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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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 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
- Diet: ALTROMIN standard diet, ad libitum):
- Water: tap water ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12h / 12h

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
aqua dest.
Details on exposure:
ADMINISTRATION:
- in a pre-experiment 500 mg/kg bw was estimated to be the maximum tolerated dose, cytotoxic reactions were not observed
- Sampling times and number of samples: 24, 48, or 72 hours after treatment
- Control groups and treatment:
negative: vehicle
positive: cyclophosphamide, dissolved in physiol. saline, 40 mg/kg bw
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:
- 1000 PCE (polychromatic erythrocytes) per animal were analysed for micronuclei
Evaluation criteria:
- Criteria for evaluating results:
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
- Criteria for selection of M.T.D.: toxic reactions without major effects on survival within 72 hours
Statistics:
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:
For the positive control a significant and clear increase in the frequency of micronucleated polychromatic erythrocytes was observed.
The mean frequencies of micronucleated polychromatic erythrocytes in the groups treated with the test item were in the same range as those of the vehicle control group. A statistically significant increase was observed at the high dose (500 mg/kg bw) at 24 hours. However, the negative control in this case was untypically low (0.02 %). The absolute result for the dosed group (0.10 %) was well within the range of the 24 hour negative controls from the preceding 12 months (0.04 - 0.12; mean 0.073 %), i.e. there was no biological relevance.

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:
Therefore, the conclusion is drawn, that Isophorone diamine is not a mutagenic substance under the in vivo conditions in this micronucleus assay
using male and female NMRI mice.
Executive summary:

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. Sampling times were 24, 48, and 72 hrs after 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.