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:
16 January 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A GLP study done according to OECD guideline 474. Although the certificate of analysis for the test item is not included, sufficient basic details including the composition are provided.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Details:
- Name of test material (as cited in study report): TRIGONOX 44B

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

NMRI BR mice (SPF) were used as test system. These mice are recommended by international
guidelines (e.g. EPA, FDA, OECD, EEC). Females were nulliparous and non-pregnant. The
animals were provided by Charles River, Sulzfeld, Germany.

In the micronucleus main test 5 male and 5 female mice were treated per sampling time In each
treatment group. Young adult animals were selected (6-8 weeks old).

The body weights of the mice at the start of the treatment are presented in table 2. An unique
number on the tail identified the mice. The animals were allocated to treatment groups as they
came to hand from the delivery boxes.

ANIMAL HUSBANDRY

Environmental Conditions
The animals were housed in an air-conditioned room with approximately 15 air changes per hour
and a conlrolled environment with a temperature of 21 ± 3°C and a relative humidity of 30-70%.
Due to cleaning procedures or performance of functional observations in the room, temporary
deviations from the maximum level for humidity (with max. 20%) occurred. Based Qn laboratory
historical data these deviations are considered not to affect the study integrity. The room was
illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Accommodation
On arrival and at the start of the treatment, all animals were clinically examined to ensure
selected animals were in a good state of health. Group housing of 5 animals per sex per cage
In labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk,
Rosenberg, Germany). Paper bedding was provided as nest material (B.M.I. Helmond, The Netherlands).
Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period
was at least 5 days before start of treatment under laboratory conditions.

Diet
Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany).
Certificates of analysis were examined and then retained in the NOTOX archives.

Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and then
retained in the NOTOX archives.

Study Plan Start: 20 October 2003; Completion: 7 December 2003

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
0.9% NaCl
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Test Substance preparation
TRIGONOX 44B was dissolved in physiological saline (B. Braun, Melsungen, Germany).
TRIGONOX 44B concentrations were dosed within 4 hours after preparation.

Reference substances:
1. Negative Control: Negative control animals were treated with physiological saline.
2. Positive Control: The positive conlrol used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0;
Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (B. Braun, Melsungen, Gennany)
dosed as a single intraperitoneal injection of 50 mg saltlkg body weight.
Duration of treatment / exposure:
Single injection
Frequency of treatment:
Treatment
The mice received an intraperitoneal Injection of a maximum tolerated (high), an intermediate and a low dose of TRIGONOX 44B.
Ttie route of administration was chosen to maximize the chance of the test article reaching the target tissue.
The dosing volume was 10 ml/kg body weight.
The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.
Post exposure period:
Micronucleus main test
Five male and five female mice were used per sampling time in each treatment group. The animals were dosed once and sampled according to the following scheme.

Treatment Dose (mg/kg body weight) Sampling time (hrs) Group

0.9% NaCI -- 24 A
TRIGONOX 44B 1500 24,48 B,C
TRIGONOX 44B 750 24 D
TRIGONOX 44B 375 24 E
Cyclophosphamide 50 48 F
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
No. of animals per sex per dose:
Five male and five female mice were used per sampling time in each treatment group. The animals were dosed once and sampled according to the table described under the section, "post exposure period".
Positive control(s):
Reference substances:

The positive conlrol used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0;
Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (B. Braun, Melsungen, Gennany)
dosed as a single intraperitoneal injection of 50 mg saltlkg body weight.

Negative control animals were treated with physiological saline.

