nutrient agar for bacterial count determination: standard I agar
- Nutrient medium for growing the overnight culture: full nutrient broth
- Nutrient agar for the main/preincubation test : minimal agar
- Nutrient medium for the main/preincubation test prior to pouring onto the nutrient agar: top agar
- Liver microsome solution (metabolic system): S9 mix and simulated S9 mix

REFERENCE SUBSTANCES
To confirm the sensitivity of the bacterial strains in the test without a metabolic system, the following positive substances were used with the individual strains:
- TA 98 and 1538: Nitrofluorene
- TA 100 and 1535: Sodium azide
- TA 1537: Aminoacridine
For the tests using a metabolic system, we used an Aroclor-activated S9 fraction whose enzyme activity we checked using amino anthracene on strain TA100. The metabolic system was produced by treatment of rat liver (rat strain: Bor : WISW (SPF/Cpb) male).

Evaluation criteria:

mutagenic effects (i.e ratio of revertant rates treated/control >= 2) at <= 5000 µg/plate with generally positive dose-response relationship in any
strain

Statistics:

The standard deviations and mean values reported were calculated using a Commodore CBM 2032 computer. These calculations were based on calculation principles usual in mathematics.

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 1000 / 5000 µg/plate with / without preincubation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION CONCENTRATION: no precipitation

Remarks on result:

other: other: Salmonella typhimurium

no further remarks

Conclusions:

Under the conditions of the study the test item proved to be non-mutagenic in the Ames Salmonella mutagenicity test, both in the presence and in the absence of Arochlor-induced liver microsomes, for all test strains used in this study.

Executive summary:

The test substance was studied in the Salmonella/microsome mutagenicity test of Ames for any mutagenic activity present. The test organisms used were five histidine auxotroph (his +) Salmonella typhimurium strains (TA 1535, TA 1537 , TA 1538 , TA 98 and TA 100). The test substance concentrations used were in the range from 8 to 5000 μg/ plate (Petri dish with nutrient agar). Substances which still cause no mutagenic effect at a concentration of 5000 μg /plate can be termed non-mutagenic according to Ames.
The test material analysed was found to be non-mutagenic towards all of the test strains, in the absence or presence of Aroclor-induced liver microsomes, even with the addition of 5000 μg of test substance/plate and when the preincubation test was used.

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:

1992-05-26 - 1992-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

mammalian cell system (CHO Chinese hamster ovary cell line)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 BH4
cell cycle length of 12 hours

Metabolic activation:

with and without

Metabolic activation system:

male Sprague-Dawley rat liver S9 from Aroclor 1254 induced animals

Test concentrations with justification for top dose:

up to 1375 ug/ml in the main experiments

Vehicle / solvent:

Ham´s F12 medium

Untreated negative controls:

yes

Remarks:

= Solvent/ vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

Ham´s F12 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

positive control with S9-mix: Cyclophosphamide Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

METHODS
CELL LINE
The Chinese hamster ovary (CHO-Kl BH4) cell line, isolated by Kao and Puck (1967) and cloned by O'Neill et al (1977) was used. Stocks of the CHO cell line are maintained at -196°C in liquid nitrogen using 10% DMSO in culture medium as the cryoprotectant. The cell line used in this study has a cell cycle length of 12 hours.

CELL CULTURE
Cells were grown in Ham's F-12 medium, supplemented with 10% foetal bovine serum and antibiotics, at 37°C with 5% CO2 in air. Stock cultures are maintained in large culture flasks containing approximately 75 ml of culture medium. The cultures are routinely subcultured when confluent which is normally every 3 - 4 days using a 1:40 dilution. Cell monolayers are suspended in culture medium after dispersion with 0.05% trypsin solution.

PREPARATION OF TEST AND CONTROL MATERIALS
The test item was accurately weighed and dissolved in Ham's Media without serum. Test material solutions were used within 60 minutes of preparation. Negative and positive controls were used in parallel with the test material. Solvent treatment groups were used as the negative controls and the positive control materials were as follows: Mitomycin C (MMC) 0.075 μg/ml and 0.05 μg/ml for cultures in the absence of metabolising enzymes. Cyclophosphamide (CP) 10 μg/ml and 5 μg/ml for cultures where S9 was included. The concentrations for the positive controls were reduced for the second experiment to improve the quality and reduce the toxicity. A response was seen at both concentrations and was therefore considered not to affect the integrity of the study.

