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:
weight of evidence
Study period:
1991-03-06 to 1991-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TCD Emulgator
- Physical state: solid
- Analytical purity: 95.7%
- Purity test date: 1990-04-26
- Lot/batch No.:90003
- Stability under test conditions: a stability test in the solvent did not detect a relevant change in the percent of active ingredient
- Expiration date of the lot/batch: 1992-01-08
- Storage condition of test material: at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 28-40 g
- Assigned to test groups randomly: yes
- Housing: females in groups of a maximum of three mice, males were kept singly
- Diet (e.g. ad libitum): Altromin 1324 standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23 °C
- Humidity (%): 45-50%
- Air changes (per hr): about 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
TCD Emulsifier was dissolved in corn oil at 40°C in a water bath using a magnetic mixer
Cyclophosphamide was dissolved in deionized water

Duration of treatment / exposure:
negative control: 24 hours
TCD Emulsifier: 16, 24 and 48 hours
Frequency of treatment:
The test substance was administered once.
Post exposure period:
Animals were sacrificed 16, 24 and 48 hours after the administration, and the femoral marrow was prepared.
Doses / concentrations
Remarks:
Doses / Concentrations:
250 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
negative control: 5
TCD Emulsifier: 15 (5 animals per sex for each of the three time points)
Control animals:
yes, concurrent vehicle
Positive control(s):
- positive control: cyclophosphamide
- Route of administration: ip
- Doses / concentrations: 20 mg/kg body weight

Examinations

Tissues and cell types examined:
Polychromatic and normochromatic erythrocytes obtained from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The selection of the TCD Emulsifier dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 250 mg/kg, 350 mg/kg, 500 mg/kg, and 2500 mg/kg TCD Emulsifier.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The volume administered was 5 ml/kg body weight for the negative and treatment groups, and for the positive control group the volume was 10 ml/kg body weight. Each respective substance was administered intraperitoneally once. Animals were sacrificed 16, 24 and 48 hours after the administration, and the femoral marrow was prepared. Negative and positive controls were all sacrificed after 24 hours.

DETAILS OF SLIDE PREPARATION:
At least one intact femur was prepared from each sacrificed animal. The femur was separated from muscular tissue. The lower-leg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g. fine scissors, making visible a small opening in the bone-marrow channel.
A suitable tube was filled with sufficient fetal calf serum. A small amount of serum was drawn from the tube suitable syringe with a thin cannula.
The cannula was pushed into the open end of the marrow cavity. The femur was then completely immersed in the calf serum and pressed against the wallof the tube, to prevent its slipping off. The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension. The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder. The sediment was mixed to produce a homogeneous suspension. One drop of the viscous suspension was placed on a well cleaned slide and spread with a suitable object, to allow proper evaluation of the smear. The labeled slides were dried overnight.
The smears were stained automatically with an Ames HemaTek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water and left to dry. Following this treatment, the smears were transferred to a holder. A cuvette was filled with xylene, into which the holder was immersed for approximately ten minutes. The slides were removed singly (e.g. with tweezers) to be covered. A small amount of covering agent was taken from a bottle with a suitable object (e.g. glass rod) and applied to the coated side of the slide. A cover glass was then placed in
position without trapping bubbles. The slides were not evaluated until the covering agent had dried.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
Evaluation criteria:
A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that range which, according to the laboratory's experience was within the range of negative controls. In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant. In this case, a second test had to be performed at the most sensitive interval.
An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory experience.
Statistics:
The TCD Emulsifier group with the highest mean (provided this superseded the negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronucleated rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2 -test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
treated animals showed signs of toxicity
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 350, 500 and 2500 mg/kg body weight
- Clinical signs of toxicity in test animals:
at 250 mg/kg: apathy, roughened fur, staggering gait, spasm, shivering and difficulty in breathing. In addition, 2 of 5 animals died in the
250 mg/kg group. 3 of 5 animals died in the 350 mg/kg group and all animals died in the 500 mg/kg and 2500 mg/kg groups.

