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Toxicological information

Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Feb 2017 to 9 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Feb 2017 to 9 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: 60700109
- Purtiy: ≥ 99%
- Date of receipt: 25 Jul 2016
- Expiration date: 14 Jul 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature (15 - 25 °C)

OTHER SPECIFICS:
- Appearance: Yellow powder
Species:
rat
Strain:
Wistar
Remarks:
(Crl:WI(Han))
Details on species / strain selection:
- Justification for strain selection: The Wistar strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The animals were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 188.5 - 234.2 g
- Assigned to test groups randomly: yes
- Housing: The animals were housed two to five animals per cage. All animals were housed in Makrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly. Animals treated with the positive controls were housed in filter top cages after dosing.
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). Each batch of diet was analyzed by the supplier for nutrients and contaminants. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that serves to prevent spillage. The feed in the feeders were replaced with fresh portions once weekly and filled up as needed.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Vitens.
- Acclimation period: 11 - 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 45 - 65 %. Occasionally, the relative humidity briefly exceeded 65% due to meteorological circumstances or because of wet cleaning activities.
- Air changes: 10 per hour
- Photoperiod: 12 hours light and 12 hours dark.
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Concentration of test material in vehicle: 1-2.5 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Batch no.: A1600985
- Purity: 100%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
For each day of the study and for each test substance group, the appropriate amount of test substance was weighed in a glass bottle. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred until visual homogeneity was obtained. All suspensions were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test substance in the vehicle.
Duration of treatment / exposure:
Three successive days.
Frequency of treatment:
Daily.
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five. One surplus animal was treated in the same way as the five other males of these groups. These animals were finally not needed in the study and killed interim.
Control animals:
yes, concurrent vehicle
Positive control(s):
INTESTINE
Methyl methane sulfonate (MMS)
- Route of administration: once intraperitoneally, ca. 3 hours before sacrifice.
- Doses / concentrations: 60 mg/kg-bw (stock concentration 6 mg/ml; dosing volume 10 ml/kg-bw)

LIVER
2-acetylaminofluorene (2-AAF)
- Route of administration: once orally (by gavage), ca. 12-16 hours before sacrifice.
- Doses / concentrations: 50 mg/kg-bw (stock concentration 2.5 mg/ml; dosing volume 20 ml/kg-bw)
Tissues and cell types examined:
Liver / hepatocytes and jejunum / intestinal cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- The highest concentration of the test substance was intended to result in toxic effects but not death or severe suffering.
- The lowest concentration was intended to produce little or no evidence of toxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Ca. 24 h interval between the first and second dose and ca. 21 h between the second and third dose. The third dose was administered ca. 3 h before scheduled sacrifice.

ISOLATION OF HEPATOCYTES
Hepatocytes were isolated from the liver of the animals of the negative control, animals treated with the test substance and the positive control using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat were perfused in situ with a HEPES buffer (0.01 M) whilst under sodium pentobarbital anaesthesia and exsanguination from the abdominal aorta, followed by an in vitro perfusion with a HEPES-buffered (0.1 M) collagenase solution. Directly after the start of the perfusion to remove the blood from the tissue, a small part of the caudate lobe was tied off using a ligature. Subsequently, part of the lobe was removed and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin). Since a positive response was not observed, these samples were not used for histological evaluation to determine whether cytotoxicity was involved. After isolation, the dissociated cells were incubated for 5-10 minutes in a shaking water bath at 37 ºC. Thereafter, they were filtered over a nylon filter (200 μm), centrifuged and resuspended in Williams medium E. Cell counts were made and the viability of the hepatocytes were determined by trypan blue exclusion.

ISOLATION OF INTESTINAL CELLS
As soon as possible after liver perfusion or sacrifice (positive control group), the first part of the jejunum (ca. 20 cm from the stomach) of each rat was collected and kept in ice-cold Krebs-Ringer bicarbonate buffer (Sigma-Aldrich Chemie B.V., pH set at ca. 7.3) until further processing. The tissue were finely minced and homogenized in 2 mL pre-warmed (37 ºC) collagenase solution (1 mg/ml in Krebs-Ringer buffer (pH ca. 7.3), supplemented with 1.26 g/L NaHCO3, 6.3 mM CaCl2, and 1% bovine serum albumin). After a short incubation (ca. 5 min) at ambient temperature, the cell suspension was filtered twice (500 μm, followed by 70 μm) and centrifuged (500 rpm, 3 min, ca. 4 ºC). The cells were resuspended in a sufficient volume for slide preparation. The viability of the cells were determined by trypan blue exclusion. In addition, a small piece of lung was preserved in neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin) for possible histopathological examination. In case of a positive response, this sample would be used for histological evaluation to determine whether cytotoxicity was involved.

