Registration Dossier

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:
14 - 17 Mar 1988 and 13 - 16 Feb 1989 (repetition of 72 h test point in females)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997
Deviations:
yes
Remarks:
samples of bone marrow was taken beyond 48h; only one dose level was used for the first sampling time (three dose levels in the range from the maximum to little or no toxicity should be used)
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1983
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1984
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: BOR:NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: males 24-32 g, females 21-26 g
- Fasting period before study: 16 h before treatment
- Housing: individually in Macrolon cages, type II
- Diet: ssniff(R) M, "Special diet for Mice", ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Mar 1988 To: 17 Mar 1988 AND From: 13 Feb 1989 To: 16 Feb 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The test material was diluted with demineralized water.
Details on exposure:
EXPERIMENTAL DESIGN:
Phase I: orientating study with doses of 2150, 2610 and 3160 mg TMT 15/kg bw
Phase II: main micronucleus test (dosing of test group (2870 mg TMT 15/kg bw), vehicle control group and positive control group)

PREPARATION OF DOSING SOLUTIONS:
The test material was diluted with demineralized water.
The positive control material (cyclophosphamide) was freshly dissolved in isotonic saline solution.
The negative control material was isotonic saline solution (0.9%).

Amount of vehicle: test material: 21.5 mL/kg bw; negative resp. positive control material: 10.0 mL/kg bw (the higher administration volume for the test material was chosen because of the local irritating effect on the gastric mucosa, as known from the orientating study)

Duration of treatment / exposure:
not applicable
Frequency of treatment:
single oral dose
Post exposure period:
24, 48 and 72 hours after treatment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2870 mg/kg bw
Basis:
other: test material TMT 15 in vehicle
Remarks:
Doses / Concentrations:
487.9 mg anhydrous TMT/kg bw
Basis:
other: recalculated dose based on 17% TMT (analytical purity)
No. of animals per sex per dose:
Test group: 21 males, 21 females (repetition group: 15 females)
Negative control group: 18 males, 18 females (repetition group: 6 females)
Positive control group: 18 males, 18 females (repetition group: 6 females)
Control animals:
yes
Positive control(s):
Cyclophosphamide (freshly dissolved in isotonic saline solution (0.9%))
- Route of administration: oral (gavage)
- Dose / concentration: 51.1 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow, immature erythrocytes
Details of tissue and slide preparation:
Removal of both femurs from each mouse; bone marrow cells flushed into labelled centrifuge tube with approx. 1.5 mL of calf serum; centrifugation at 1000 rpm for 5 minutes; supernatant serum discarded and bone marrow cells suspended upon a thin layer of serum; small drop of marrow serum suspension smeared on a slide and allowed to dry overnight.
Evaluation criteria:
If a test material produced no stat. sign. and reproducible positive response at any of the test points compared to the negative control group, it was considered non-mutagenic in this system (significance level: 5%, p<=0.05).
Statistics:
Poisson test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
An orientating study of the acute toxicity of the test material was performed with concentrations of 2150, 2610, 3160 mg TMT 15/kg bw. All animals receiving 3160 mg TMT 15/kg bw died. Animals administered 2610 mg TMT 15/kg bw showed an indistinct pattern of intoxication, whereas in animals administered 2150 mg TMT 15/kg bw no clinical symptoms were observed. Therefore 2870 mg TMT 15/kg bw was considered to the maximum tolerated dose (males and females).

RESULTS OF DEFINITIVE STUDY
After single administration of the test material at 2870 mg TMT 15/kg bw by gavage, no significant test material-related increase in micronucleated polychromatic erythrocytes was observed in either male or female animals, respectively males and females combined, when compared with corresponding negative control group animals. These results were found at each of the three examination times, 24, 48 and 72 h after administration, respectively. A reduction in the ratio of polychromatic to normochromatic erythrocytes occurred in both single male and female test group animals at all sampling times. The positive control group animals, which received cyclophosphamide, exhibited a significant increase in the number of micronucleated polychromatic erythrocytes and thus validated the test system.

Any other information on results incl. tables

Mortality:

Seven animals of the main test group administered the test substance died. Two females died on the second day after administration and two males and three females on the third day after test substance administration, respectively. Due to the high mortality in females, a repetition group containing 15 females was implemented. Four mice of the repetition group died on day 2 or three after administration of the test substance. Necropsy of the dead animals revealed reddening of the glandular stomach and/or the small intestine and filling of the small intestine with yellowish or black-reddish fluid.

 

Clinical signs:

Animals receiving the test substance exhibited hypokinesia (slight to moderate, 6/21 males, 2/36 females), clonic convulsions (slight to moderate, 5/21 males, 12/36 females), decrease of muscle tone (1/21 males, 2/36 females), tear formation (1/21 males, 1/36 females), piloerection (5/21 males, 11/36 females) and reduced body temperature (2/21 males, 3/36 females). Restricted to females were clinical signs as tremor (moderate, 4/36), tonic convulsions of the hind legs (3/36), stilted gait (1/36), ptosis (4/36), sunken sides (3/36), loss of righting, pinna, pain and corneal reflex (each 1/36). Animals of the negative and positive control group did not show any abnormal clinical signs.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
TMT 15 at the maximum tolerable dose of 2870 mg/kg bw (equivalent to 487.9 mg anhydrous TMT/kg bw) (single oral administration) is non-clastogenic in the reported in vivo mouse micronucleus test under the described conditions.