CYTOTOXICITY TEST
A cytotoxicity test was performed on cell cultures using a 4-hour exposure time with metabolic activation followed by an 8 and 16- hour culture period in treatment free media. Treatment without metabolic activation was continuous with cell harvest at 12 hours and 20 hours. Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent negative control value.

MICROSOMAL ENZYME FRACTION
Obtained from the British Industrial Biological Research Association on 12/05/92 and
09/06/92 respectively. They were prepared from the livers of male Sprague-Dawley rats weighing ~200g. These had received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation. The S9 was stored at -196°C in a Statebourne liquid N2 freezer, model SXR 34. 10% S9 mix was freshly prepared by mixing 2 ml of S9 with 1 ml of 0,1M NADP, 1 ml of 0,1M G6P, 2 ml of 330mM KCl/80mM MgCl2 and 14 ml of 0,1M Phosphate buffer (pH 7.4). The S9 mix was stored at 4°C until used, and used within 4 hours of preparation.

CULTURE CONDITIONS
Cultures were established using 0.5 x 10^6 cells per flask approximately 24 hours prior to treatment. Flasks were exposed to either one of the dose levels of the test material, negative or positive controls, both with and without metabolic activation, in duplicate (A + B). All exposures were in the presence of serum complete media. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air .
The treatment regimens were as follows:
a) Without Metabolic Activation:
i ) 12 hours continuous exposure to the test material.
i i ) 20 hours continuous exposure to the test material.
b)With Metabolic Activation:
i) 4 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium, of 10% S9 in standard cofactors). A phosphate buffered saline wash and then a further 8 hours in treatment-free media prior to cell harvest.
ii) 4 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium, of 10% S9 in standard cofactors). A phosphate buffered saline wash and then a further 16 hours in treatment-free media prior to cell harvest.

CELL HARVEST
Mitosis was arrested by addition of demecolcine (0.1 ug/ml) approximately two hours before the required harvest time. After incubation with demecolcine, the cells were trypsinised to detach them from the tissue culture flask and suspended in culture medium. A sample of the cell suspension from each harvest time was counted to measure growth inhibition at each concentration. 0.5 ml of cell suspension was diluted in 10 ml of Isoton and counted on a Coulter ZF cell counter. Each suspension was counted twice and the mean value was calculated and expressed as a percentage of the vehicle control value. The cells were centrifuged (approximately 1000-1500 rpm for five minutes), the culture medium drawn off and discarded, and the cells resuspended in 4 ml 0.075M KCl. After fifteen minutes (including five minutes centrifugation), all but approximately 0.5 ml of hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCl suspension into 3 ml fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least twice and the cells stored at 4°C for at least four hours to ensure complete fixation.

PREPARATION OF METAPHASE SPREADS
The cells were resuspended in 3.0 ml of fresh necessary before centrifugation and resuspension fixative if in 0.5 ml of fixative. Three or four drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

STAINING
When the slides were dry they were stained in 2% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and mounted in Depex mounting medium.

CODING
After checking that the slide preparations were of good quality, the slides were coded using a computer generated random number code.

SCORING OF CHROMOSOME DAMAGE
Three of the four dose levels were selected on the basis of toxicity for evaluation of chromosome damage for each experiment of the aberration study. Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 18 to 22 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS guidelines for mutagenicity testing. If the cell had more than 22 chromosomes then it was recorded on a separate sheet as an aneuploid or polyploid cell, chromosome aberrations in such cells were not recorded. Endoreduplicated cells are included as polyploid cells but can also be evaluated separately if necessary. All chromosome aberrations were checked by a senior cytogeneticist prior to decoding the slides.

MITOTIC INDEX
A total of 1000 cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

EXPERIMENT 2
The experimental conditions used for Experiment 1 were repeated with minor changes to the dose ranges used in order to establish the presence or absence of a dose-response relationship.

CONFIRMATORY EXPERIMENT
A further experiment was performed to investigate the positive response seen in Experiment 1 and 2. Cultures were exposed to the test item at three dose levels (1125, 1250 and 1375 μg/ml) for 4 hours in the presence of S9, washed and reincubated for a 16 hours before harvest in the normal way. Two sets of cultures were exposed, one set as in the earlier experiments and the second set with the addition of Hepes buffer to normalise the pH. Initial pH values were recorded and cell counts taken at cell harvest. The solvent control and 1250 μg/ml cultures were evaluated for chromosome aberrations and mitotic index.

