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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:
1984-03-07 to 1984-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984
Reference Type:
publication
Title:
Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone
Author:
O'Donoghue JL, Haworth SR, Curren RD, Kirby PE, Lawlor T, Moran EJ, Phillips RD, Putnam DL, Rogers-Back AM, Slesinski RS and Thilagar A
Year:
1988
Bibliographic source:
Mutat. Res. 206, 149-161

Materials and methods

Principles of method if other than guideline:
Method: Micronucleus Cytogenetic Assay
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Isophorone of Exxon Corp., > 97 % pure
- pale yellow liquid;
- stored at room temperature blanketed with nitrogen

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Age: 6-8 weeeks
- Source: Charles River Breeding Laboratories, Kingston, NY (USA)
- Weight at randomization: males 33-36 g, females 22-29 g
- No. of animals per dose: 5 males + 5 females per dose and sampling  time; 1 replacement animal per treated group
- Diet: ad libitum
- Water: ad libitum
- Housing: up to six animals per cage in plastic autoclavable cages with wire lids
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3 ° C
- Humidity (%): 50 +/- 20 %
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
corn-oil
Details on exposure:
ADMINISTRATION: 
- Vehicle: corn oil
- Control groups and treatment:   
negative: vehicle   
positive: 0.25 mg triethylenemelamine (TEM, CAS RN 51-18-3)/kg bw,  24  hours
- Frequency of treatment: single application
- Total volume applied: 10 ml/kg bw
- Duration of test: 12; 24; 48 hours
- Sampling times and number of samples: 12: 24; 48 h post dosing
Duration of treatment / exposure:
1 injection
Frequency of treatment:
1 time
Post exposure period:
12; 24; 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
498 mg/kg bw
Basis:
other: test dose was the LD20 of the test substance, determined in a preliminary toxicity study
No. of animals per sex per dose:
test substance: 5
vehicle control: 5
positive control: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
0.25 mg triethylenemelamine (TEM, CAS RN 51-18-3)/kg bw,  24  hours

Examinations

Tissues and cell types examined:
polychromatic erythrocytes of the bone marrow from femur
Details of tissue and slide preparation:
- Criteria for selection of M.T.D.: LD20
EXAMINATIONS: 
- Clinical observations: after dose administration
- Organs examined: femur bone marrow   
- Preparation: At sample collection, the femur was exposed and bone marrow aspirated into a syringe containing fetal calf serum. The cells was
washed, centrifuged, resuspended and smears prepared. May-Gruenwald-Giemsa stain was used to stain the bone marrow prparations.
- 1000 PCE (polychromatic erythrocytes) per animal were analysed for micronuclei; micronucleated normocytes were also counted
Evaluation criteria:
- Criteria for evaluating results: Statistically significant increase  (1-way analysis of variance and Duncan's multiple range test; P <= 0.05)  in 
micronucleated PCE, assessed considering reproducibility and sound science
Statistics:
t-test statistics

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
No significant increase in the frequency of micronucleated polychromatic erythrocytes over the control was found with any group treated  with the test substance.
Toxicity:
yes
Remarks:
Heavy sedation immediately following test substance administration, no other clinical signs.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- Main test: 3/18 dosed males and 3/18 dosed females died.
CLINICAL SIGNS: Heavy sedation immediately following test substance administration, no other clinical signs.

-see: " Remarks on results including tables"

Any other information on results incl. tables

MORTALITY: 
- Dose finding: Dead/total animals within 14 days
--------------------------------------------------------
  ml/kg bw    mg/kg bw    Males    Females    Total
--------------------------------------------------------
   0.00           0        0/5       0/5        0/10
   0.42         387        1/5       0/5        1/10
   0.46         424        0/5       0/5        0/10
   0.50         461        0/5       1/5        1/10
   0.52         479        0/5       1/5        1/10
   0.54         498        1/5       0/5        1/10
   0.56         516        2/5       2/5        4/10
   0.58         534        0/5       3/5        3/10
   0.60         553        2/5       3/5        5/10
   0.62         571        2/5       2/5        4/10
   0.64         590        3/5       4/5        7/10
--------------------------------------------------------
  LD20 = 0.54 +- 0.02 ml/kg bw
- Main test: 3/18 dosed males and 3/18 dosed females died.
CLINICAL SIGNS: Heavy sedation immediately following test substance  

administration, no other clinical signs.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: insignificant in all groups  

including positive control
GENOTOXIC EFFECTS: 
For the positive control a significant increase in the frequency of  

micronucleated polychromatic erythrocytes was observed. 

No significant  increase over the control was found with any group treated 

with the test  substance.
--------------------------------------------------------
Treatment     Sex   Time   Micron. PCE/1000PCE    % PCE
--------------------------------------------------------
Neg. contr.    m    12 h       0.6 +- 0.5           51
Neg. contr.    f    12 h       1.2 +- 1.3           51
Neg. contr.    m    24 h       0.8 +- 1.1           50
Neg. contr.    f    24 h       0.8 +- 0.8           51
Neg. contr.    m    48 h       0.8 +- 0.4           50
Neg. contr.    f    48 h       1.2 +- 1.1           51
Test subst.    m    12 h       1.4 +- 0.5           50
Test subst.    f    12 h       1.2 +- 1.3           51
Test subst.    m    24 h       0.4 +- 0.5           50
Test subst.    f    24 h       0.8 +- 1.1           50
Test subst.    m    48 h       1.4 +- 0.5           51
Test subst.    f    48 h       1.2 +- 1.3           52
Pos. contr.    m    24 h      37.6 +- 4.3 **        50
Pos. contr.    f    24 h      35.4 +- 3.7 **        52
--------------------------------------------------------
% PCE refers to total erythrocytes; * P<0.05; ** P<0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative No significant increase in the frequency of micronucleated polychromatic erythrocytes over the control was found with any group treated  with the test substance.
The results of this study indicate that under the test conditions, isophorone did not induce micronucleated polychromatic erythrocytes in male and
female mice.
Executive summary:

In this in vivo mouse micronucleus assay, 498 mg/kg isophorone was administered i.p. to 5 male and 5 female CD-1 mice per group. This dose was selected as the maximum tolerated dose (= LD20 = MTD) based upon a preliminary toxicity study. Bone marrow polychromatic erythrocytes, collected 12, 24, and 48 hours after single treatment, were examined microscopically for micronucleated polychromatic erythrocytes (PCE). No significant increase in the frequency of PCE over the control was found with any group treated  with the test substance. For the positive control (triethylene melamine, TEM) a significant increase in the frequency of PCE was observed.

Therefore, the conclusion is drawn, that isophorone is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female CD-1 mice.