PREPARATION OF SLIDES
Microscopic slides was prepared by mixing an aliquot of the cell suspension with a low-melting agarose solution (0.5 % (w/v) in Phosphate Buffered Saline). Subsequently, this mixture was loaded on a glass slide, pre-coated with normal-melting agarose (1.5 % (w/v) in PBS), and mounted with a coverslip. Three slides per animal were prepared (one slide was kept in reserve). The slides were stored on a cold plate until the agarose has solidified. Subsequently, the coverslip was removed and the slide was incubated in lysis buffer (2.5 M NaCl, 0.1 M Na2EDTA, 0.175 M NaOH, 0.01 M Tris in Milli-Q water, supplemented with 1 % Triton X-100 (w/v), pH 10) overnight at 2-10 ºC. Subsequently, slides were incubated in ice-cold electrophoresis buffer (0.3 M NaOH, 0.001 M Na2EDTA in Milli-Q water, pH >13) for 30 ± 1 min, following electrophoresis (ca. 32V and 360 mA) for 30 ± 1 min in ice-cold electrophoresis buffer, while cooled on ice. After incubation in neutralisation buffer (0.4 M Tris in Milli-Q water, pH 7.5) for at least 5 min, slides were dehydrated by incubating in ethanol at room temperature and air-dried.

SLIDE ANALYSIS AND COUNTING
Slides were coded by a qualified person not involved in analysing the slides to enable ‘blind’ scoring. Slides were stained with ethidium bromide solution (20 μg/mL in Milli-Q water) which was directly pipetted on the slide and covered with a coverslip just before analysis. A fluorescent microscope connected to a camera and Comet Assay IV software (Perceptive Instruments) was used for the analysis of the slides. Seventy-five cells (randomly selected starting from the center of the slide) per slide and two slides per animal were analysed to yield a total number of 150 cells per animal, except that for two animals (one in mid dose of test substance group and one in positive control MMS group) in total 123 and 162 intestinal cells were analysed from three slides due to very low cell density on the two comet slides that were initially analysed, respectively. For the animal in the mid dose group, only 105 cells analysed from two slides were used for calculation of the animal mean.

Ghost cells, with a small head and a diffuse and large tail, were excluded from analysis, but their presence was recorded. Microscopic slides will be retained for at least one month after finalisation of the report and then discarded.
Evaluation criteria:
EVALUATION CRITERIA
- The viability of the hepatocytes and intestinal cells of the negative control animals should be at least 50 %.
- The assay was considered valid if the group mean tail intensity of the positive control groups (for intestine and liver) showed a statistically significant increase compared to the group mean of the negative control group and if the group mean tail intensity of the negative control was comparable to the historical range.
- The test substance was considered to be positive in the in vivo comet assay if a statistically significant increase was observed at one or more dose levels compared to the group mean of the negative control group and/or if a dose related increase in the group mean tail intensity was observed. Positive results from the in vivo comet assay indicated that the test material has the potential to induce primary DNA damage in vivo in the tissue evaluated, under the conditions used in this study.
- The test substance was considered to be negative in the in vivo comet assay if no statistically significant increase was observed at any of the dose levels compared to the group mean of the negative control group. Negative results indicated that the test material did not have the potential to induce DNA damage in vivo in the tissue evaluated, under the test conditions used in this study.
- Biological relevance was taken into account for interpretation of the results. If a positive response in the comet assay was obtained, the possibility that the increase in DNA migration was not associated with genotoxicity, but with severe toxicity, was assessed. Furthermore, the likelihood that the test substance or its metabolites reached the target tissues is discussed.
Statistics:
Two ANOVA models were applied. In the first ANOVA model it was tested if the positive control differs from the negative control (unpaired t test). In a second ANOVA model, it was tested if the negative control differed from the test substance (different doses) using the Dunnett’s multiple comparisons test. For both models and for both tissues the ANOVA assumptions were met (i.e. variances were equal). In all statistical tests, a significance level of 5% was used (α = 0.05). All statistical tests were performed using GraphPad Prism®, Version 6.04, January 17, 2014. 1992-2014 GraphPad Software, Inc., CA, USA.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Tail intensity of the test substance was comparable to the negative control and did not demonstrate a statistically significant increase in tail intensity both in intestinal and liver cells at any of the concentrations tested. However, tail intensity of the test substance was just outside the historical range for intestinal cells at the lowest concentration tested. Since this increase was observed at the lowest concentration only and was not dose-related, the response was considered not biologically relevant.

Table: Mean tail intensity of intestinal and liver cells of rats after oral exposure to the test substance.