DOSING
- preliminary toxicity test: 0-5000 mg/l
- Experiment 1: 312.5-937.5 mg/l without S9; 312.5-1250 mg/l with S9
- Experiment 2:156.25-625 mg/l without S9; 625-1250 mg/l with S9
- Confirmatory experiment: 1125, 1250, and 1375 mg/l with S9, both with and without Hepes buffer
- Number of replicates: 2

Rationale for test conditions:

The dose range for Experiment 1 was selected on the results of a preliminary toxicity test and was 312.5 to 937.5 μg/ml for the treatments without metabolic activation and 312.5 to 1250 μg/ml for the treatments with metabolic activation for both the 12 and 20-hour treatments. Experiment 2 used a dose range similar to Experiment l but with slight changes to gain additional information. The dose range used was 156.25 to 625 μg/ml for treatments without metabolic activation and 625 to 1250 μg/ml with metabolic activation for both 12 and 20-hour treatments. A further confirmatory experiment was performed using dose levels of 1125, 1250 and 1375 μg/ml with S9 and both with and without the addition of Hepes buffer to normalise the pH.

Evaluation criteria:

Positive responses were also recorded if the% cells with aberrations (gaps excluded) was statistically significantly greater than the concurrent control level, even if it was below historical levels, but only if there was an indication of a dose response. However, consideration is given to a number of factors, such as the frequency of chromosome exchange events which are comparatively rare in control cultures, and the ultimate designation must rely upon experience and sound scientific judgement (UKEMS Guidelines for Mutagenicity Testing, 1983).

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >= 2500 ug/ml (+ S9); >= 1250 ug/ml (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CLASSIFICATION OF CHROMOSOME STRUCTURAL ABERRATIONS
GAPS
Gaps are small areas of the chromosome which are unstained. The chromatids remain aligned as normal and the gap does not extend along the chromatid for a distance greater than the width of a chromatid. If the gap occurs on one chromatid only it is a chromatid gap (g). If a gap appears in both chromatids at the same position it is a chromosome gap (G).

CHROMATID BREAKS
Chromatid breaks (ct) vary in appearance. The chromatid may remain aligned but show a gap which is too large to classify as a gap. Alternatively, the chromatid may be broken so that the broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid fragment (f) should be scored if the chromosome of origin cannot be identified. Very small fragments are scored as minutes (m).

CHROMOSOME BREAKS
Chromosome breaks (CS) are breaks in both chromatids of the chromosome. A fragment with two chromatids is formed and this may be displaced by varying degrees. Breaks are distinguished from gaps by the size of the unstained region. A chromosome break is scored if the fragment is associated with a chromosome from which it was probably derived. However, fragments are often seen in isolation and are then scored as chromosome fragments (F). Very small fragments are scored as minutes (M).

EXCHANGE
Exchanges are formed by faulty rejoining of broken chromosomes and may be of the chromosome or chromatid type. Chromatid exchanges (c/c,r) have numerous different forms but are generally not further classified. Where multiple exchanges have occurred each exchange point is counted as one chromatid exchange. Chromosome exchanges generally appear as either a dicentric (D) or a ring (R) form, either of which can be associated with a fragment, which if possible should be scored as part of the exchange.

MULTIPLE ABERRATIONS
If many aberrations are present in one metaphase, the exact details may not be scorable. This is particularly the case when chromosome pulverisation occurs. If the number of aberrations is 10 or more then the cell is classified as X.

CHROMOSOME NUMBER
If the chromosome (centromere) number is between 18 and 22 inclusive then it is classified as a diploid cell and scored for aberrations. If less than 18 chromosomes are counted then the cell is ignored under the assumption that some chromosomes may have been lost for technical reasons. If greater than 22 but less than 26 chromosomes are scored then the count is recorded and the cell classified as an aneuploid cell. Cells with greater than 25 chromosomes are classified as polyploid cells. Aberrations are not scored in non-diploid cells. If the chromosomes are arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell is scored as endoreduplicated (E) and included in the total of polyploid cells.

