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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
EC Number:
220-666-8
EC Name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
Cas Number:
2855-13-2
Molecular formula:
C10H22N2
IUPAC Name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
Constituent 2
Reference substance name:
Isophorone diamine
IUPAC Name:
Isophorone diamine
Details on test material:
Isophorone diamine of Hüls AG, produced 20 March 1990, ID No. 3641/81172; purity 99.85 % (GC)

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

--------------------------------------------------------
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

Applicant's summary and conclusion

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.