Treatment

Tail intensity intestinal cells (mean ± SD)

Tail intensity liver cells (mean ± SD)

Negative control (corn oil)

6.71 ± 2.10

3.07 ± 1.56

5 mg/kg bw/day

15.12 ± 7.37

1.90 ± 0.75

15 mg/kg bw/day

8.50 ± 5.38

2.05 ± 1.16

45 mg/kg bw/day

9.79 ± 12.09

2.06 ± 1.04

Positive control (intestine, MMS)

48.67 ± 4.95*

Not determined

Positive control liver, 2-AAF)

Not determined

15.92 ± 1.66*

*Statistically significantly different from negative control, P-value <0.0001 (t-test)

CLINICAL SIGNS AND BODY WEIGHT

No mortality was observed and no treatment-related clinical signs were observed in the animals during the study period. The few clinical signs noted (skin encrustations, and irregular respiration (a single animal of the negative control group only)) occurred randomly throughout the groups in a few animals only and are not treatment related.

Group mean body weights in all groups were considered within the normal range as expected for healthy rats of this age and strain. There were no statistically significant effects on mean body weight following treatment with the test substance.

VALIDITY OF THE RESULTS

- The percentage viability of the isolated intestinal cells was 94%, 92%, 98% and 95% for the negative control, 5 mg/kg bw, 15 mg/kg bw and 45 mg/kg bw of the test substance, respectively, whereas the percentage viability of the isolated hepatocytes was 93%, 91%, 94% and 93%, respectively. Based on the observed viability, all cell suspensions were considered suitable for the comet assay.

- The positive control substances MMS and 2-AAF demonstrated a statistically significant increase in tail intensity compared to the negative control for intestine and liver, respectively (p-value: <0.0001 for both tissues). Mean tail intensity of the negative control was within the historical range for both intestine and liver. Therefore, the comet assay was considered valid.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrakis(diethyldithiocarbamato-S,S')tellurium
EC Number:
244-121-9
EC Name:
Tetrakis(diethyldithiocarbamato-S,S')tellurium
Cas Number:
20941-65-5
Molecular formula:
C20H40N4S8Te
IUPAC Name:
tetrakis(diethyldithiocarbamato-S,S')tellurium
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: 60700109
- Purtiy: ≥ 99%
- Date of receipt: 25 Jul 2016
- Expiration date: 14 Jul 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature (15 - 25 °C)

OTHER SPECIFICS:
- Appearance: Yellow powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
(Crl:WI(Han))
Details on species / strain selection:
- Justification for strain selection: The Wistar strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The animals were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 188.5 - 234.2 g
- Assigned to test groups randomly: yes
- Housing: The animals were housed two to five animals per cage. All animals were housed in Makrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly. Animals treated with the positive controls were housed in filter top cages after dosing.
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). Each batch of diet was analyzed by the supplier for nutrients and contaminants. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that serves to prevent spillage. The feed in the feeders were replaced with fresh portions once weekly and filled up as needed.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Vitens.
- Acclimation period: 11 - 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 45 - 65 %. Occasionally, the relative humidity briefly exceeded 65% due to meteorological circumstances or because of wet cleaning activities.
- Air changes: 10 per hour
- Photoperiod: 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Concentration of test material in vehicle: 1-2.5 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Batch no.: A1600985
- Purity: 100%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
For each day of the study and for each test substance group, the appropriate amount of test substance was weighed in a glass bottle. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred until visual homogeneity was obtained. All suspensions were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test substance in the vehicle.
Duration of treatment / exposure:
Three successive days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five. One surplus animal was treated in the same way as the five other males of these groups. These animals were finally not needed in the study and killed interim.
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C (MMC)
- Route of administration: intraperitoneally, ca. 24 h before sacrifice
- Doses / concentrations: 1.5 mg/kg bw (stock concentration 0.15 mg/mL in physiological saline; dosing volume 10 mL/kg bw)

Examinations

Tissues and cell types examined:
Bone marrow / bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- The highest concentration of the test substance was intended to result in toxic effects but not death or severe suffering.
- The lowest concentration was intended to produce little or no evidence of toxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Ca. 24 h interval between the first and second dose and ca. 21 h between the second and third dose. The third dose was administered ca. 3 h before scheduled sacrifice.

BONE MARROW COLLECTION AND PROCESSING
Immediately following liver perfusion (control and treatment groups) or sacrifice at ca. 24 h after intraperitoneal injection (positive control), the bone marrow cells of one of the femurs were collected into foetal calf serum and processed into glass-drawn smears according to the method described by Schmid (1976). Four bone marrow smears per animal were made, air-dried and fixed in methanol. Two fixed smears were stained with a May-Grünwald Giemsa solution. The other fixed smears were kept in reserve. If needed, these reserve slides would be stained with May-Grünwald Giemsa solution and used for microscopic examination.