GENOTOXIC EFFECTS:
- With metabolic activation: highly statistically significant, but only at maximum dose level and 20 hour treatment. This was demonstrated to be an artefact caused by the interaction of a high pH value and the S9 metabolic activation system.
- Without metabolic activation: no
- Positive controls: positive

MITOTIC INDEX: mean of 2 counts each
- WITH METABOLIC ACTIVATION:
-Experiment 1:
0 mg/l, 12 h: 7.15; 20 h: 9.5
312.5 mg/l, 12 h: 7.8; 20 h: 9.25
625 mg/l, 12 h:10.75; 20 h:10.65
1250 mg/l, 12 h: 4.8; 20 h: 6.15
-Experiment 2:
0 mg/l, 12 h: 9.15; 20 h: 8.8
625 mg/l, 12 h: 4.3; 20 h:11.5
937.5 mg/l, 12 h:10.0; 20 h: 7.35
1250 mg/l, 12 h: 3.4; 20 h: 2.85
- WITHOUT METABOLIC ACTIVATION:
- Experiment 1:
0 mg/l, 12 h: 7.45; 20 h: 3.8
312.5 mg/l, 12 h: 8.65; 20 h: 3.75
625 mg/l, 12 h: 6.45; 20 h: 4.75
937.5 mg/l, 12 h: 2.0; 20 h: 2.3
- Experiment 2:
0 mg/l, 12 h: 9.55; 20 h: 9.8
156.25mg/l, 12 h: 8.35; 20 h:12.3
312.5 mg/l, 12 h: 7.6; 20 h: 8.7
625 mg/l, 12 h: 6.9; 20 h: 6.8

CHROMOSOMAL ABERRATIONS (cells with aberrations - gaps):
mean of 2 replicates each
- WITH METABOLIC ACTIVATION:
- Experiment 1:
0 mg/l, 12 h: 0; 20 h: 1
312.5 mg/l, 12 h: 2; 20 h: 0
625 mg/l, 12 h: 2; 20 h: 1
1250 mg/l, 12 h: 4; 20 h: 14
- Experiment 2:
0 mg/l, 12 h: 1; 20 h: 1
625 mg/l, 12 h: 2; 20 h: 4
937.5 mg/l, 12 h: 3; 20 h: 4
1250 mg/l, 12 h: 2; 20 h: 21
- Confirmatory experiment
0 mg/l, 20 h: 4 (with hepes buffer)
0 mg/l, 20 h: 7 (without hepes buffer)
1250 mg/l, 20 h: 6 (with hepes buffer)
1250 mg/l, 20 h: 13 (without hepes buffer)
- WITHOUT METABOLIC ACTIVATION:
- Experiment 1
0 mg/l, 12 h: 2; 20 h: 0
312.5 mg/l, 12 h: 1; 20 h: 4
625 mg/l, 12 h: 1; 20 h: 6
937.5 mg/l, 12 h: 5; 20 h: 1
- Experiment 2
0 mg/l, 12 h: 3; 20 h: 3
156.25mg/l, 12 h: 2; 20 h: 2
312.5 mg/l, 12 h: 3; 20 h: 3
625 mg/l, 12 h: 3; 20 h: 6

CYTOTOXIC CONCENTRATION
- With metabolic activation:
total absence of metaphase cells at >= 2500 mg/l
- Without metabolic activation:
total absence of metaphase cells at >= 1250 mg/l
- A dose-related increase was observed. Since addition of a buffer in the confirmatory experiment reduced toxicity, pH seems to be decisive for cytotoxicity

HISTORICAL ABERRATION RANGES FOR VEHICLE AND UNTREATED CONTROL CULTURES
Many experiments with the CHO cell line have established a range of aberration frequencies acceptable for control cultures, these are presented below:
i) % Diploid cells with aberrations (gaps included)
Untreated controls , range: 3 - 14
DMSO controls, range: 2 - 10
Combined, range: 2 - 14
S9 controls, range: 2 - 6
S9/DMSO controls, range: 1 - 10
Combined, range: 1 - 10
All controls, range: 1 - 14
ii) % Diploid cells with aberrations (gaps excluded)
Untreated controls, range: 1 - 10
DMSO controls, range: 0 - 7
Combined, range: 0 - 10
S9 controls, range: 1 - 3
S9/DMSO controls, range: 0 - 9
Combined, range: 0 - 9
All Controls, range: 0 - 10

Remarks on result:

other: strain/cell type: CHO (Chinese hamster ovary) cells

no further remarks

Conclusions:

The test substance is considered to be non-clastogenic to CHO cells in vitro under conditions of this study.

Executive summary:

Chinese hamster ovary (CHO} cells, treated with the test item, were evaluated for chromosome aberrations at three dose levels, in duplicate, together with negative and positive controls. Three treatment conditions were used. Exposure was for 4 hours with the addition of an induced rat liver homogenate metabolising system at 10% in standard cofactors with cell harvest at 12 hours and 20 hours after initiation of exposure. Exposure in the absence of metabolic activation was continuous with cell harvest at 12 hours and 20 hours after culture initiation.