Examinations

Tissues and cell types examined:
Bone marrow of TRIGONOX 44B-treated groups was sampled 24 or 48 hrs after dosing. Bone marrow of the negative and positive control groups were isolated 24 and 48 hrs after dosing, respectively. The bone was flushed and the cell suspension prepared. Bone marrow smears were prepared from these suspensions.The slides were automatically stained using the "Wright-stain-procedure". The dry slides were dipped in xylene, embedded in MicroMount and mounted with a coverslip. Slides were scored at 1000X magnification. The number of micronuclealed polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by simultaneously counting and differentiating the first 1000 erythrocytes. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

In-life Observations:
The systemic toxic signs were recorded at least once a day. The animals were weighed just prior to dosing.
Details of tissue and slide preparation:
Bone marrow cells: Bone marrow of TRIGONOX 44B-treated groups was sampled 24 or 48 hrs after dosing. Bone marrow of the negative and positive control groups were isolated 24 and 48 hrs after dosing, respectively. Animals were sacrificed, both femurs removed and both ends of the bone were shortened until the marrow canal became visible. The bone was flushed with fetal calf serum (2 mL), and the cell suspension centrifuged at 1000 rpm (~100 g) for 5 min.

Bone marrow smears: The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum with the remaining serum. A drop of the cell suspension was placed on the end of a slide (2 slides / animal), which was previously cleaned (24
h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether) and marked with study identification and the animal numbers. The drop was spread by a clean slide at an angle of ~45· over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight.

Bone marrow smears: The slides were automatically stained using the "Wright-stain-procedure". The dry slides were dipped in xylene, embedded in MicroMount and mounted with a coverslip.

Analysis of the micronuctei: All slides were randomly coded before examination. An adhesive identification label (with a code) was stuck over the marked slide. At first the slides were screened at 100x magnification for regions of suitable technical quality (cells well spread, undamaged and well stained). Slides were scored at 1000X magnification. The number of micronuclealed polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by simultaneously counting and differentiating the first 1000 erythrocytes. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
Acceptability of the assay:
A micronucleus test is considered acceptable if it meets the following criteria:

a) The positive control substance induced a statistically signifIcant (Wilcoxon Rank Sum Test,
two-sided test at P < .0.05) increase In the frequency of micronucleated polychromatic
erythrocytes.

b) The incidence of micronucleated polychromatic erythrocytes in the control animals should
reasonably be within the laboratory historical control data range (mean ± three times the
standard deviation): Males: 1.2%o:i: 3.6%0 indicated are means for n=229. Females: 1.3% ±
3.9% indicated are means for n=150).

Statistics:
Data evatuatlon and statistical procedures:

Equivocal results should be clarified by further testing using modification of experimental
conditions.
A test substance Is considered positive In the micronucfeus test if:
It induced a biologically as well as a statistically signWicant (Wilcoxon Rank Sum Test; twosided
test at P < 0.05) increase In the frequency of micronucleated polychromatic erythrocytes
(at any dose or at any sampling time) In the combined data for both sexes or in the data for
male or female groups separately.

A test Substance is considered negative In the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P <
0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the
combined data for both sexes nor In the data for male or female groups separately.
The preceding criteria are not absolute and other modifying factors may enter into the final
evaluation decision.

Please see Appendix III (Statitics) under "Any other results" section.

Results and discussion

Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: In a dose range finding study 8 animals (group A: 1 male and 1 female, group B: 3 males and 3 females) were dosed intraperitoneally (single dose) with 2000 and 1500 mglkg body weight (groups A and B respectively).

- Solubility: No data provided.
- Clinical signs of toxicity in test animals: The following toxic signs were observed in the animals dosed with 2000 mg/kg body weight: lethargy, ataxia, no reaction to stimulus (only observed at one hour after dosing), ventre-lateral recumbency, a hunched posture and a rough coat. At the dose level of 1500 mg/kg body weight the same toxic signs were observed as in Ihe animals treated with 2000 mg/kg, but the time that the animals showed no reaction to stimulus was shorter in these animals. Therefore, a dose level of 1500 mg/kg body weight was used as the highest dose level for the main study.

- Rationale for exposure: The group comprising 3 males and 3 females were dosed with the highest concentration that was used for the main study. The study duration per dosing was one to four days. During this period mortality and physical condition were recorded at least once daily. At the dose level of 1500 mg/kg body weight the same toxic signs were observed as in Ihe animals treated with 2000 mg/kg, but the time that the animals showed no reaction to stimulus was shorter in these animals. Therefore, a dose level of 1500 mg/kg body weight was used as the highest dose level for the main study.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay) is described

Micronucleus main test results:
Based on the results of the dose range finding study dose levels of 375, 750 and 1500 mg/kg, body weight were selected as appropriale doses for the micronucleus main test. Five male and five female animals were used in each treatment group. The mean body weights per group recorded immediately prior to dosing are presented in Table 2.