MICROSCOPIC EXAMINATION OF THE BONE MARROW SMEARS
- Slides were coded by a qualified person not involved in analysing the slides to enable ‘blind’ scoring. Slides (two per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation are evenly distributed over the whole smear.
- The numbers of polychromatic erythrocyte (PE) and normochromatic erythrocyte (NE) were recorded in a total of at least 500 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 500 E (PE + NE) have been scored, an additional number of PE was scored for the presence of micronuclei until a total of 4000 PE has been scored. In case of high cytotoxicity (<10% PE per total E) scoring of all E was continued until a total of 6000 E has been reached.
Evaluation criteria:
SCORING CRITERIA
- A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
- A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
- A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.

EVALUATION CRITERIA
- The study was considered valid if the positive control group showed a statistically significant increase in the mean number of MPE/4000 PE compared to the negative control group, and if the negative control group was within the historical range.
- The test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a dose related positive response or a statistically significant increase of micronucleated cells in one or more dose groups when compared to the negative control group.
- The test substance was considered to be negative in the micronucleus test if it did not produce a positive response at any of the dose levels analysed.
- Biological relevance and the likelihood that the test substance or its metabolites reached the target tissues was taken into account for interpretation of the results.
Statistics:
The mean ratio PE/E and MPE/4000 PE were calculated for each group.
Two ANOVA models were applied for both PE/500E and MPE/4000PE. The first ANOVA model is used to test if the positive control differed from the negative control (t test). In a second ANOVA model, it was tested if the negative control differed from the test substance (different doses). For both models it was checked if the ANOVA assumptions were met (i.e. if variances were equal). This was the case for the second ANOVA model, but not for the first. Therefore, non-parametric testing was performed using the Mann Whitney test to if the positive control differed from the negative control. In all statistical tests a significance level of 5% was used (α = 0.05). All statistical tests were performed using GraphPad Prism®, Version 6.04, January 17, 2014. 1992-2014 GraphPad Software, Inc., CA, USA.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There was no statistically significant increase in the mean numbers of MPE/4000 PE in the groups treated with the test substance when compared to the negative control group. This indicated that oral administration of the test substance, up to 45 mg/kg-bw (by gavage), did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells. Statistical analysis of the test results indicated that there was no statistically significant difference in the mean number of PE/500E when comparing the animals treated with the test substance when compared to the negative control group.

Any other information on results incl. tables

Table: Mean numbers of the PE/500 E and MPE/4000 PE observed in the bone marrow micronucleus test in rats after oral exposure to the test substance.

Treatment

PE/500 E (mean ± SD)

MPE/4000 PE (mean ± SD)

Negative control (corn oil)

259 ± 49

2.4 ± 1.8

5 mg/kg bw/day

221 ± 65

6.0 ± 3.4

15 mg/kg bw/day

197 ± 61

4.4 ± 2.3

45 mg/kg bw/day

223 ± 49

5.8 ± 2.3

Positive control (MMC)

202 ± 22*

49.6 ± 51.9**

* Statistically significantly different from negative control, P-value <0.0430 (t-test)

** Statistically significantly different from negative control, P-value 0.0397 (Mann-Whitney)

CLINICAL SIGNS AND BODY WEIGHT

No mortality was observed and no treatment-related clinical signs were observed in the animals during the study period. The few clinical signs noted (skin encrustations, and irregular respiration (a single animal of the negative control group only)) occurred randomly throughout the groups in a few animals only and are not treatment related.

Group mean body weights in all groups were considered within the normal range as expected for healthy rats of this age and strain. There were no statistically significant effects on mean body weight following treatment with the test substance.

VALIDITY OF THE RESULTS

A statistically significant decrease (P-value: 0.0430) in the mean number of PE/500 E in the positive control animals was observed, when compared to the negative control animals. This indicated that the positive control substance Mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male rats. The mean numbers of MPE/4000 PE in the negative control group were within the historical range when related to 2000 PE. Furthermore, the positive control animals demonstrated a statistical significant increase in the mean number of MPE compared to the negative control animals (P-value: 0.0397). The mean number of MPE found in the positive control Mitomycin C was within historical range when related to 2000 PE. However, two out of five animals of the positive control groups showed a response in the range of the negative control. The reason for the absence of a response in these animals is unknown (probably resulted from incorrect dosing with the positive control substance, although there were no observations to confirm a dosing error). Since the other three animals clearly showed a positive response, the micronucleus test was considered valid.

Applicant's summary and conclusion