The negative (solvent) controls gave frequencies of aberrations within the range expected for the CHO cell line. Both of the positive control chemicals gave significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
The test item demonstrated a highly statistically significant increase in the frequency of cells with aberrations at the maximum dose level in the 20-hour with metabolic activation treatment only. However, this was demonstrated to be an artefact caused by the interaction of a high pH value and the S9 metabolic activation system. The test item was shown to be non-clastogenic to CHO cells in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In this micronucleus assay with NMRI mice according to OECD TG 474, 5 male/female animals per group were orally administered single doses of 50, 150, or 500 mg/kg bw isophorone diamine. The highest dose was considered the maximum tolerable dose (MTD) based on the induction of toxic effects without major effects on survival within 72 hours of test substance administration. Isophorone diamine treatment did not result in an increase in the number of micronucleated polychromatic erythrocytes (PCE), nor did it negatively affect the PCE/NCE ratio. Therefore, isophorone diamine is considered to be non-mutagenic under the conditions of this micronucleus assay (Cytotest Cell Research 1990).

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:

1990-04-17 - 1990-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

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 ORGANISMS:
- Supplier: BRL Tierfarm Füllinsdorf (Switzerland)
- Age: minimum 10 weeks + 5 days acclimatization
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: single, Makrolon Type I, with wire mesh top
- Diet: ALTROMIN standard diet, ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12h / 12h

Route of administration:

oral: gavage

Vehicle:

aqua dest.

Details on exposure:

PRE-EXPERIMENT FOR TOXICITY
500 mg/kg bw was estimated to be the maximum tolerated dose, cytotoxic reactions were not observed

TREATMENT
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

doses: 0, 50, 150, 500 mg/ kg b.w.
positive control: cyclophosphamide, dissolved in physiol. saline, 40 mg/kg bw
negative control: vehicle

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

24, 48 or 72 hours

Remarks:

Doses / Concentrations:
50, 150, or 500 mg/kg, dissolved in 10 ml/kg bw dose volume
Basis:
actual ingested

No. of animals per sex per dose:

6 per dosage group and sex with 3 cases of post-treatment duration for negative control and treated groups totals 4 x 3 + 1 groups x 6 animals x 2 sexes = 156 animals; only 5 animals per group and sex were evaluated

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide, dissolved in physiol. saline, 40 mg/kg bw

Tissues and cell types examined:

femora, bone marrow

Details of tissue and slide preparation:

PREPARATION OF THE ANIMALS::
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grilnwald (MERCK, D-6100 Darmstadt, F.R.G.) /Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

ANALYSIS OF CELLS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed as normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be
considered together.

Statistics:

Statistical significance at the five per cent level (p < 0. 05) was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Maximum tolerated dose determined in pre-experiment

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

EFFECT ON PCE/NCE RATIO: not affected (PCE = polychromatic erythrocytes, NCE = normochromatic erythrocytes)
GENOTOXIC EFFECTS:
In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at preparation intervals 48 hours and 72 hours. The mean values of micronuclei observed after treatment with Isophorondiamine (50, 150, and 500 mg/kg b.w.) were in the same range as compared to the corresponding negative control groups. At preparation interval 24 hours a statistically significant increase of the micronucleus frequency after administration of 500 mg/kg b.w. Isophorondiamine was proved by biometric analysis ( p< 0.05, Mann-Whitney test).
This statistical significance is considered to be of minor importance:
1. The corresponding actual negative control rate in this study was very low (0.02%). The mean historical negative control value obtained within the last 12 months was 0.073 %. The range was 0.04% - 0.12%.
2. The frequency of 0.10 % PCEs with micronuclei after treatment with 500 mg/kg b.w. Isophorondiamine is within the range of the historical control data presented.
3. The value of 0.10 deviates not substantially from the obtained at preparation intervals 48 h (0.06 %) and (0.09 %).

Therefore, the statistical significance is not considered to be an indication for an induced mutagenic effect due to the test article.
40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase in induced micronucleus frequency.

