Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-06-24 to 1996-09-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1992)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
2,2,4(or 2,4,4)-trimethyl-1,6-hexanediol of Hüls AG
purity 94.6 % (GC-FID area)
batch No. Pt. 15570/3033 of August 1995; sample ID 0637/81 770

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Source: Harlan Winkelmann, Borchen (Germany)
- Age: young adults
- Weight at study initiation:   27.8 +/- 5.6 g (males); 25.9 +/- 5.2 g (females)
- No. of animals per dose: 5 males + 5 females per test duration
- Housing: max 5 animals per sex per cage
- Diet (e.g. ad libitum): Ssniff R 10, complete feed for rats, Ssniff Spezialfutter GmbH, 59494 Soest, Germany
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark rhythm

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle: corn oil
Details on exposure:
ADMINISTRATION: 
- Vehicle: corn oil
- Control groups and treatment:    
negative: vehicle    
positive: 100 mg cyclophosphamide (CPA)/kg bw in 0.9 % aqueous NaCl   additional treated satellite group to replace mortalities
- Total volume applied: 10 ml/kg bw
- Duration of test: 24 hours; 48 hours
- Sampling times and number of samples: 24 hours; 48 hours
Duration of treatment / exposure:
Duration of test: 24 hours; 48 hours
Frequency of treatment:
single dose
Post exposure period:
Sampling times and number of samples: 24 hours; 48 hours
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males + 5 females 
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
positive: 100 mg cyclophosphamide (CPA)/kg bw in 0.9 % aqueous NaCl, oral gavage

Examinations

Tissues and cell types examined:
EXAMINATIONS: 
- Organs examined at necropsy: femur bone marrow; others not specified in  report approx. 2000 PCE (polychromatic erythrocytes) per animal were analysed  for micronuclei  (5000 in re-evaluation of 48 hour vehicle and test  compound slides from males)
- Clinical observations: yes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Criteria for selection of M.T.D.: maximum dose <= 2000 mg/kg bw without  mortalities within 48 hours

TREATMENT AND SAMPLING TIMES:
- 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.
- The bone marrow was suspended in fetal calf serum (FCS) and erythrocytes were purified by means of a cellulose chromatography column.
- The eluate (3 x 1.5 ml) was centrifuged (5 min, 750 x g) and the pellet was again suspended in FCS/EDTA.

DETAILS OF SLIDE PREPARATION:
- The 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 May­Grunwald/Giemsa.

METHOD OF ANALYSIS:
- The MIAMED image analyzer equipped with the "Micronucleus Test V 4.00" software was used for fully automated slide scoring.
- Where possible, a total of approximately 2000 PCE (i.e. 10,000 PCE per treatment group) were analyzed for micronuclei.
- The corresponding NCE were also scored for micronuclei. In addition, as an indicator for chemical-induced bone marrow toxicity, for each animal the PCE/NCE ratio was
determined on the basis of 2000 PCE scored.

Evaluation criteria:
- Criteria for evaluating results: Statistically significant and  biologically relevant increase in frequency of micronucleated  polychromatic erythrocytes of at least one test group as 
compared to the  negative control group of the same sampling time

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MORTALITY: 
- Phase 1 of dose finding = 2000 mg/kg bw: No mortalities within 48 hours  among 5 males + 5 females. No further phases were required.
- Main test: No mortalities
CLINICAL SIGNS: Predominant signs were hunched posture (only males),  slight sedation (only males), piloerection.
NECROPSY FINDINGS: No necropsy reported.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: 
- The average PCE/NCE ratio of the positive control groups was  significantly lower than that of the corresponding vehicle controls 
(0.27  +- 0.09 vs 0.83 +- 0.20 for males, 0.32 +- 0.13 vs 0.96 +- 0.29 for  females).   
- The PCE/NCE ratio of the male 48 hour dosed group (0.45 +- 0.14) was  significantly lower than that of the corresponding vehicle control (0.93  +- 0.30).

GENOTOXIC EFFECTS:    
- The micronucleus frequencies of the negative controls were within the  range of historical control data of the performing laboratory.  - For the positive control a significant increase in the frequency of  micronucleated polychromatic erythrocytes was observed 
(4.78 +- 0.88 vs  0.22 +- 0.09 for males; 5.90 +- 1.03 vs 0.06 +- 0.07 for females). - No significant increase in the frequency of micronucleated  polychromatic erythrocytes over the control was found with the females  treated with the test substance. - In males of the 48 hour sampling time a weak but statistically  significant increase was observed (0.33 +- 0.12 vs 0.12 +- 0.10; 0.001 <  p < 0.01).
- Statistical significance was reduced to 0.01 < p < 0.05 and  the increase in micronucleus frequency was reduced to 0.26 +- 0.08 vs  0.16 +- 0.07 by scoring 5000 instead of 2000 PCE. According to Richold et  al. (1990), the "statistical significance should increase with increased  sample size ... if the response is real and not due to sampling error".  Since an opposite trend was observed, and since in addition the final  micronucleus frequency of 0.26 % is well within the range of historical  negative controls of the performing laboratory (0.00 - 0.40 %; 0.20 +-  0.10), the present observation was not considered of being indicative of  a clastogenic activity of the test compound. -The clinical symptoms indicate that the test substance or its metabolites  had reached the blood and hence the target organ, i.e. the bone marrow.  
Additional evidence for that comes from the reduction of the PCE/NCE  ratio in male animals of the 48 hours sampling time, which is indicative  of of bone marrow toxicity.

Treatment Sex Time % Micron PCE/NCE
in PCE
2000 T.S.   m    24 h    0.25 +- 0.07     0.60 +- 0.26
2000 T.S.   m    48 h    0.33 +- 0.12 **  0.45 +- 0.14 **
2000 T.S.   m    48 h    0.26 +- 0.08 * (1)
2000 T.S.   f    24 h    0.16 +- 0.19     0.76 +- 0.21
2000 T.S.   f    48 h    0.14 +- 0.10     1.21 +- 0.23  
Vehicle    m    24 h    0.22 +- 0.09     0.83 +- 0.20  
Vehicle    m    48 h    0.12 +- 0.10     0.93 +- 0.30  
Vehicle    m    48 h    0.16 +- 0.07   (1)  
Vehicle    f    24 h    0.06 +- 0.07     0.96 +- 0.29  
Vehicle    f    48 h    0.13 +- 0.08     1.47 +- 0.68  
100 CPA    m    24 h    4.78 +- 0.88 *** 0.27 +- 0.09 ***  
100 CPA    f    24 h    5.90 +- 1.03 *** 0.32 +- 0.13 ***
--------------------------------------------------------
T.S. = test substance (trimethylhexanediol; mg/kg bw)
CPA = cyclophosphamide (mg/kg bw)
* p < 0.05; ** p < 0.01; *** p < 0.001
(1) based on 5000 PCE/animal; other data based on 2000 PCE/animal

Any other information on results incl. tables

no other information

Applicant's summary and conclusion

Conclusions:
From the results obtained, it is concluded, that the test item shows no clastogenic activity in this in vivo mouse micronucleus assay.
Executive summary:

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

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

In the main study, 2000 mg of test item/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 (LEITZ Miamed). One slide per animal was examined for the presence of micronuclei in at least 2000 PCEs (5000 PCEs in a re-evaluation of vehicle and test compound slides from the 48 hrs sampling time).

The ratio of PCE to normochromatic erythrocytes (NCE) as well as the incidence of micronucleated NCE was determined in parallel. At both sampling times, male and female mice treated with test item 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 the test item shows no clastogenic activity in this in vivo test system.