Mortality and systemic toxic signs - In-life Observations:
The systemic toxic signs were recorded at least once a day. The animals were weighed just prior to dosing. The animals of the groups treated with 375 mg TRIGONOX 44B /kg body weight and the animals of the negative and positive control groups showed no abnormalities. (Appendix I (Tables 1 and 2). The following clinical observations were made in the groups treated with 750 and 1500 mg TRIGONOX 44B /kg body weight:
During the first hour after dosing all animals of the groups treated wilh 1500 mg/kg body weight were lethargic, showed ventre-lateral recumbency and no reaction to stimulus. In the group treated with 750 mg!kg body weight all animals were lethargic and showed ataxia. Within 2 hours after dosing in the groups treated with 1500 mg/kg body weight all animals were lethargic. Thirteen animals additionally had a rough coat. Six animals also showed a hunched posture. The animals dosed with 750 mg/kg body weight showed no abnormalities. Within 42 hours after dosing all animals had recovered from the treatment.

Micronucleated polychromatic erythrocytes
The mean number of micronucleated polychromatic erythrocyles per group and the mean ratio of polychromatic to normochromatic eryihrocyles are presented in Table 3 (Appendix I). The individual data are described in Appendix II. The mean number of micronucleated polychromatic erythrocytes scored in TRIGONOX 44B treated groups were compared with the corresponding solvent control group.

No increase in the frequency of micronucleated polychromatic erythrocyles was observed in the polychromatic erythrocytes of the bone marrow of TRIGONOX 44B treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.

Any other information on results incl. tables

APPENDIX I

 

TABLES

 

Table1. Mortality and systemic toxic signs after treatment with TRIGONOX44Bin the dose range finding study

Group 

 

 Sex 

 

 Animal 

number 

 Dose 

 mgIkg 

                                 Systemic toxic signs 

           day1 within 

 day· 2 

 day 3 

 day4 

10 min.

1 hr

4 hrs

 A 

 Male 

 1 

 2000 

 FC 

 FGX 

 FNY 

 B 

 B 

 B 

 A 

 Female 

 2 

 2000 

 FC 

 FGX 

 FX 

 B 

 B 

 B 

 B 

 Male 

 3 

 1500 

 FC 

 FGX 

 N 

 B 

 B 

 

 B 

Female 

 4 

 1500 

 FC 

 FGX 

 FNY 

 B 

 B 

 

 B 

 Male 

 5 

 1500 

 FC 

 FGX 

 FNY 

 B 

 B 

 

 B 

 Male 

 6 

 1500 

 FC 

 FGX 

 FNY 

 B 

 B 

 

 B 

 Female 

 7 

 1500 

 FC 

 FGX 

 FN 

 B 

 B 

 

 B 

 Female 

 8 

 1500 

 FC 

 FGX 

 NY 

 B 

 B 

 

• Legend 'Mortality and systemic toXic signs':

B=showed no abnormalities; C=ataxia; Flethargy; Gno reaction to stimulus; N=rough coat; X=

ventro-lateral recumbency; Y=hunched posture

Table 2 Meanbody weightimmediately prior to dosingwithTRIGONOX44B

Group

Body weight (g) (mean±S.D.)(1)

 

MALES                                  FEMALES

A

32.8± 1.5

26.4±1.5

B

31.8±1.8·

27.2±1.3

C

32.0 ± 2.2

27.4±1.1

D

32.4±2.3

26.6±0.9

E

32.0±2.0

27.2±1.3

F

32.0±2.4

25.6:t0.9

(1) Five animals per treatment group

Table 3. Mean number of micronucleated polychromatic erythrocytesper2000

polychromatic erythrocyte and ratio of polychromatic/normochromatic erythrocytes

Group 

 

 

 

 Treatment 

 

 

 

 Dose (mg/kg 

 body weight) 

 

 