--------------------------------------------------------
Treatment           Time   % Micron. in PCE    PCE/NCE
--------------------------------------------------------
 Vehicle            24 h     0.02 (0 - 2)    1000 / 970
  50 mg TS/kg bw    24 h     0.07 (0 - 2)    1000 / 917
 150 mg TS/kg bw    24 h     0.06 (0 - 2)    1000 / 892
 500 mg TS/kg bw    24 h     0.10 (0 - 3) *  1000 / 908
  40 mg CPA/kg bw   24 h     1.08 (2 -22) *  1000 /1127
 Vehicle            48 h     0.04 (0 - 1)    1000 / 699
  50 mg TS/kg bw    48 h     0.06 (0 - 2)    1000 / 676
 150 mg TS/kg bw    48 h     0.06 (0 - 3)    1000 / 702
 500 mg TS/kg bw    48 h     0.09 (0 - 3)    1000 / 781
 Vehicle            72 h     0.14 (0 - 5)    1000 / 744
  50 mg TS/kg bw    72 h     0.07 (0 - 3)    1000 / 681
 150 mg TS/kg bw    72 h     0.13 (0 - 4)    1000 / 754
 500 mg TS/kg bw    72 h     0.16 (0 - 4)    1000 / 758
--------------------------------------------------------
TS = test substance; CPA = cyclophosphamide; * p<0.05

Conclusions:

It can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

The test articlewas assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test article was dissolved in aqua dest .. This solvent was used as negative control. The volume administered orally was 10 ml/kg body weight (b.w.). 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were
collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei in PCEs. Per animal 1000 PCEs
were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as number of NCEs per 1000 PCEs.
The following dose levels of the test article were investigated:
24 h preparation interval: 50, 150 and 500 mg/kg b.w.
48 h preparation interval: 50, 150 and 500 mg/kg b.w.
72 h preparation interval: 50, 150 and 500 mg/kg b.w.
In pre-experiments 500 mg/kg b.w. was estimated to be the maximum tolerated dose. The animals expressed toxic reactions.
The ratio of normochromatic to polychromatic erythrocytes was not affected by the treatment with Isophorondiamine, indicating that the test article had no cytotoxic properties at the dose levels tested.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Studies in animals

In vivo studies

Cited from SIAR for SIAM 18 (Paris, April 2004):

"In a micronucleus assay with NMRI mice according to OECD TG 474, 5 male/female animals per group were orally administered single doses of 50, 150, or 500 mg/kg bw 3-aminomethyl-3,5,5- trimethylcyclohexylamine. The highest dose was considered the maximum tolerable dose (MTD) based on the induction of toxic effects without major effects on survival within 72 hours of test substance administration. Sampling times were 24, 48, and 72 hrs after test substance administration. 3-Aminomethyl-3,5,5-trimethylcyclohexylamine treatment did not result in an increase in the number of micronucleated polychromatic erythrocytes (PCE), nor did it negatively affect the PCE/NCE ratio (Cytotest Cell Research, 1990). This result was confirmed in another study with a slightly different design (Hüls AG, 1988a). Here a single dose of 100 mg/kg bw was administered, which – based on the absence of cytotoxic effects – was determined as the MTD. Sampling times were 24, 48, and 72 hrs after test substance administration."

In vitro studies

Cited from SIAR for SIAM 18 (Paris, April 2004):

"In an Ames test performed according to Directive 84/449/EEC B.14 (1984) with Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, test substance concentrations of up to 5,000 μg/plate were employed in the presence and absence of Aroclor 1254-induced rat liver S9 mix. At non-toxic test substance concentrations, a significant increase in mutant frequency was not observed (Hüls AG, 1990). The same result was obtained in another test with comparable design (Hüls AG, 1988b; method according to the original publication by B. Ames, 1975).

In a HPRT test with Chinese Hamster ovary (CHO) cells according to OECD TG 476 (1984), 3- aminomethyl-3,5,5-trimethylcyclohexylamine concentrations of 20 - 2,000 mg/l (+/- S9 mix from Aroclor 1254 induced rat livers) did not significantly increase the mutant frequency of treated cells. Cytotoxicity was not observed at any of the concentrations tested (Hüls AG, 1992c). In a cytogenetic assay with Chinese Hamster Ovary (CHO) cells (according to OECD TG 473), concentrations of 156.25 – 1,375 mg/l (+/-S9 mix from Aroclor 1254-induced rat livers) were employed. A significant, test compound related increase in chromosomal aberrations was not observed (Safepharm, 1992)."

Justification for classification or non-classification

Because of the results of the in vitro and in vivo mutagenicity studies the substance isophorone diamine is not classified according to CLP regulation (1272/2008).