RESULTS OF DEFINITIVE STUDY
- signs of toxicity: animals treated with 250 mg/kg showed the following compound related symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm, twitching, shivering and difficulty in breathing. Their feeding behavior was normal. One treated animals died during the test period, due to the acute toxicity of 250 mg/kg TCD Emulsifier.
- Induction of micronuclei (for Micronucleus assay): negative
- Ratio of PCE/NCE (for Micronucleus assay): the ratio was altered by the treatment with TCD Emulsifier; being 1000: 899 in the negative control, 1000: 1587 in the 16 hours group, 1000: 1716 in the 24 hours group and 1000: 1305 in the 48 hours group

Any other information on results incl. tables

Table 1
Negative Control i.p. application, Sacrifice 24 hours after treament
 
randon number and sex number of evaluated polychromatic erythrocytes number of normochromatic erythrocytes per 1000 PCE micronucleated cells per 1000
normochromatic erythrocytes polychromatic erythrocytes
 
9   1000 975 0 4
14 ♂ 1000 1017 1.0 2
15 ♂ 1000 1419 0.7 1
16 ♂ 1000 457 0 1
28 ♂ 1000 443 0 3
44 ♀ 1000 877 1.1 1
50 ♀ 1000 1030 1.0 2
52 ♀ 1000 605 1.7 1
53 ♀ 1000 1089 0.9 2
56 ♀ 1000 1073 0 6
Mean 1000 899 0.6 2.3
1s   310 0.6 1.6

Table 2
TCD Emulsifier 250 mg/kg i.p. application, Sacrifice 16 hours after treament
 
randon number and sex number of evaluated polychromatic erythrocytes number of normochromatic erythrocytes per 1000 PCE micronucleated cells per 1000
normochromatic erythrocytes polychromatic erythrocytes
 
 13 ♂ 1000 1604 1.2 1
17 ♂ 1000 2488 0.8 1
19 ♂ 1000 843 3.6 2
23 ♂ 1000 1031 1.0 0
25 ♂ 1000 2363 1.3 1
32 ♀ 1000 1339 0 2
39 ♀ 1000 1509 0 2
41 ♀ 1000 1406 2.8 2
46 ♀ 1000 1485 0 0
58 ♀ 1000 1798 0.6 1
Mean 1000 1587 1.1 1.2
1s   520 1.2 0.8

Table 3
TCD Emulsifier 250 mg/kg i.p. application, Sacrifice 24 hours after treament
 
randon number and sex number of evaluated polychromatic erythrocytes number of normochromatic erythrocytes per 1000 PCE micronucleated cells per 1000
normochromatic erythrocytes polychromatic erythrocytes
 
 3 ♂ 1000 2231 2.2 1
20 ♂ 1000 1792 0.6 1
21 ♂ 1000 1550 1.3 0
26 ♂ 1000 1882 1.1 0
30 ♂ 1000 1993 1.5 0
34 ♀ 1000 2287 0.4 2
35 ♀ 1000 717 0 1
36 ♀ 1000 1567 0 1
51 ♀ 1000 936 2.1 2
55 ♀ 1000 2204 0.9 0
Mean 1000 1716 1.0 0.8
1s   537 0.8 0.8

Table 4
TCD Emulsifier 250 mg/kg i.p. application, Sacrifice 48 hours after treament
 
randon number and sex number of evaluated polychromatic erythrocytes number of normochromatic erythrocytes per 1000 PCE micronucleated cells per 1000
normochromatic erythrocytes polychromatic erythrocytes
 
 1 ♂ 1000 673 1.5 0
2 ♂ 1000 1061 1.9 2
6 ♂ 1000 3858 1.3 0
8 ♂ 1000 1634 0.6 0
22 ♂ 1000 648 0 1
31 ♀ 1000 1857 0.5 2
37 ♀ 1000 1163 0 0
42 ♀ 1000 666 0 1
54 ♀ 1000 505 0 1
60 ♀ 1000 981 2.0 3
Mean 1000 1305 0.8 1.0
1s   1000 0.8 1.1

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was no indication of chromosomal damage after an intraperitoneal application of 250 mg/kg TCD Emulsifier in the in vivo mouse micronucleus test.
Executive summary:

In a mouse bone marrow micronucleus assay, 5 animals/sex/dose and timepoint were treated intraperitoneally with TCD Emulsifier (95.7% a.i.) at doses of 0 and 250 mg/kg bw. Bone marrow cells were harvested at 16 hours (treated animals), at 24 hours (negative/positive control and treated animals) and at 48 hours (treated animals) post-treatment. The vehicle was corn oil and the applied volume was 5 ml/kg bw for the negative control and treatment groups and 10 ml/kg bw for the positive control group.

Animals treated with the test compound showed the following clinical symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm, twitching, shivering and difficulty in breathing. One animal died due to acute toxicity of 250 mg/kg bw TCD Emulsifier Additionally, the ratio of polychromatic to normochromatic erythrocytes was decreased by the treatment in comparison to the negative control value.

TCD Emulsifier was tested at an adequate dose, as the chosen dose of 250 mg/kg bw was based on the results of pilot study. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time tested.

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.