Sampling

time 

 (hours) 

 

 No. of micronucleatedpolychromatic erythrocytes  per 2000 polychromatic erythrocytes (mean±SD)(1) 

 Ratio polychromatic/ 

 nonnochromatic 

 erythrocytes 

 (mean±S.D.)(1) 

MALES

 A 

 Solvent control 

 0 

 24 

 1.6±0.5 

 0.97 :to.02 

 B 

 TRIGONOX 44B 

 1500 

 24 

 2.0 :t 1.6 

 0.95 ±0.05 

 C 

 TRIGONOX 44B 

 1500 

 48 

 2.4 :t 1.5 

 0.96 :to.06 

 D

TRIGONOX 44B 

 750 

 24 

 2.4 ± 1.1 

 0.96 :to.03 

 E 

 TRIGONOX 44B

 375 

 24 

 1.4 ± 0.5 

 0.96 :to.05 

 F 

 CP 

 50 

 48 

 24.8 ± 8.0(2) 

 0.51 to.08 

FEMALES

 A 

 Solvent control 

 0 

 24 

 1.2 ± 0.8 

 0.95 ±0.01 

 8 

TRIGONOX 44B

 1500 

 24 

 3.4±1.1 

 0.97 ±0.04 

 C 

TRIGONOX 44B

 1500 

 48 

 1.4 ± 1.5 

 0.91 :to.05 

 D

TRIGONOX 44B

 750 

 24 

 3.2 ± 1.5 

 0.96 :to.04 

 E 

TRIGONOX 44B 

 375 

 24 

 1.6±0.5 

 0.97 :to.03 

 F 

 CP 

 50 

 48 

 23.0 ± 4.7(2) 

 0.51 :to.10 

Solvent control=physiological saline

CP=Cyclophosphamide

(1) Five animalspertreatment group

(2) Significantiy different from corresponding control group (Wilcoxon Rank Sum Test, P<0.01).

APPENDIX II INDIVIDUAL DATA

Individual data (males)

(groupA :intraperitoneal injection of physiological saline)

(group B & C: intraperitoneal injection of TRIGONOX 44B at 1500mg/kgbody weight)

(group D:intraperitoneal injection of TRIGONOX44Bat 750mglkgbody weight)

(groupE :intraperitoneal injection ofTRIGONOX44Bat 375 mg/kg body weight)

(groupF :intraperitoneal injection of cyclophosphamide)

Group

 

 

Animal number 

Number of mlcronucleated 

 polychromatic erythrocytes / 2000 polychromatic erythrocytes 

 Ratio polychromatic/normochromatlc 

 erythrocytes 

 

 A 

 1 

 2 

 0.99 

 A 

 2 

 1 

 0.98 

 A 

 3 

 2 

 0.97 

 A 

 4 

 1 

 0.97 

 A 

 5 

 2 

 0.94 

 B 

 11 

 1 

 0.90 

 B 

 12 

 2 

 0.96 

 B 

 13 

 4 

 0.93 

 B 

 14 

 0 

 0.91 

 B 

 15 

 3 

 1.03 

 C 

 21 

 3 

 0.96 

 C 

 22 

 2 

 1.03 

 C 

 23 

 3 

 0.99 

 C 

 24 

 0 

 0.91 

 C 

 25 

 4 

 1.00 

 0 

 31 

 2 

 0.95 

 0 

 32 

 4 

 0.99 

 0 

 33 

 2 

 0.93 

 0 

 34 

 3 

 0.93 

 0 

 35 

 1 

 0.98 

 E 

 41 

 1 

 0.88 

 E 

 42 

 2 

 1.01 

 E 

 43 

 1 

 0.98 

 E 

 44 

 2 

 0.92 

 E 

 45 

 1 

 0.99 

 F 

 51 

 34 

 0.43 

 F 

 52 

 19 

 0.56 

 F 

 53 

 18 

 0.45 

 F 

 54 

 20 

 0.63 

 F 

 55 

 33 

 0.48 

Individual data (females)

(groupA :intraperitoneal injection of physiological saline)

