3,5,5-trimethylhexanal

Genetic toxicity: in vivo

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:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylhexanal

EC Number:

226-603-0

EC Name:

3,5,5-trimethylhexanal

Cas Number:

5435-64-3

Molecular formula:

C9H18O

IUPAC Name:

3,5,5-trimethylhexanal

Details on test material:

Date of production: February/March 1996
Properties: colourless, liquid, homogenous
Stability: > 1 year
ph: not determinate
Purity: 91,2 mass-% (GC-analysis)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

young, adult animals with a bodyweight within 31.0+/-6.2 grams (male) and 24.5+/-4.9 grams (female)
Identification: color code applied to the back of each animal, cage cards
Housing: conventional, 5 animals/sex/Makrolon cage Type III
Room temperature: 22 +/- 3 ° C
Rel. humanidity: 30 - 70 %
(remark: eventual short periods of higher humidity due to room cleaning procedures generally do not affect the outcome of the study)
Air supply: approx. 15 changes of air per hour
Lighting: artificial light for 12 hours per day
Water: drinking water ad libitum
Diet: Sniff R 10 - Alleindiät für Ratten (Sniff Spezialfutter GmbH, Soest, Germany), the animals were allowed free access to the diet
expect for a period approx. 16 - 18 hrs prior to the dosing procedure

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Male and female mice received a single oral dose of either 2000 mg/kg Trimethylhexanal, 100 mg/kg Cyclophosphamide or 10 ml/kg b.w. corn oil

Duration of treatment / exposure:

48 h

Frequency of treatment:

single dose

Post exposure period:

Erythrocyte preparations were obtained from the negative and test compound groups at two sampling times, 24 and 48 hours after dosing.
Erythrocytes from the positive control group were prepared 24 hours after dosing only

No. of animals per sex per dose:

5 male and 5 female animals

Positive control(s):

The positive control, Cyclophosphamide, caused significant increases in the frequencies of micronucleated polychromatic erythrocytes of male and
female mice, thus demonstrating the sensitivity of the test system to clastogenic agents.

Examinations

Tissues and cell types examined:

Accoding to the group assignment, animals were killed by cervical dislocation 24 and 48 hours after test compound administration.
Both femurs were dissected out from each animal, cleared of tissue and one epiphysis removed from each bone.

Details of tissue and slide preparation:

The bone marrow was suspended in fetal calf serum (FCS) and erythrocytes were purified by means of a cellulose chromatography column (8).
The eluate (3 x 1.5 ml) was centrifuged (5 min, 750 x g) and the pellet was again suspended in FCS/EDTA. This pure erythrocyte suspension was used to prepare "flat" cells on glass slides by means of a Shandon Cytospin 3 (2 slides per animal). Slides were air dried and stained with
May-Grünwald/Giemsa.

Evaluation criteria:

Means and standard deviations of the following parameters were calculated for each treatment group
the number of PCE with micronuclei, the number of NCE with micronuclei, the PCE/NCE ratio.
A test compound is considered an inducer of micronuclei in PCE, if at least one group of mice treated with the test compound reveals a statistically (as compared to the negative control group of the same sampling time). and biologically relevant increase in micronucleated PCE.

Statistics:

Statistical analysis of the data was performed using the "Statgraphics" statistical software package. The compound groups were compared with
negative controls of the same sampling time. The first stage of this analysis is to test for evidence of heterogeneity between animals of the same
dose group. This is done by calculating the heterogeneity x2 for the diffences between the proportions of micronuclei for each animal. In case all
the groups are homogeneous, comparisons can be made between the control and test groups using Pearson's contingency 2 x 2 x2 test with one
degree of freedom and inclusing a Yates correction factor. In the case on inhomogeneity between groups, after transformation of the data, a
one-sided two-sample t-test ist performed. The t-test is also used to compare the micronucles frequencies of the positive control with that of negative control groups and for comparison of PCE/NCE ratios.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

signs of toxicity but no deaths

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Treatment with the test compound did result in clincial symptoms in all animals, indicating that TRIMETHYLHEXANAL was absorbed from the gastro-
intestinal tract into the blood. As there is no known barrier between blood and bone marrow, it must be concluded that TRIMETHYLHEXANAL reached
the target organ ot this test system (the bone marrow). This can also be concluded from the reduction of the PCE/NCE ratio in female animals of the 48 hrs sampling time, which is indicative of test compound induced bone marrow toxicity.
In female mice, TRIMETHYLHEXANAL did not exhibit any mutagenity resp. clastogenicity detectable as a significant increase in micronucleated
polychromatic erythrocytes.
In male mice of the 24 hrs sampling time, there was a weak but statistically significant increase in the number of micronucleated PCEs (0.23 +/- 0.03) as compared to the concurrent negative control (0.08+/- 0.03). The statistical significance was only achieved, because the micronucleus frequency
of the negative control was unusually low. Compared to the histroical negative control value of this laboratoy (0.20 +/- 0.10) a frequency of 0.23 is
clearly within the range of normal assay variation and can not be taken as being indicative of any clastogenic potential of the test compound.
Therefore, a biological relevance ot his increase seems not to be given.
Cyclophosphamide, a known clastogen induced significant increases in micronucleated PCE, demonstrating the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: non-clastogen under in vivo-conditions.
Based on the results of this study, TRIMETHYLHEXANAL is considered a non-clastogen under in-vivo conditions.

Executive summary:

In the in vivo mouse micronucleus assay, TRIMETHYLHEXANAL was tested for its potential to induce micronuclei in polychromic erythrocytes (PCE) of NMRI mice.

In a preliminary toxicity test, 2000 mg/kg of TRIMETHYLHEXANAL were determined as the maximum tolerable dose.

In the main study, 2000 mg of TRIMETHYLHEXANAL/kg bodyweight were administered to male and female mice as a single oral dose (gavage). The negative control group received the vehicle, corn oil. Animals of the positive control group were administered cyclophosphamide, at 100 mg/kg bodyweight. Administration of each control substance was done by oral gavage, too. Five animals/sex/dose/sampling time were employed.

Erythrocyte preparations were obtained from the negative and test compound groups at two sampling times, 24 and 48 hours after dosing. Erythrocytes from the positive control group were prepared 24 hours after dosing only. Slides were screened with an automatic image analyzer. One slide per animal was examined for the presence of micronuclei in (if possible) at least 2000 PCEs. The ratio of PCE to normochromatic erythrocyates (NCE) as well as the incidende of micronucleated NCE was determined in parallel. At both sampling times, male and female mice treated with TRIMETHYLHEXANAL did not reveal biologically significant increases in the frequencies of micronucleated PCE.

The positive control compound, cyclophosphamide, induced highly significant increases in the frequency of micronucleated PCE, demonstrating the sensitivity of the test system.

From the results obtained, it is concluded, that TRIMETHXYLHEXANAL shows no glastogenic activity when administered as a single oral dose in this in vivo test system.