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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08/23/1994 (start date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A non-GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data.

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Principles of method if other than guideline:
Deviations from guideline - separation of split dose treatment.
Not mentioned - details on animals and environmental conditions.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): cyclohexene oxide

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
No further information on test animals and environmental conditions.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
no data
Duration of treatment / exposure:
Injections at 3 and 72 hours.
Frequency of treatment:
Injections at 3 and 72 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
19.5, 39.06, 78.13, 156.25, 312.5, 625 and 1250 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 males per dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
Dose: 10 mg/kg
No. test animals: 5

Examinations

Tissues and cell types examined:
Erythrocytes in the bone marrow of the femurs.
Details of tissue and slide preparation:
- Animals were euthanized by CO2 inhalation and the femurs were removed.
- The bone marrow was flushed from the femurs and spread onto slides.
- The slides were air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminated any micronuclei that may be present.
Evaluation criteria:
- Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) were scored per animal for frequency of micronucleated cells in each of test animals.
- In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as an indicator of chemically-induced toxicity.
- In non-treated healthy rats, the %PCE in bone marrow is usually around 50-60%.
- If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the typical normal level.
- Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes may be observed.
Statistics:
A formal statistical analysis of the data was performed that includes a trend test, to determine if there was an overall increase across all doses in the frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group was statistically different from the control group in frequency of micronucleated cells.

- A positive trend test is one in which the P value is equal to or less than 0.025.
- For the slide-based micronucleus data, the micronucleus frequency in any dose group is considered significantly elevated over the control group if the P value is equal to or less than 0.025 divided by the number of chemical-treatment groups.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
All test animals died after receiving 625 and 1250 mg/kg of the test material and one animal died after receiving a dose of 312.5 mg/kg.

Any other information on results incl. tables

  Dose Animal Polychromatic Erythrocytes        
(mg/kg) Number Trend P = 0.638  
    No. Examined Total MN Cells Percent PCE MN Cells  
    per 1000  
Vehicle Control CRO  0      1219 2000 2 40 1  
 
   0      1250 2000 0 45 0  
 
   0      1261 2000 3 32 1.5  
 
   0      1620 2000 1 48.5 0.5  
 
   0      1689 2000 2 40 1  
 
Average ± SEM   41.10 ± 2.79 0.80 ± 0.25  
   
Test Material   19.5    1150 2000 3 36 1.5  
 
  19.5    1161 2000 3 40 1.5  
 
  19.5    1220 2000 3 36 1.5  
 
  19.5    1289 2000 5 41 2.5  
 
  19.5    1419 2000 3 38.5 1.5  
 
Average ± SEM   38.30 ± 1.02 1.70 ± 0.20  
Pairwise P   0.0358  
   
Test Material   39.06   1121 2000 2 42 1  
 
  39.06   1185 2000 1 51.5 0.5  
 
  39.06   1210 2000 1 39 0.5  
 
  39.06   1235 2000 6 57.5 3  
 
  39.06   1284 2000 7 45.5 3.5  
 
Average ± SEM   47.10 ± 3.33 1.70 ± 0.64  
Pairwise P   0.0358  
   
Test Material   78.13   1327 2000 1 42.5 0.5  
 
  78.13   1340 2000 5 40 2.5  
 
  78.13   1382 2000 1 35 0.5  
 
  78.13   1385 2000 4 41 2  
 
  78.13   1390 2000 4 36 2  
 
Average ± SEM   38.90 ± 1.45 1.50 ± 0.42  
Pairwise P   0.0721  
   
Test Material   156.25   1205 2000 2 35.5 1  
 
  156.25   1207 2000 4 45 2  
 
  156.25   1226 2000 4 49 2  
 
  156.25   1277 2000 2 42.5 1  
 
  156.25   1279 2000 2 47.5 1  
 
Average ± SEM   43.90 ± 2.37 1.40 ± 0.24  
Pairwise P   0.1003  
   
Test Chemical   312.5    1181 2000 3 37 1.5  
 
  312.5    1306 2000 1 35.5 0.5  
 
  312.5    1323 2000 2 30 1  
 
  312.5    1426 2000 3 36 1.5  
 
  312.5    1448d 0 0 0 0  
 
Average ± SEM   34.63 ± 1.57 1.13 ± 0.24  
Pairwise P   0.2403  
   
Test Material   625      1320d 0 0 N/A 0  
 
  625      1362d 0 0 N/A 0  
 
  625      1364d 0 0 N/A 0  
 
  625      1371d 0 0 N/A 0  
 
  625      1383d 0 0 N/A 0  
 
Average ± SEM   N/A ± N/A 0.00 ± 0.00  
Pairwise P   *  
   
Test Chemical   1250      1105d 0 0 N/A 0  
 
  1250      1307d 0 0 N/A 0  
 
  1250      1326d 0 0 N/A 0  
 
  1250      1377d 0 0 N/A 0  
 
  1250      1379d 0 0 N/A 0  
 
Average ± SEM   N/A ± N/A 0.00 ± 0.00  
Pairwise P   *  
   
Positive Control CPA 10      1285 2000 28 40 14  
 
  10      1290 2000 25 26.5 12.5  
 
  10      1363 2000 30 31.5 15  
 
  10      1640 2000 26 32 13  
 
  10      1682 2000 28 37 14  
 
Average ± SEM   33.40 ± 2.34 13.70 ± 0.44  
Pairwise P   < 0.0001  
   
Abbreviations:
CRO = Corn Oil
d = dead
* = Insufficient animals scored to calculate p-value.
CPA = Cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The genetic toxicity of the test material was determined to be negative in an in vivo micronucleus study conducted in male Fischer 344 rats.
Executive summary:

The genetic toxicity of the test material was determined to be negative in an in vivo micronucleus study. The study was conducted to a protocol which is similar to the standard guideline OECD 475; there were only minor deviations. Five male Fischer 344 rats per dose were exposed to the test material via intraperitoneal injection. The test material was administered up to the level of toxicity. Under the conditions of the test, the genotoxicity of the test material was determined to be negative.