(group B & C: intraperitoneal injection of TRIGONOX 44B at 1500mg/kgbody weight)

(group D:intraperitoneal injection of TRIGONOX44Bat 750mglkgbody weight)

(groupE :intraperitoneal injection ofTRIGONOX44Bat 375 mg/kg body weight)

(groupF :intraperitoneal injection of cyclophosphamide)

Group 

Animal number 

Number of mIcronucleated 

 polychromatic erythrocytes / 2000 polychromatic erythrocytes 

 Ratio polychromatic/normochromatlc 

 erythrocytes 

 

 A 

 6 

 1 

 0.95 

 A 

 7 

 2 

 0.94 

 A 

 8 

 0 

 0.94 

 A 

 9 

 2 

 0.97 

 A 

 10 

 1 

 0.95 

 B 

 16 

 5 

 0.96 

 B 

 17 

 3 

 0.94 

 B 

 18 

 4 

 1.01 

 B 

 19 

 3 

 0.93 

 B 

 20 

 2 

 1.03 

 C 

 26 

 1 

 0.B9 

 C 

 27 

 0 

 0.B5 

 C 

 2B 

 0 

 0.B9 

 C 

 29 

 3 

 0.97 

 C 

 30 

 3 

 0.96 

 D 

 36 

 3 

 0.90 

 D 

 37 

 1 

 0.99 

 D 

 36 

 5 

 0.96 

 D 

 39 

 4 

 0.96 

 D 

 40 

 3 

 0.95 

 E 

 46 

 1 

 0.94 

 E 

 47 

 2 

 0.96 

 E 

 4B 

 2 

 1.00 

 E 

 49 

 2 

 1.01 

 E 

 50 

 1 

 0.94 

 F 

 56 

 31 

 0.47 

 F 

 57 

 23 

 0.39 

 F 

 5B 

 21 

 0.59 

 F 

 59 

 19 

 0.46 

 F 

 60 

 21 

 0.63 

APPENDIX III  STATISTICS

Wilcoxon Rank Sum Test.

 

Number of micronucleated polychromatic erythrocytes per2000polychromatic erythrocytes;

Treatment/conlrol comparison.

Group 

 

 

 Treatment 

 

 

 Dose 

 mg/kgbody weight 

 Sex 

 

 

P-value  (two-sided) 

 

Decision at 95%  confidence level 

 

 F 

cyclophosphamide 

 50 

males 

<0.01 

 significant 

 F 

cyclophosphamide 

 50 

females 

<0.01 

 significant 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
II is concluded that this test Is valid and that TRIGONOX 44B is not mutagenic in the
micronucleus test under the experimental conditions described in this report.
Executive summary:

TRIGONOX 44B was tested in the Micronucleus Test in mice, to evaluale its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the following guidelines:

• European Economic Community (EEC), Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.12: "Mutagenicity: In Vivo Mammalian Erythrocyte Micronucleus Test" .

• OECD Guidelines for the Testing of Chemicals, Guideline No. 474: Mammalian Erythrocyte Micronucleus Test (adopted 21st July 1997).

Batch 0410306045344 of TRIGONOX 44B was a clear colourless liquid. The test substance was dissolved in 0.9% NaCI. Five male and fIve female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with TRIGONOX 44B at 1500 (two groups), 750 (one group), and 375 (one group) mg/kg body weight. After dosing the animals of the dose level of 1500 mglkg body weight showed the following toxic signs: lethargy, no reactlon to stimulus, ventra-lateral recumbency, rough coat and a hunched posture. All animals of lhe dose level of 750 mglkg body weight were lethargic and showed ataxia after dosing. The animals of the dose level of 375 mglkg body weight showed no abnormalities after dosing. Bone marrow of the groups treated with TRIGONOX 44B was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and postive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with TRIGONOX 44B. The incidence of micro nucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromalic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met. The groups that were treated wilh TRIGONOX 448 showed no decrease in the ratio of polychromatic to normochromalic erythrocytes compared to the vehicle conlrols, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromallc erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is cencluded that TRIGONOX 44B is not mutagenic in the micronucleus test under the experimental conditions described in this report.